Local Organizing Committee Welcome to the 18th ISMAR 2013/14th NMR Users Meet- Jose Daniel Figueroa Villar ing! IME, Chair I am very proud of your participation at this event, which Sonia Cabral de Menezes is the most important magnetic resonance event in Brazil PETROBRAS, Vice-Chair and Latin America in the last 27 years. The scientific Anita J. Marsaioli development of the world strongly depends on this topic, UNICAMP and each participant is certainly contributing with science and technology to improve human life condition. Claudia Jorge do Nascimento UNIRIO This meeting is organized including ten Plenary Lectures, four Parallel Sessions with more than 150 lectures and oral Dorila P. Veloso presentations, three Poster Sessions and exhibit booths of UFMG 17 companies. The event is financed by 24 industries and Fábio C.L. Almeida 7 Brazilian foment agencies. The Student Grants were UFRJ financed by 13 companies, a very important contribution Fernando Hallwass for this event, helping economically 37 students. UFPE Several actions necessary for all participants are financed by the event, for the comfort Jairton Dupont of participants like the wireless Internet system, freely available in the whole event UFRGS region and transportation to the meeting, which is free and arranged for all the event Jerson Lima da Silva participants and accompanying people. The respective information on transportation UFRJ and internet is part of this book. Luiz Alberto Colnago In this event we also have two satellite meetings, the Vth Iberoamerican NMR Meeting EMBRAPA (Friday afternoon) and the Biomedical Imaging Symposium (Wednesday afternoon). To see the programs and to participate at the satellite meetings, it is necessary to Naira M. da Silva Ruiz register for them at the event’s secretary desk. We strongly encourage everyone to PUC/RJ join! Rosane A. da Silva San Gil UFRJ The social activities will start with a Welcome Reception on Sunday after the opening, honor and prizes lectures. On Thursday evening we will have the Brazilian Night at Jochen Junker the Fogo de Chão Brazilian Steak House, including dinner, music shows and dancing. FIOCRUZ We strongly encourage your participation at this party, which will be a special op- Sandra Mello portunity for fraternization for all participants. Free transportation will be provided Event Production Coordination by the Meeting Organization. Rio de Janeiro is a nice city, with many attractive sites, including mountains, beaches, forests, parks and districts, restaurants, museums, shopping and show cen- ters. Wednesday and Friday afternoon/evening the ISMAR2013/14th NMR Users Meeting is also offering some excursions to the major touristic sites. I hope you will enjoy this event and Rio de Janeiro!

José Daniel Figueroa-Villar Chairman 18th ISMAR 2013/14th NMR Users Meeting

Contents

Committees ...... ii Program ...... 1

Honor and Prizes ...... iii Abstracts of Talks ...... 21

General Information ...... iv Poster Abstracts ...... 69

Map of the Event Locations ...... vii Index of Authors ...... 161

Event Sponsors ...... viii Directory of Participants ...... 172

Student Travel Awards ...... ix Late Participants ...... 186

i Committees

Committees

Scientific Committee ISMAR Officers

Ad Bax President Hans Wolfgang Spiess NIH, USA Germany Alejandro Vila Vice President Daniella Goldfarb IRB, Argentina Israel Angelo da Cunha Pinto Past President Ad Bax IQ/UFRJ, Brazil USA Antonio José da Costa Filho Secretary General Roxanne Deslauriers USP-RP, Brazil Canada Bob Griffin Treasurer David Ailion MIT, USA USA Carlos Geraldes Univ. of Coimbra, Portugal Eduardo Ribeiro Azevêdo USP-SC, Brazil Gunnar Jeschke ETH, Switzerland Lucio Frydman Weizmann Institute, Israel Miquel Pons Univ. of Barcelona, Spain Warren Warren Duke Univ., USA

ii Honor and Prizes

Honor and Prizes

ISMAR awards Prizes that recognize important accomplishments and developments in Magnetic Resonance and its applications to various fields of science. All awardees will present plenary lectures in the opening session on Sunday, May 19th. The principal Prize of ISMAR is the ISMAR Prize sponsored by Cambridge Isotope Laboratories for world renowned scientists, who have made outstanding contributions in the field of magnetic resonance. Former Prize winners include Anatole Abragam, Erwin Hahn, Jean Jeener, Charles Slichter and John Waugh to name just a few - see http://www.ismar.org/ismar-prize. This year’s Awardee is Professor Jack H. Freed, Cornell University, Ithaca, USA. Professor Freed is honored for the foundation of modern EPR through an extraor- dinary range of contributions from mathematics and theory to methodology and instrumentation; and for the application of his ingenious methods of pulsed EPR spectroscopy to fundamental problems in areas from chemistry to biophysics. In 2011 ISMAR established the Anatole Abragam Prize sponsored by Bruker Biospin to pay tribute to the outstanding achievements of the late Anatole Abragam who made seminal contributions to both NMR and EPR and was himself a recipient of the ISMAR Prize and a Fellow of ISMAR. In addition to his contributions to science, education through his written books and other works, and the fostering of science and people in science was clearly a valuable component of his sensibility and legacy. With this in mind, ISMAR has established an award bearing his name for young investigators within 5 years of completing their PhD. The award aims to recognize and encourage outstanding young scientists at an important early point of their career. The first Awardee is Vikram S. Bajaj, Ph.D. University of California & Lawrence Berkeley National Laboratory, Berkeley, USA He is honored for his achievements in magnetic resonance methodology, including contributions to high field DNP, remote detection of microfluidic flow, optical encoding and detection of magnetic resonance, and new implementations of the Xenon biosensor. Both Awardees were selected by the ISMAR Prize committee, chaired by Alex Pines, and I thank all members very much, see http://www.ismar.org/organization. Paul Callaghan Lecture sponsored by Magritek ISMAR has a particular responsibility and privilege to celebrate the legacy of the late Paul Callaghan who made singular contri- butions to science in general and to the function and welfare of ISMAR in particular. Professor Callaghan served as Vice Presi- dent, President and Past President until his death in March 2012. To honor his memory, a lecture in his name was introduced, the first one to be given by Professor Lucio Frydman, The Weizmann Institute of Science, Rehovot, Israel. During the course of his career, Lucio Frydman has made ex- citing and significant contributions to NMR methodology and instrumentation and to the applications of his techniques to many fields of science. Among them are inno- vative studies of the flow behavior of complex fluids, an area of research at the center of Paul Callaghan’s wide-ranging interests. Hans Wolfgang Spiess ISMAR President

iii General Information

General Information

Detailed information is available at the conference website: http://www.ismar2013.net

Conference site:

Royal Tulip Rio de Janeiro - Rua Aquarela do Brasil, 75, São Conrado - 22610-010, Rio de Janeiro, Brazil. Tel: +55 21 3323 2200 - Fax: +55 21 3322 5500

Program:

All lectures and posters abstracts are in this ISMAR 2013 Book. The electronic version of the book, including last submitted abstracts, is available on the conference website.

Special Activities:

Sunday, May 19th – Pre-conference meetings 8:30 - 15:00 We-NMR Workshop (Room: Água Marinha) 8:30 - 15:10 WW-NMR Workshop (Room: Ágata) 12:00 - 14:40 Bruker Users Meeting (Room: Turmalina/Topázio) 9:00 - 13:00 Agilent Users Meeting (Room: Ônix)

During ISMAR Meeting: Monday, May 20th, 12:30 - 14:00 MESTRELAB Users Meeting (Room: Água Marinha) Tuesday, May 21st, 12:30 - 13:30 AUREMN GENERAL ASSEMBLY (Room: Ônix) Wednesday, May 22nd, 14: 15 - 18:00 Satellite Meeting - Biomedical Imaging Symposium (Room: Ônix) Friday, May 24th, 14:30 - 18:20 Satellite Meeting - Vth Iberoamerican NMR Meeting (Room: Turmalina/Topázio)

ISMAR Activities:

Tuesday, May 21st, 19:30 ISMAR Executive Committee Meeting (Room: Água Marinha) Wednesday, May 22nd, 19:30 ISMAR Council Meeting (Room: Água Marinha)

Posters:

Poster Sessions: Room Quartzo A/B

Poster Set Up: until 10:30 of Monday, May 20th.

Poster removal: until 10:30, Friday, May 24th.

Posters to be presented on Monday are labeled: MOxxx

Posters to be presented on Tuesday are labeled: TUxxx

Posters to be presented on Thursday are labeled: THxxx

Talks:

Speakers must arrive 1/2 hour prior to the start of their sessions to set up their laptops or talks in the media available.

Internet Access:

Free WiFi is provided in all conference area

Select wireless network: ISMAR

Password: ismar2013

Lunch and Dinner:

There are restaurants available at the conference hotel, further restaurants, cafes and fast foods are at "Fashion Mall", located in walking distance from the conference site. A list of restaurants and locations is provided in the conference bag.

iv General Information

Social Events:

Sunday, May 19th Welcome Reception (18:30 at Foyers) Thursday, May 23rd Brazilian Night, the Conference Banquet (20:00) Fogo de Chão Brazilian Steak House: Address: Av. Rep. Nestor Moreira, 12, Botafogo, Phone: ++ 55 21 2542 1545 Free transportation will be provided for the conference at- tendees from the Royal Tulip and back (following the hotels routes). Tickets for the Brazilian Night are required.

Tours:

Wednesday and Friday afternoon are free for sightseeing. There will be a Travel Agency offering tours to the main tourist attractions in Rio throughout the meeting. If you are interested, please get tickets at the Tour Desk.

Exhibition and Sponsorship:

Exhibitions will be held during the Conference period. Exhibits of many companies will be available. The map of exhibitors is available on this book.

Transportation:

The conference attendees can use regular city buses and taxi.

Regular City Buses from Copacabana to São Conrado (conference site):

• Number 522 (São Conrado, R$2.75) at BRS 2 stops at Barata Ribeiro St.

• Number 557 (Rio das Pedras, R$2.75) at BRS 3 stops at Barata Ribeiro St.

• Number 318 (Recreio dos Bandeirantes, R$3.10) at Atlântica Av.

Taxi cabs- Estimated prices to the event location from: Leme R$ 70, Copacabana: R$ 50, Ipanema R$ 35; rates increase by 20% after 9 PM and sundays. Yellow cabs are also available at the Royal Tulip Hotel for your return trip.

Shuttle buses are free provided for the attendees during the conference.

Morning Route and stops:

1. Golden Tulip Continental Hotel, main entrance (Leme)

2. Barata Ribeiro St. (1), Bus stop BRS3 close to #204 Barata Ribeiro St., at the Metro station Cardeal Arco Verde (Copacabana)

3. Barata Ribeiro St. (2), Bus stop BRS2 close to #426 Barata Ribeiro St., at the intersection of Siqueira Campos and Barata Ribeiro, close to Metro station Siqueira Campos (Copacabana)

4. Barata Ribeiro St. (3), Bus stop BRS1 close to #774 Barata Ribeiro St., at the intersection of Bolivar and Barata Ribeiro (Copacabana)

5. Prudente de Moraes St., Bus stop in front of #149 Prudente de Moraes St., at General Osório Square (Ipanema)

6. Bartolomeu Mitre Av., Bus stop close to #246 Bartolomeu Mitre Av., in front of Antero de Quental Square (Leblon)

7. Royal Tulip Hotel (São Conrado), ISMAR 2013 venue

Return Route and stops:

1. Royal Tulip Hotel, main entrance (São Conrado)

2. Delfin Moreira Av., at the intersection with Bartolomeu Mitre Av. (Leblon)

3. Vieira Souto, at the intersection with Texeira de Mello St. (close to General Osório Square, Ipanema)

4. Atlantica Av. (1), intersection with Miguel Lemos St. (Copacabana)

5. Atlantica Av. (2), intersection with Siqueira Campos St. (Copacabana)

6. Atlantica Av. (3), intersection with Rodolfo Dantas St. (Copacabana)

7. Golden Tulip Continental Hotel (Leme)

v General Information

Timetable:

Sunday - May 19th From Golden Tulip Cont. Hotel to Royal Tulip Afternoon 12:00 13:00 14:00 From Royal Tulip to Golden Tulip Cont. Hotel Evening 19:30 20:00 20:30 21:15 Monday - May 20th From Golden Tulip Cont. Hotel to Royal Tulip Morning 7:00 7:20 7:40 From Royal Tulip to Golden Tulip Cont. Hotel Evening 18:30 19:00 22:15 Tuesday - May 21st From Golden Tulip Cont. Hotel to Royal Tulip Morning 7:00 7:20 7:40 From Royal Tulip to Golden Tulip Cont. Hotel Evening 19:15 20:30 22:00 Wednesday - May 22nd From Golden Tulip Cont. Hotel to Royal Tulip Morning 7:00 7:20 7:40 From Royal Tulip to Golden Tulip Cont. Hotel Afternoon 12:30 From Royal Tulip to Golden Tulip Cont. Hotel Evening 18:15 Thursday - May 23rd From Golden Tulip Cont. Hotel to Royal Tulip Morning 7:00 7:20 7:40 From Royal Tulip to Fogo de Chão Hotel Evening 18:30 From Fogo de Chão to Hotels Night 22:30 24:00 Friday - May 24th From Golden Tulip Cont. Hotel to Royal Tulip Morning 7:00 7:20 7:40 From Royal Tulip to Golden Tulip Cont. Hotel Afternoon 12:30 From Royal Tulip to Golden Tulip Cont. Hotel Evening 18:40 Please note that buses will only leave if a minimum occupancy of 30 is reached!

Other Information:

Message Board: Available at the poster room (Quartzo A/B). Regulations: • Badges are required at all time on event premisses, specially conference sessions, including posters and social events. • Exhibitor badges are restricted to the exhibition area. • The whole event site is a smoking free environment. NO SMOKING is permitted. • Please turn off your cell phones during oral sessions. • NO photography or recording is permitted in any session, including posters.

vi Map of the Event Locations

Map of the Event Locations

vii Event Sponsors

Event Sponsors

Main Sponsors

Funding Agencies and Universities

CAPES CNPq CRQ-RJ FCC/UFRJ IQ/UFRJ PUC-Rio

Sponsor Category Company

Diamond

Bruker

Sapphire

Agilent Tecnologies

Platinum

Cambridge Isotope Labs Schlumberger SIGMA-ALDRICH/ISOTEC Tedia Brazil

Gold

Anasazi/Superlab Magritek Limited Oxford/dpUNION Philips Spinlock

Silver

ACD Labs Cryogenic Doty Scientific, Inc. Halliburton Jeol Merck-Millipore Mestrelab Research Shimadzu Tecmag

Bronze

Baker Hughes Daedalus Innovations Euriso-top

viii Student Travel Awards

Student Travel Awards

Graduate Student Travel Recipients funded by the sponsors above:

Elias Akoury Mariano M. Gonzalez Damjan Pelc Max-Planck-Institute for Biophysical IBR - CONICET University of Zagreb, Faculty of Chemistry Sheetal Kumar Jain Science Rohan Alvares Aarhus University Daniel C Pizetta University of Toronto Danielle Kaminski Instituto de Fisica de São Carlos - Yang Bai University of Oxford USP Nanyang Technological University, Natalia V Kulminskaya Katherine M Poole School of Biological Sciences Aarhus University University of Florida Emeline Barbet-Massin Zhao Li Matthew P Renshaw Centre de RMN à Très Hauts Champs University of California, Los Angeles University of Cambridge Otonye Braide María Eugenia Llases Benjamin Schomburg University of Florida Institute of Molecular and Cell Max-Planck-Institute for Biophysical Paula Burdisso Biology of Rosario; Max-Planck Chemistry Instituto de Biología Molecular y Laboratory of Rosario Emilia V. Silletta Celular de Rosario Antonio Marchi Netto FAMAF – Universidad Nacional de Tonci Cvitanic Rheinisch-Westfaelische Technische Córdoba, IFEG –CONICET University of Zagreb Hochschule Aachen Camila P Silveira David M Dias Irene Marco-Rius UFRJ - UNICAMP University of Cambridge University of Cambridge and Cancer Romana Spitzer Yesu Feng Research UK University of Innsbruck Duke University Marco C Miotto Sebastian Täubert Alexander M Funk Institute of Molecular and Cell Max-Planck-Institute for Biophysical Durham University Biology of Rosario; Max-Planck Chemistry Piotr Garbacz Laboratory of Rosario Bruno L S Vicentin University of Warsaw Marcos N Morgada Universidade Estadual de Londrina Eduard M. Gataullin Instituto de Biología Molecular y Alexandre Zagdoun Kazan (Volga Region) Federal Celular de Rosario CRMN ENS Lyon University

Undergraduate Student Travel Recipients funded by the sponsor above:

João Felipe Alves da Cruz Federal University of São Carlos William S. Nunes André S. Carvalho Mato Grosso do Sul University Federal University of São Carlos

ix Program

Program

Sunday – May 19th Pre-Conference Meetings

8:30-15:00 We-NMR Workshop, Room Água Marinha, coordinated by Roberto Salinas (USP) 8:30-15:10 WW-NMR/COSMOS Meeting, Room Ágata, coordinated by Fábio Almeida (UFRJ) and Ulrich Günther (University of Birmingham) 12:00-14:40 Bruker Users Meeting, Rooms Turmalina/Topázio 9:00-13:00 Agilent Users Meeting, Room Ônix

12:00-15:30 Registration

Sunday – May 19th Opening Ceremony Room Gávea A

15:30-16:00 Welcome José Daniel Figueroa President of AUREMN Chair of the 18th ISMAR 2013/14th NMR Users Meeting

Sunday – May 19th Honor and Prizes Session Presiding: Hans Spiess Room Gávea A

page 16:00-16:45 Anatole Abragam Prize Lecture Winner 2013: Vikram Bajaj, UCLA & Lawrence Berkeley National Laboratory, USA 16:45-17:30 ISMAR Price Lecture Winner 2013: Jack Freed, Cornell University, USA 17:30-18:15 Paul Callaghan Lecture PL001: Emerging Frontiers in Ultrafast Multidimensional NMR and MRI Rita Schmidt, Eddy Solomon, Avi Leftin, ∗Lucio Frydman; Department of Chemical Physics, Weizmann Institute of Science, Israel ...... 21 18:30 Welcome Reception (Foyer) 18:30-21:00 Vendors Exhibit Booths (Foyer)

1 Program

Monday – May 20th - Plenary Session Chair: Daniella Goldfarb Room Gávea A

page

8:30-9:15 PL002: Biomolecular NMR at Very Low Temperatures ∗Robert Tycko, Kent Thurber, Alexey Potapov, Wai-Ming Yau, Eric Moore; National Institutes of Health...... 21

9:15-10:00 PL003: Sensitivity enhancement in High Field NMR and EPR Vasyl Denysenkov, Petr Neugebauer, Phillip Spindler, Burkhard Endeward, ∗Thomas F. Prisner; Institute of Physical and Theoretical Chemistry and Center of Biomolecular Mag- netic Resonance, Goethe University Frankfurt, Germany ...... 21

10:00-10:30 Coffee Break

Monday – May 20th - Dynamics and Catalysis Chair: Jarbas Resende Room Gávea A

page

10:30-10:55 OP004: Control of Periplasmic Interdomain Thiol: Disulfide Exchange in the Transmembrane Oxidoreductase DsbD 1Despoina A.I. Mavridou, 2Emmanuel Saridakis, 1Paraskevi Kritsiligkou, 1Lukas Stelzl, 1Erin C. Mozley, 1Alan D. Goddard, 1Julie M. Stevens, 1Stuart J. Ferguson, 1∗Christina Redfield; 1Department of Biochemistry, University of Oxford, 2National Center for Scien- tific Research Demokritos ...... 21

10:55-11:20 OP005: Hydration of yeast Thioredoxin 1 Modulates Slow Dynamics ∗Fabio C. L. Almeida; Institute of Medical Biochemistry, National Center of Nuclear Magnetic Resonance, Federal University...... 22

11:20-11:35 UP006: The magnitude of a pre-existing equilibrium tunes adenylate kinase ac- tivity 1Jörgen Åden, 2Abhinav Verma, 2Alexander Schug, 1∗Magnus Wolf-Watz; 1Umeå Uni- versity, 2Karlsruhe Institute of Technology ...... 22

11:35-11:50 UP007: Structural and dynamic insights into substrate binding and catalysis in human Lipocalin Prostaglandin D synthase 1∗Konstantin Pervushin, 1,2Pär Nordlund, 1,2Sing Mei Lim, 1Dan Chen, 1Hsiangling Teo, 2Annette Roos, 2Tomas Nyman, 2Lionel Trésaugues; 1Nanyang Technological University, 2Karolinska Institute ...... 22

11:50-12:15 OP008: EPR Studies of Radical SAM Enzymes ∗R. David Britt; University of California, Davis ...... 22

2 Program

Monday – May 20th - General Solids Chair: Bernhard Blümich Room Gávea B

page

10:30-10:55 OP009: Structure Determinations of Molecularly Ordered, Non-Crystalline Sil- icate, Borosilicate, and Aluminosilicate Frameworks by Combined NMR, Scat- tering, and DFT Analyses 1∗Bradley F. Chmelka, 2Sylvian Cadars, 3Darren H. Brouwer, 1Ming-Feng Hsieh, 1Robert J. Messinger, 1Matthew T. Aronson, 2Mounesha N. Garaga, 2Zalfa Nour; 1University of Cal- ifornia, Santa Barbara, USA, 2CNRS, Université d’Orléans, France, 3Redeemer College, Ancaster, Ontario, Canada...... 22

10:55-11:20 OP010: Zero- and low-field NMR spectroscopy 1,2∗Dmitry Budker; 1Department of Physics, University of California, Berkeley, 2Nuclear Science Division, E. O. Lawrence Berkeley National Laboratory, Berkeley ...... 23

11:20-11:35 UP011: Use of 1H Chemical Shifts to Determine Structural Motifs in π- Conjugated Polymers ∗Michael Ryan Hansen; Max Planck Institute for Polymer Research...... 23

11:35-11:50 UP012: NMR Crystallography, from “Pas de deux” to Spin Choreography. Nanoporous Crystal Structures of Powders : Methods and Hardware. 1∗Francis Taulelle, 2Frank Engelke, 2Frank Decker, 1Boris Bouchevreau, 1Charlotte Matineau; 1University of Versailles, 2Bruker-Biopspin GmbH ...... 156

11:50-12:15 OP013: High Frequency Dynamic Nuclear Polarization ∗Robert G. Griffin; Francis Bitter Magnet Laboratory and Department of Chemistry, MIT 24

Monday – May 20th - NMR in Medicinal Chemistry Chair: Anita Marsaioli Rooms Turmalina/Topázio

page

10:30-10:55 OP014: HTS by NMR of combinatorial libraries: a new approach to peptide and ligand discovery ∗Maurizio Pellecchia; Chemical Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA ...... 24

10:55-11:20 OP015: Enzyme Kinetics and Inhibition by NMR ∗José Daniel Figueroa-Villar; Mititary Institute of Engineering ...... 24

11:20-11:35 UP016: Integrated structural biology shows how scarce sequence elements con- trol the function of single ß-thymosin/WH2 domains in actin assembly 1François-Xavier Cantrelle, 2Dominique Didry, 1Célia Deville, 1Jean-Pierre Placial, 2Clotilde Husson, 3Javier Perez, 2Marie-France Carlier, 2Louis Renault, 1Carine van Heijenoort, 1∗Eric Guittet; 1CNRS-ICSN, 2CNRS-LEBS, 3Synchrotron SOLEIL ...... 24

11:35-11:50 UP017: Human Macrophage Lectin (CLEC10A) recognition of monosaccharides related to tumor marker Tn-antigen studied by 1H and 19F NMR. 1∗Francisco Javier Cañada, 1Anneloes Oude Vrielink, 1,2Filipa Marcelo, 1Ana Manzano, 1Pilar Blasco, 1Jesús Jiménez-Barbero, 3Sabine Andre, 3Hans-Joachim Gabius; 1Centro de Investigaciones Biológicas, CIB-CSIC Chemical and Physical Biology Department. Ramiro de, 2REQUIMTE, CQFB, DQ, FCT-UNL, 2829-516 Caparica, Portugal, 3Institute of Phys- iological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, . . . . . 158

11:50-12:15 OP018: High-yield expression and NMR structural studies of Antimicorbial Pep- tides Ji-Ho Jung, Ji-Sun Kim, ∗Yongae Kim; Depart of Chemistry and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Korea ...... 25

3 Program

Monday – May 20th - EPR and New Materials Chair: Eduardo Di Mauro Room Ônix

page

10:30-10:55 OP019: EPR of thermo- and photoswitchable copper-nitroxide based molecular magnets 1∗Matvey Fedin, 1Sergey Veber, 1Irina Drozdyuk, 1Ksenia Maryunina, 1Evgeny Tretyakov, 2Hideto Matsuoka, 2Seigo Yamauchi, 1Renad Sagdeev, 1Victor Ovcharenko, 1,3Elena Bagryanskaya; 1International Tomography Center SB RAS, Novosibirsk, Russia, 2Tohoku University, Sendai, Japan, 3N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, Russia ...... 25

10:55-11:20 OP020: Theoretical EPR Spectroscopy of Open-Shell Transition Metal Com- plexes with Strong Spin Orbit Coupling ∗Frank Neese, Mihail Atanasov, Michael Römelt, Kantharuban Sivalingam; Max-Planck- Institut für Chemische Energiekonversion ...... 26

11:20-11:35 UP021: Multifunctional in vivo EPR-based spectroscopy and imaging of param- agnetic probes ∗Valery V. Khramtsov; The Ohio State University Medical Center, Columbus, OH, USA 26

11:35-11:50 UP022: New Kind of Mesoscopic EPR In Some Itinerant Undoped Ferromagnets ∗Vladimir A. Ivanshin, Eduard M. Gataullin; Kazan (Volga region) Federal University . 26

11:50-12:15 OP023: Elucidating Smart Polymeric Materials Using Electron Paramagnetic Resonance (EPR) Spectroscopy ∗Dariush Hinderberger; Max Planck Institute for Polymer Research...... 27

12:15-14:15 Lunch Break 12:30-13:30 Mestrelab Users Meeting, Room Água Marinha

Monday – May 20th - Membrane Proteins Chair: Ana Carolina Zeri Room Gávea A

page

14:15-14:40 OP024: Structural studies of membrane proteins in membrana ∗Francesca M. Marassi; Sanford-Burnham Medical Research Institute, La Jolla CA, USA 27

14:40-15:05 OP025: Brain Fatty-acid Binding Protein and its Interactions with Membrane Model Systems 1Fábio H. Dyszy, 1Ítalo A. Calvini, 1Daniel F. Silva, 1Andressa P. A. Pinto, 1Ana P.U. Araújo, 2Hans Robert Kalbitzer, 1Claudia Elisabeth Munte, 1,3∗Antonio José Costa- Filho; 1Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Car- los, USP, 2Department of Biophysics and Physical Biochemistry, University of Regensburg, 3Laboratório de Biofísica Molecular, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP ...... 27

15:05-15:20 UP026: Defining the Flexible and Fixed Sides of a Protein Channel by EPR ∗Betty J. Gaffney; Florida State University ...... 158

15:20-15:35 UP027: Conformational selection of GPCR ligands upon binding to their recep- tors as observed by NMR 1∗Laurent Catoire, 2Marc Baaden, 1Jean-Luc Popot, 3Eric Guittet, 4Jean-Louis Banères; 1Laboratory of Biology and Physico-Chemistry of Membrane Proteins - UMR 7099 CNRS & University Paris, 2Laboratory of theoretical Biochemistry - UPR 9080 CNRS, 3ICSN- UPR 2301 CNRS, 4IBMM - UMR 5247 CNRS, Universite Montpellier 1 & 2...... 28

15:35-16:00 OP028: Structural Biology by DNP MAS NMR and Investigations on the Trans- port Cycle of an ABC Transporter ∗Hartmut Oschkinat; Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany . 28

4 Program

Monday – May 20th - Dynamics and Recognition Chair: Robert Kaptein Room Gávea B

page

14:15-14:40 OP029: Dynamic Aspects of Indirect DNA Readout by Nucleoid Associated Proteins 2Tiago Cordeiro, 2Jesús García, 3Oscar Millet, 1∗Miquel Pons; 1University of Barcelona, 2Institute for Research in Biomedicine, 3CIC-bioGUNE ...... 28

14:40-15:05 OP030: Protein Kinase A and Conformational Dynamics: ‘Not too fast. . . not too slow. . . but just right’ ∗Gianluigi Veglia; University of Minnesota...... 29

15:05-15:20 UP031: Sliding and target location of DNA-binding proteins: an NMR view of the lac repressor system 1∗Rolf Boelens, 2Karine Loth, 4Manuel Gnida, 5Julija Romanuka, 1,3Robert Kaptein; 1Utrecht University, 2University of Orleans, 3Novosibirsk State University, 4Paderborn Uni- versity, 5Shell Research Laboratories ...... 29

15:20-15:35 UP032: RNA regulation - A new Twist ∗Harald Schwalbe; Goethe-University Frankfurt ...... 29

15:35-16:00 OP033: Protein interactions and function studied by novel NMR methods ∗S. Grzesiek, M. Gentner, J.-r. Huang, S. Morin, L. Nisius, H.-.J. Sass, L. Skora, N. Vajpai, M. Wiktor; Biozentrum, University of Basel, Switzerland...... 29

Monday – May 20th - Methods for Inorganic Materials Chair: Bradley Chmelka Rooms Turmalina/Topázio

page

14:15-14:40 OP034: NMR Non-Crystallography 1∗Philip J. Grandinetti, 1Kevin Sanders, 1Eric Keeler, 2Jay H. Baltisberger; 1Ohio State University, Columbus, USA, 2Berea College, Berea, KY, USA ...... 30

14:40-15:05 OP035: Hunting for Hydrogen in Wadsleyite: Multinuclear Solid-State NMR and First-Principles Calculations 1∗Sharon E. Ashbrook, 1John M. Griffin, 2Andrew J. Berry, 3Stephen Wimperis; 1School of Chemistry and EaStCHEM, University of St Andrews, UK, 2Research School of Earth Sciences, Australia National University, Canberra, Australia, 3School of Chemistry and WestCHEM, University of Glasgow, UK ...... 30

15:05-15:20 UP036: Uniform excitation and inversion of broad NMR spectra in rotating solids using DANTE 1Veronika Vizthum, 1Marc Caporini, 1Diego Carnevale, 1Simone Ulzega, 2Julien Trébosc, 2Olivier Lafon, 1Geoffrey Bodenhausen, 2∗Jean Paul Amoureux; 1Ecole Polytechnique Fédérale de Lausanne, Switzerland, 2Lille University, France ...... 30

15:20-15:35 UP037: A homonuclear version of REDOR for recoupling dipolar interactions in multi-spin systems: Applications to inorganic phosphorus clusters and networks 1,2∗Hellmut Eckert, 2Jinjun Ren; 1University of Sao Paulo, 2WWU Münster ...... 31

15:35-16:00 OP038: Topologic, Geometric, and Chemical Order in Materials: Insights from Solid-State NMR Dominique Massiot, Sylvian Cadars, Michael Deschamps, Emmanuel Véron, Mounesha N.Garaga, Robert J. Messinger, Mathieu Allix, ∗Pierre Florian, Franck Fayon; CEMHTI UPR3079 CNRS, France ...... 31

5 Program

Monday – May 20th - Hyperpolarization/ Instrumentation Chair: Dmitry Budker Room Ônix

page

14:15-14:40 OP039: Imaging Pulmonary Gas Exchange from Mouse to Human with Hyper- polarized 129Xe MRI ∗Bastiaan Driehuys; Department of Radiology, Duke University Medical Center, Durham 32

14:40-15:05 OP040: Hyperpolarized Carbon-13 MR Pre-Clinical and Human Research Stud- ies 1∗Daniel B. Vigneron, 1Sarah Nelson, 2Andrea Harzstark, 3Marcus Ferrone, 1Robert Bok, 1John Kurhanewicz; 1Department of Radiology & Biomedical Imaging, University of Califor- nia, San Francisco, 2Investigational Therapeutics, University of California, San Francisco, 3Clinical Pharmacy, University of California, San Francisco ...... 32

15:05-15:20 UP041: Universal tags to sustain long-lived hyperpolarized signal: Extension of hyperpolarization to broad range of biomarkers Yesu Feng, Thomas Theis, Ryan M. Davis, Kevin Claytor, Qiu Wang, Pei Zhou, ∗Warren S. Warren; Duke University ...... 32

15:20-15:35 UP042: Avalanching Amplification of NMR/MRI Sensitivity by Solvent- Generated Feedback Fields Zhao Li, Susie Y. Huang, Jamie D. Walls, ∗Yung-Ya Lin; Department of Chemistry and Biochemistry, UCLA ...... 32

15:35-16:00 OP043: An Implantable Wireless Amplified NMR Detector for High Resolution MRI Chunqi Qian, Xin Yu, Der-Yow Chen, Stephen Dodd, Joseph Murphy-Boesch, ∗Alan P. Koretsky; Laboratory of Functional and Molecular Imaging, NINDS, NIH, Bethesda, MD 33

16:00-18:30 Poster Session I Rooms Quartzo A/B, Authors with poster numbers starting "MO" present

all day Vendors Exhibit Booths (Foyer) 19:00-22:00 Bruker’s Night (Gávea A)

6 Program

Tuesday – May 21st - Plenary Session Chair: Ad Bax Room Gávea A

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8:30-9:15 PL044: Singlet Nuclear Magnetic Resonance ∗Malcolm H. Levitt; School of Chemistry, University of Southampton, UK ...... 33

9:15-10:00 PL045: Visualizing Transient Structures of DNA and RNA using NMR ∗Hashim M. Al-Hashimi; Department of Chemistry and Biophysics, University of Michi- gan ...... 33

10:00-10:30 Coffee Break

Tuesday – May 21st - Dynamics and Recognition Chair: Jose Pires Room Gávea A

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10:30-10:55 OP046: Protein dynamics and interaction mediated by local water dynamics at the ps timescale ∗Songi Han; Departement of Chemistry and Biochemistry, University of California Santa Barbara ...... 34

10:55-11:20 OP047: Molecular Recognition and Dynamics: NMR Studies of Defensins and Epitopes ∗Ana Paula Valente; Federal University of Rio de Janeiro ...... 34

11:20-11:35 UP048: Bacterial transmembrane signalling through a heme/hemophore recep- tor 1,2∗Nadia Izadi-Pruneyre; 1Institut Pasteur, Paris, France, 2CNRS UMR 3528 ...... 34

11:35-11:50 UP049: NMR Mapping of PCNA Interaction with Translesion Synthesis DNA Polymerase Rev1 Mediated by Rev1-BRCT Domain Yulia Pustovalova, Mark W. Maciejewski, ∗Dmitry M. Korzhnev; University of Connecti- cut Health Center ...... 155

11:50-12:15 OP050: Structural basis of epigenetic regulation ∗Yunyu Shi; School of Life Sciences, University of Science and Technology of China, Hefei, China ...... 34

7 Program

Tuesday – May 21st - IDPs, Folding and Misfolding Chair: Gianluigi Veglia Room Gávea B

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10:30-10:55 OP051: New methods based on 13C direct detection to study intrinsically disor- dered proteins ∗Isabella C. Felli; CERM and Department of Chemistry “Ugo Schiff”, University of Flo- rence, Italy...... 35

10:55-11:20 OP052: NMR-based Structural Biology in Brain Diseases 1,2∗Claudio O. Fernández; 1Universidad Nacional de Rosario (UNR), Instituto de Bi- ologia Molecular y Celular de Rosario (IBR-CON, 2Max PLanck Laboratory of Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR) ...... 35

11:20-11:35 UP053: Caught in Action: Selecting Peptide Aptamers Against Intrinsically Disordered Proteins in Live Cells 1Jacqueline Washington, 1Sergey Reverdatto, 1David Burz, 2Kathleen McDonough, 1∗Alexander Shekhtman; 1State University of New York at Albany, Albany, NY, USA, 2Wadsworth Center, NY Department of Health, Albany, NY...... 35

11:35-11:50 UP054: Phosphorylation Of The Intrinsically Disordered Unique Domain Of c- Src Studied By In-Vivo Real-Time NMR 1,2Irene Amata, 2Mariano Maffei, 1Ana Igea, 1Angel R. Nebreda, 2∗Miquel Pons; 1Signaling and Cell Cycle Laboratory, Institute for Research in Biomedicine, Barcelona, Spain, 2Biomolecular NMR, Organic Chemistry Department, University of Barcelona, Barcelona, Spain ...... 35

11:50-12:15 OP055: NMR of Intrinsically Disordered Proteins: Tools for Increasing Sensi- tivity and Resolution 1Zsofia Solyom, 1Melanie Schwarten, 1Sophie Feuerstein, 2Dieter Willbold, 3Michael Plevin, 1∗Bernhard Brutscher; 1Institut de Biologie Structurale, CEA-CNRS-UJF, Grenoble, France, 2Institute of complex systems, Structural Biochemistry, FZ Jülich, Germany, 3Department. of Biology, University of York, UK ...... 36

Tuesday – May 21st - Methods for Organic Materials Chair: Klaus Schmidt-Rohr Rooms Turmalina/Topázio

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10:30-10:55 OP056: 14N-1H Solid-State NMR Spectroscopy of Organic Solids ∗Steven P. Brown; Department of Physics, University of Warwick, UK ...... 36

10:55-11:20 OP057: Separated Local Field NMR as a tool for probing molecular dynamics in organic solids 1Marcio F. Cobo, 2Anja Achilles, 1Gregório C. Faria, 2Detlef Reichert, 2Kay Saalwächter, 1∗Eduardo Ribeiro de Azevêdo; 1Universidade de São Paulo, São Carlos-SP, Brazil„ 2Martin-Luther-University Halle Wittenberg, Halle, Germany ...... 36

11:20-11:35 UP058: Low-field 1H-NMR investigations to reveal local properties of polymer networks 1∗Maria Ott, 2Roberto Pérez-Aparicio, 1Horst Schneider, 2Paul Sotta, 1Kay Saalwächter; 1Institut für Physik – NMR, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann- Str. 7, D-0, 2Laboratoire Polymères et Matériaux Avancés, CNRS/Rhodia,85 avenue des Frères Perret, F-69192 Sai ...... 37

11:35-11:50 UP059: Efficient Heteronuclear Cross Polarization Techniques for Solid State NMR Sheetal Kumar Jain, Morten Bjerring, ∗Niels Christian Nielsen; Aarhus University . . . . . 37

11:50-12:15 OP060: NMR crystallography of small molecules: from ionic solids to pharma- ceuticals ∗Luís Mafra, Sérgio M. Santos; Department of Chemistry, University of Aveiro, CICECO, Portugal ...... 37

8 Program

Tuesday – May 21st - MRI/In-Vivo Chair: Michael Garwood Room Ônix

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10:30-10:55 OP061: Parallel imaging with nonlinear gradient fields ∗Gigi Galiana, Leo Tam, Dana C. Peters, R. Todd Constable; Yale University ...... 37

10:55-11:20 OP062: Relaxivity Optimized Metal Based MRI Molecular Imaging Agents ∗Carlos F.G.C. Geraldes; Department of Life Sciences, Center of Neurosciences and Cell Biology and Coimbra Chemistry Center, University of Coimbra ...... 38

11:20-11:35 UP063: Magnetic Resonance Microimaging of a Swelling Gel 1Wilson Barros Jr., 1Eduardo N. de Azevedo, 2∗Mario Engelsberg; 1Universidade Federal de Pernambuco, Recife, 2Universidade Federal de Pernambuco, Caruaru ...... 38

11:35-11:50 UP064: Early Detection of Pancreatic Cancers & Brain Tumors by Active- Feedback Controlled MR 1Zhao Li, 2Chaohsiung Hsu, 2Lian-Pin Hwang, 1∗Yung-Ya Lin; 1Department of Chemistry and Biochemistry, UCLA, 2Department of Chemistry, National Taiwan University ...... 38

11:50-12:15 OP065: Detecting Tumor Treatment Response using Metabolic Imaging with hyperpolarized 13C-labeled Cell Substrates ∗Kevin M. Brindle, Mikko I. Kettunen, Tiago B. Rodrigues, Eva M. Serrao, Brett W.C. Kennedy, De-en Hu; Department of Biochemistry and CRUK Cambridge Institute, Univer- sity of Cambridge, UK ...... 39

12:15-14:15 Lunch Break 12:30-13:30 AUREMN General Assembly (AUREMN members only, lunch box included), Room Ônix

Tuesday – May 21st - Plenary Session Chair: José Figueroa-Villar Room Gávea A

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14:15-15:00 PL066: Recent contributions for Zero-, Low-, and High-field NMR ∗Tito J. Bonagamba; Instituto de Física de São Carlos - Universidade de São Paulo, São Carlos, Brazil ...... 39

15:00-15:25 OP067: Magnetic resonance studies of topological materials ∗Louis Bouchard; UCLA ...... 40

15:25-15:50 OP068: Hyperpolarized Molecular Sensing and Detection 1,2∗Vikram S. Bajaj; 1Department of Chemistry and California Institute of Quantita- tive Biosciences, University of California, Berkeley, 2Materials Sciences Division, Lawrence Berkeley National Laboratory ...... 40

15:50-16:35 PL069: Structures and mechanisms of viral ion channels and fusion peptides from solid-state NMR ∗Mei Hong; Iowa State University ...... 40

16:35-19:30 Poster Session II Rooms Quartzo A/B, Authors with poster numbers starting "TU" present all day Vendors Exhibit Booths (Foyer)

9 Program

Wednesday – May 22nd - Amyloids Chair: Ana Paula Valente Room Gávea A

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8:30-8:55 OP070: Amyloid aggregates and large soluble protein complexes 1,2,3Sam Asami, 1,2,3Juan-Miguel Lopez del Amo, 1,2,3Andi Mainz, 1,2,3Muralidhar Dasari, 3Uwe Fink, 4Marcus Fändrich, 5Erich E. Wanker, 5Jan Bieschke, 6Tomasz Religa, 6Lewis E. Kay, 1,2,3∗Bernd Reif; 1Technische Universität München (TUM), Department Chemie, 2Helmholtz-Zentrum München (HMGU), 3Leibniz-Institut für Molekulare Phar- makologie (FMP), 4Max-Delbrück Centrum für Molekulare Medizin (MDC), 5Max-Planck- Forschungsstelle für Enzymologie der Proteinfaltung, Martin-Luther Universität Halle- Wittenberg, 6University of Toronto, Department of Medical Genetics and Microbiology . . . . 40

8:55-9:20 OP071: Amyloid Fibrils: Towards a Molecular Level Picture with Solid-State Nuclear ∗P. K. Madhu; Tata Institute of Fundamental Research...... 41

9:20-9:35 UP072: Metallobiology of neurodegenerative diseases: Structural and mechanis- tic basis behind the acceleration of amyloid protein assembly 1,2M.C. Miotto, 1,2A.A. Valiente Gabioud, 3Andres Binolfi, 4L. Quintanar, 5Christian Griesinger, 1,2∗Claudio O. Fernández; 1Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, 2Max Planck Laboratory of Struc- tural Biology, Chemistry and Molecular Biophysics of Rosario (MPLPbioR, 3Department of NMR-assisted Structural Biology, In-cell NMR, Leibniz Institute of Molecular Pharmacology, 4Centro de Investigación y de Estudios Avanzados (Cinvestav), 5Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry ...... 155

9:35-10:00 OP073: Structural studies of an engineered mimic of neurotoxic amyloid-β protofibrils 1Christofer Lendel, 2Morten Bjerring, 3Andrei Filippov, 3,4Oleg N. Antzutkin, 2Niels Chris- tian Nielsen, 1∗Torleif Härd; 1Dept. of Molecular Biology, Swedish University of Agricul- tural Sciences (SLU), Uppsala, Sweden., 2Dept. of Chemistry, Aarhus University, Aarhus, Denmark., 3Chemistry of Interfaces, Luleå University of Technology, Luleå, Sweden., 4Dept. of Physics, Warwick University, Coventry, United Kingdom...... 41

Wednesday – May 22nd - Dynamics and Recognition Chair: Rodolfo Rasia Room Gávea B

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8:30-8:55 OP074: Probing Protein Conformational Flexibility with Pulsed EPR Spec- troscopy Xi Huang, I.M.S. de Vera, Manuel Britto, Mandy E. Blackburn, Angelo M. Veloro, Jamie L. Kear, ∗Gail E Fanucci; University of Florida ...... 42

8:55-9:20 OP075: NMR Spectroscopy reveals alternative Ground States in Copper Pro- teins ∗Alejandro J. Vila, Luciano A. Abriata, Andrés Espinoza-Cara, Marcos N. Morgada, María-Eugenia Zaballa; Institute for Molecular and Celular Biology (IBR), University of Rosario, Argentina ...... 42

9:20-9:35 UP076: NMR Studies of the Loop Dynamics of the Nitrophorins ∗F. Ann Walker, Dhanasekaran Muthu, Robert E. Berry, Hongjun Zhang; The University of Arizona ...... 42

9:35-10:00 OP077: EPR Method Development for Addressing Protein Structure and Dy- namics 1∗Gunnar Jeschke, 1Yevhen Polyhach, 1Tona von Hagens, 2Carsten Dietz, 2Harald Paulsen; 1ETH Zurich, 2University of Mainz ...... 43

10 Program

Wednesday – May 22nd - Workshop: NMR on Porous Media Chair: Vinicius Machado Rooms Turmalina/Topázio

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8:30-8:55 OP078: Improved Estimation of Oil and Gas Reserves Using Magnetic Reso- nance Imaging “MRI” Technology - Applications and Case Histories ∗Maged Fam; HALLIBURTON, Bogota, Colombia ...... 43

8:55-9:20 OP079: Transport and Reaction in Porous Media: New Information Obtained Using Compressed Sensing and Bayesian Methods ∗Lynn F. Gladden, D. J. Holland, Mick D. Mantle, J. Mitchell, Andrew J. Sederman, A. B. Tayler; University of Cambridge, Department of Chemical Engineering and Biotechnology . 43

9:20-9:35 UP080: Pore Sizes Distribution of Unconsolidated Geomaterials 2,3∗Marcos Montoro, 1,3Lucas C. Cerioni, 3Daniel José Pusiol; 1Spinlock SRL, 2Universidad Nacional de Córdoba, 3CONICET...... 160

9:35-10:00 OP081: Diffusion dynamics in porous media ∗Yi-Qiao Song; Schlumberger ...... 44

Wednesday – May 22nd - Small Molecules and Methods Chair: Jochen Junker Room Ônix

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8:30-8:55 OP082: Structure Determination Using Anisotropic NMR Parameters ∗Burkhard Luy; Karlsruhe Institute of Technology ...... 44

8:55-9:20 OP083: New NMR strategies for the accurate measurement of small heteronu- clear coupling constants ∗Teodor Parella; Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, Catalonia ...... 44

9:20-9:35 UP084: Experimental measurement and theoretical assessment of fast lanthanide electronic relaxation in solution with four series of isostructural complexes 1Alexander M. Funk, 1∗David Parker, 1Peter Harvey, 1Alan M. Kenwright, 2Pascal H. Fries; 1Durham University, 2CEA, Grenoble ...... 44

9:35-10:00 OP085: Structural Analysis of Small Organic Molecules Assisted by Residual Dipolar Couplings ∗Roberto R. Gil; Carnegie Mellon University ...... 45

10:00-10:30 Coffee Break

11 Program

Wednesday – May 22nd - Challenging Systems Chair: Roberto Salinas Room Gávea A

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10:30-10:55 OP086: Protein Dynamics: Its Kinetics and Implications for Function 1David Ban, 1,2Colin Smith, 1Michael Sabo, 3R. Bryn Fenwick, 1Korvin F. A. Walter, 1Claudia Schwiegk, 1Stefan Becker, 3Xavier Salvatella, 1Berend L. de Groot, 1Donghan Lee, 1∗Christian Griesinger; 1Max Planck Institute for Biophysical Chemistry - NMR based Structural Biology, 2Max Planck Institute for Biophysical Chemistry - Theoretical and computational biophysics, 3ICREA and Institute for Research in Biomedicine Barcelona . . 45

10:55-11:20 OP087: Distance measurements in biomolecules using Gd3+ spin labels ∗Daniella Goldfarb; Weizmann Institute of Science, Rehovot, Israel ...... 45

11:20-11:35 UP088: Detection of light induced intermediates of photoreceptor membrane proteins by in-situ photo-irradiated solid-state NMR 1∗Akira Naito, 1Yuya Tomonaga, 1Tetsuro Hidaka, 1Hiroki Yomoda, 1Teruki Makino, 1Izuru Kawamura, 2Yuki Sudo, 3Akimori Wada, 3Takashi Okitsu, 4Naoki Kamo; 1Yokohama National University, 2Nagoya University, 3Kobe Pharmaceutical University, 4Matsuyama University...... 155

11:35-11:50 UP089: High resolution conformational description of Alpha-synuclein inside neurons using mammalian In-cell NMR Andres Binolfi, Beata Bekei, Francois-Xavier Theillet, Honor M. Rose, ∗Philipp Selenko; Leibniz Institut für Molekulare Pharmakologie (FMP-Berlin) ...... 158

11:50-12:15 OP090: NMR studies of the structure and dynamics of protein-RNA interac- tions in gene regulation 1,2∗Michael Sattler; 1Institute of Structural Biology, Helmholtz Zentrum München, Ger- many, 2Munich Center for Integrated Protein Science and Biomolecular NMR, Department Chemie, TUM, Germany...... 46

Wednesday – May 22nd - Metabolomics and Chemometrics Chair: Ana Gil Room Gávea B

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10:30-10:55 OP091: Deciphering Cancer Metabolism for Drug Discovery ∗Ulrich L. Günther, Christian Ludwig, Katarzyna Koczula, Farhat Khanim, Chris Bunce; University of Birmingham, UK...... 47

10:55-11:20 OP092: Food NMR applied to typical Brazilian food products ∗Antonio Gilberto Ferreira; Federal University of São Carlos - Chemistry Department. 47

11:20-11:35 UP093: Visualizing metabolic system catalyzed by microbial ecosystem using statistical correlation analysis 1,2,3,4∗Jun Kikuchi; 1RIKEN Plant Science Center, 2Biomass Engineering Program, RIKEN, 3Nagoya University, 4Yokohama City University ...... 47

11:35-11:50 UP094: The Use of Magnetic Resonance in Pollution Assessment: Theory and Applications ∗Oliver A.H. Jones; RMIT University ...... 47

11:50-12:15 OP095: Rapid-dissolution 13C DNP in kinetics of cellular transmembrane ex- change 1∗Philip W. Kuchel, 2Guilhem Pagès, 1Max Puckeridge; 1University of Sydney, 2Singapore Bioimaging Consortium ...... 48

12 Program

Wednesday – May 22nd - Workshop: NMR on Porous Media Chair: Vinicius Machado Rooms Turmalina/Topázio

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10:30-10:55 OP096: Earth’s-field Surface NMR for Groundwater Characterization 1,2∗Elliot Grunewald, 2Rosemary Knight, 1David Walsh, 2Denys Grombacher, 2Jan O. Walbrecker, 2Katherine Dlubac; 1Vista Clara, Inc., 2Stanford University ...... 48

10:55-11:20 OP097: Magnetic Resonance Imaging of Fluids in Porous Media ∗Bruce J. Balcom; MRI Research Centre, Department of Physics, University of New Brunswick, Canada ...... 48

11:20-11:35 UP098: Single-scan T1-T2 relaxation correlation experiment ∗Susanna Ahola, Ville-Veikko Telkki; University of Oulu ...... 48

11:35-11:50 UP099: High Pressure Magic Angle Spinning NMR Capability 1,2∗Flaviu R.V. Turcu, 1David W. Hoyt, 1Jesse A. Sears, 1John S. Loring, 1Kevin M. Rosso, 1Jian Z. Hu; 1Pacific Northwest National Laboratory, 2"Babes-Bolyai" University, Romania ...... 49

11:50-12:15 OP100: Efficient measurement of T1/T2 ratio in porous media ∗Martin D. Hürlimann; Schlumberger – Doll Research, USA ...... 49

Wednesday – May 22nd - Materials and Applications Chair: Hellmut Eckert Room Ônix

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10:30-10:55 OP101: Following Function in Real Time: New In situ NMR Methods for Study- ing Structure and Dynamics in Batteries and Supercapacitors 1Lina Zhou, 1Michal Leskes, 1Hao Wang, 1Alex Forse, 1John Griffin, 2Nicole M. Trease, 1Elodie Salager, 1∗Clare. P. Grey; 1Chemistry Department, Cambridge University, UK, 2Chemistry Department, Stony Brook University, USA ...... 49

10:55-11:20 OP102: Unique Insights into Carbon Capture and Geosequestration of CO2 from In Situ High-Pressure, High-Temperature NMR J. Andrew Surface, Jeremy Moore, Mark Conradi, ∗Sophia E. Hayes; Washington Univer- sity in St. Louis ...... 50

11:20-11:35 UP103: Direct Alcohol Fuel Cells Investigated In Situ and Ex Situ by NMR Spectroscopy 1,2∗Oc Hee Han; 1Korea Basic Science Institute, 2Chungnam National University ...... 50

11:35-11:50 UP104: Local Order and Cation Distribution in Mixed-Alkali Phosphate Glasses 1∗José Fabian Schneider, 1Jefferson Tsuchida, 2Hellmut Eckert, 2Rashmi Deshpande; 1Instituto de Física de São Carlos, Universidade de São Paulo, 2Institut für Physikalische Chemie, Westfälische Wilhelms-Universität Münster ...... 50

11:50-12:15 OP105: Nanoscale Imaging of Electron and Nuclear Spins ∗Jörg Wrachtrup; 3rd Institute of Physics, University of Stuttgart, Stuttgart, Germany . 50

12:15 -14:15 Lunch Break

page Free for Excusions 14:15-18:00 Satellite Meeting: Biomedical Imaging Symposium, Chair: Fernanda Moll, Room: Ônix. Requires 14:15-18:00 separate registration. all day Vendors Exhibit Booths (Foyer)

13 Program

Thursday – May 23rd - Plenary Session Chair: Beat Meier Room Gávea A

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8:30-9:15 PL106: Spectroscopy in the Clinic – from dreams to reality ∗Jeremy K. Nicholson; Department of Surgery and Cancer, Faculty of Medicine, Imperial College London ...... 51

9:15-10:00 PL107: New contraptions/gadgets/techniques for solid-state NMR ∗K. Takegoshi; Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan ...... 51

10:00-10:30 Coffee Break

Thursday – May 23rd - DNP Chair: Christian Griesinger Room Gávea A

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10:30-10:55 OP108: Magnetic resonance is no longer the world’s least sensitive form of spec- troscopy 1,2∗Geoffrey Bodenhausen; 1Ecole Normale Supérieure, Paris, 2Ecole Polytechnique Fédérale, Lausanne ...... 51

10:55-11:20 OP109: Dissolution Dynamic Nuclear Polarization of Large Molecules ∗Christian Hilty, Mukundan Ragavan, Hsueh-Ying Chen, Youngbok Lee, Haifeng Zeng, Hlaing Min; Texas A&M University, Chemistry Department, USA ...... 52

11:20-11:35 UP110: DNP Surface Enhanced NMR (SENS): a Tool for Structural Investiga- tion of Supported Catalysis 1∗Moreno Lelli, 1Alexandre Zagdoun, 1David Gajan, 1Aaron J. Rossini, 4Olivier Ouari, 4Paul Tordo, 3Chloé Thieuleux, 2Christophe Copéret, 1Anne Lesage, 1Lyndon Emsley; 1Centre de RMN à Très Hauts Champs, Université de Lyon (CNRS/ENS Lyon/UCB Lyon 1), 2Department of Chemistry, ETH Zürich, Switzerland, 3Institut de Chimie de Lyon, Université de Lyon (CNRS-Université Lyon 1-ESCPE Lyon), 4Aix-Marseille Université, CNRS, Institut de Chimie Radicalaire (ICR), Marseille ...... 159

11:35-11:50 UP111: Sensitivity enhancement in solution NMR through dynamic nuclear po- larization of encapsulated proteins 1Kathleen G. Valentine, 2Guinevere Mathies, 1Nathaniel V. Nucci, 1Igor Dodevski, 1Sabrina Bédard, 1Matthew Stetz, 2Thach V. Can, 2Robert G. Griffin, 1∗A. Joshua Wand; 1University of Pennsylvania, 2Massachusetts Institute of Technology ...... 159

11:50-12:15 OP112: Dissolution DNP: Theoretical Considerations and Experimental Imple- mentation Grzegorz Kwiatkowski, Alexander Karabanov, ∗Walter Köckenberger; University of Not- tingham ...... 53

14 Program

Thursday – May 23rd - Dynamics and Recognition Chair: Martin Billeter Room Gávea B

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10:30-10:55 OP113: Use of Solution NMR for Studies of Proteins and Protein Complexes Involved in Protein and Peptide Synthesis ∗Gerhard Wagner; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston...... 53

10:55-11:20 OP114: Allosteric Motions in periplasmic binding Proteins characterized by NMR Spectroscopy Gabriel Ortega, Tammo Diercks, ∗Oscar Millet; CIC bioGUNE, Parque Tecnológico de Vizcaya, Spain ...... 53

11:20-11:35 UP115: Atomistic Descriptions of Protein Dynamics on Multiple Timescales From NMR Chemical Shifts ∗Paul Robustelli; Columbia University ...... 157

11:35-11:50 UP116: Viral RNAs and Infection Joseph D. Puglisi, Aaron Coey, ∗Elisabetta Viani; Department of Structural Biology & Stanford Magnetic Resonance Laboratory, Stanford University...... 54

11:50-12:15 OP117: High-Resolution Protein Structures by Magic-Angle Spinning NMR ∗Chad M. Rienstra, Marcus D. Tuttle, Gemma Comellas, Andrew J. Nieuwkoop, Ming Tang, Lindsay J. Sperling, Robert B. Gennis, Julia M. George; University of Illinois at Urbana Champaign...... 54

Thursday – May 23rd - New Solution NMR Methods Chair: Julien Wist Rooms Turmalina/Topázio

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10:30-10:55 OP118: Broadband Inversion of 1JCC Correlations in 1,n-ADEQUATE Spectra 1∗Gary E Martin, 1Mikhail Reibarkh, 1R. Thomas Williamson, 2Wolfgang Bermel; 1Merck Research Laboratories, 2Bruker BioSpin ...... 54

10:55-11:20 OP119: Highlighting Change 1∗Eriks¯ Kupče, 2Ray Freeman; 1Bruker Ltd, Coventry, UK, 2Jesus College, Cambridge, UK ...... 55

11:20-11:35 UP120: Measurements of Nuclear Magnetic Shielding ∗Karol Jackowski; Faculty of Chemistry, University of Warsaw, Poland ...... 55

11:35-11:50 UP121: Optimizing Ionic Liquids for CO2 Capture: an NMR Approach 1Marta Corvo, 1João Sardinha, 2Sonia Maria Cabral de Menezes, 3Jairton Dupont, 3Graciane Marin, 4Sandra Einloft, 4Marcus Seferin, 1Teresa Casimiro, 1∗Eurico J. Cabrita; 1REQUIMTE,CQFB, Dept. Química, Fac. Ciências e Tecnologia, Univ. Nova Lisboa, Portugal, 2PETROBRAS/CENPES, RJ Brazil, 3Dept. Química Orgânica, Inst. Química, Univ. Federal do Rio Grande do Sul, Brazil, 4Fac. Química Pontificia Univ. Católica Rio Grande do Sul, Brazil ...... 156

11:50-12:15 OP122: Old Dog, New Tricks: Methodological NMR Developments to Charac- terize Solution-State Structure and Dynamics Pieter ES Smith, Gonzalo A Álvarez, Noam Shemesh, Gershon Kurizki, ∗Lucio Frydman; Department of Chemical Physics, Weizmann Institute of Science, Israel ...... 56

15 Program

Thursday – May 23rd - New BioSolid Methods Chair: Stanley Opella Room Ônix

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10:30-10:55 OP123: Designing efficient re- and decoupling experiments for biological solid- state NMR using optimal control and effective Hamiltonian methods ∗Niels Christian Nielsen; Aarhus University, Aarhus, Denmark ...... 56

10:55-11:20 OP124: Spectrally Edited 2D NMR of Carbon Materials and Proteins 1∗Klaus Schmidt-Rohr, 1Robert L. Johnson, 1Keith J. Fritzsching, 2Jason Anderson, 2Brent Shanks, 1Mei Hong; 1Department of Chemistry, Iowa State University, 2Department of Chemical and Biological Engineering, Iowa State University ...... 56

11:20-11:35 UP125: Recording Quantitative One- and Two-Dimensional 13C NMR Spectra of Microcrystalline Proteins with Improved Sensitivity 1,2,3∗Piotr Tekely, 1,3Rudra Narayan Purusottam, 1,2,3Geoffrey Bodenhausen; 1Ecole Nor- male Superieure (ENS), 2Centre National de la Recherche Scientifique (CNRS), 3Université Pierre et Marie Curie (UPMC) ...... 57

11:35-11:50 UP126: Molecular interactions with the bacterial cell wall by liquid state, stan- dard and DNP solid state NMR. 1Catherine Bougault, 1Lauriane Lecoq, 2Sabine Hediger, 2Hiroki Takahashi, 2Gaël De Paëpe, 3Michel Arthur, 1∗Jean-Pierre Simorre; 1Institut de Biologie Structurale CEA-CNRS- UJF Grenoble, 2Laboratoire de Chimie Inorganique et Biologique, CEA Grenoble, 3Centre de Recherche des Cordeliers, INSERM, Paris ...... 57

11:50-12:15 OP127: The HIPER project – Very High Sensitivity Pulse EPR 1∗Graham Smith, 1Hassane El Mkami, 1Paul Cruickshank, 1Robert Hunter, 1David Bolton, 1Duncan Robertson, 1Bela Bode, 2Olav Schiemann, 3Richard Wylde, 4David Kee- ble, 4David Norman; 1University of St Andrews, Scotland, 2University of Bonn, Germany, 3Thomas Keating Ltd, Billingshurst England, 4University of Dundee, Scotland ...... 57

12:15-14:15 Lunch Break

Thursday – May 23rd - New BioNMR Methods Chair: Gisele Amorim Room Gávea A

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14:15-14:40 OP128: High Sensitivity Pulse Dipolar ESR and Protein Structure ∗Jack H. Freed; Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA ...... 58

14:40-15:05 OP129: Harnessing the Synergies between NMR and Computation for the Un- derstanding of Protein Function 1,2Mioara Larion, 1,2,3Roberto Salinas, 4Vitali Tugarinov, 1,2Dawei Li, 1,2Dong Long, 1,2Yina Gu, 2Lei Bruschweiler-Li, 1Brian Miller, 1,2∗Rafael Brüschweiler; 1Department of Chemistry and Biochemistry, Florida State University, 2National High Magnetic Field Laboratory, Tallahassee, FL, 3Institute of Chemistry, University of São Paulo, 4Department of Chemistry and Biochemistry, University of Maryland ...... 58

15:05-15:20 UP130: A unified conformational selection and induced fit approach to the mod- elling of protein-peptide interactions. Mikael Trellet, Adrien Melquiond, ∗Alexandre M. J. J. Bonvin; Utrecht University, Fac- ulty of Science, Bijvoet Center ...... 58

15:20-15:35 UP131: Replica Averaging-Replica Exchange (RARE) Molecular dynamics sim- ulations with NMR chemical shifts ∗Carlo Camilloni, Andrea Cavalli, Michele Vendruscolo; University of Cambridge...... 59

15:35-16:00 OP132: Extending the Empirical Limits of Chemical Shifts Yang Shen, Alex Maltsev, Alexey Mantsyzov, Jinfa Ying, ∗Ad Bax; Laboratory of Chemical Physics, NIDDK, NIH ...... 59

16 Program

Thursday – May 23rd - Bio-Solids Chair: Mônica Freitas Room Gávea B

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14:15-14:40 OP133: Type-III secretion needles studied by solid-state NMR 1Jean-Philippe Demers, 1Antoine Loquet, 1Veniamin Shevelkov, 2Nikolaos Sgourakis, 3Rashmi Gupta, 3Michael Kolbe, 1Karin Giller, 1Dietmar Riedel, 1Christian Griesinger, 2David Baker, 1Stefan Becker, 1∗Adam Lange; 1Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, 2Department of Biochemistry, University of Washington, USA, 3Max Planck Institute for Infection Biology, Berlin, Germany ...... 59

14:40-15:05 OP134: Solid-State NMR Structural Studies of Proteins Using Paramagnetic Probes 1Ishita Sengupta, 1Philippe S. Nadaud, 1Min Gao, 1Rajith J. Arachchige, 2Charles D. Schwi- eters, 1∗Christopher P. Jaroniec; 1The Ohio State University, Columbus, USA, 2National Institutes of Health, Bethesda, USA ...... 59

15:05-15:20 UP135: 100 kHz Magic-Angle Spinning for Proteins 1∗Beat H. Meier, 2Ago Samoson, 3Anja Böckmann, 1Matthias Ernst, 1Vipin Agar- val, 2Marie-Laure Fogeron, 1Matthias Huber, 1Susanne Penzel, 1Francesco Ravotti; 1ETH Zurich, Physical Chemistry, Switzerland, 2Tallinn University of Technology, Estonia, 3IBCP, CNRS, Lyon, France ...... 60

15:20-15:35 UP136: Determining supramolecular organisation of ion channels by solid-state NMR and computational methods 1∗Markus Weingarth, 1Elwin van der Cruijsen, 1,2Alexander Prokofyev, 1Eline Koers, 1Alexandre M. J. J. Bonvin, 2Olaf Pongs, 1Marc Baldus; 1Utrecht University, Bijvoet Cen- ter for Biomolecular Research, Utrecht, The Netherlands, 2Saarland University, Faculty of Medicine, Department of Physiology, Homburg, Germany ...... 157

15:35-16:00 OP137: Membranes, crystals, sediments: solid-state NMR of large proteins 1∗Anja Böckmann, 2Beat H. Meier, 1Britta Kunert, 2Andreas Hunkeler, 2Susanne Pen- zel, 2Anne Schütz, 1Laurent Terradot Alexandre Bazin, 1Pierre Falson, 3Jean-Michel Jault, 1Carole Gardiennet; 1Institut de Biologie et Chimie des Protéines, CNRS-Université de Lyon, France, 2ETH Zürich, Physical Chemistry, CH-8093 Zürich, Switzerland, 3IBS, CNRS-CEA-Université de Grenoble, France ...... 60

Thursday – May 23rd - Contrast Agents/ Instrumentation Chair: Carlos Geraldes Rooms Turmalina/Topázio

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14:15-14:40 OP138: New Directions in Dipolar Imaging to Enhance Endogenous Contrast ∗Warren S. Warren, Thomas Theis, Yi Han, Zijian Zhou; Duke University ...... 60

14:40-15:05 OP139: Capturing Signals from Ultrafast Relaxing Spins with SWIFT MRI ∗Michael Garwood; Center for Magnetic Resonance Research, University of Minnesota, USA ...... 61

15:05-15:20 UP140: Molecular imaging of tumors using CEST MRI of 2DG and FDG 1Michal Rivlin, 2Galia Tzarfaty, 1Judith Horev, 1Ilan Tzarfaty, 1∗Gil Navon; 1Tel Aviv University, 2Chaim Sheba Medical center...... 61

15:20-15:35 UP141: Physiological Model for the Determination of rCMR(O2) and rCBF by 17O MRI in the Human ∗Daniel Fiat; Universityof Illinois at Chicago ...... 61

15:35-16:00 OP142: Magnetic Susceptibility Contrast in Human Brain ∗Jeff Duyn, Peter van Gelderen, Pascal Sati, Afonso Silva, Daniel Reich, Hellmut Merkle, Jacco de Zwart; Laboratory of Functional and Molecular Imaging, National Institutes of Health, Bethesda ...... 62

17 Program

Thursday – May 23rd - Physics Chair: Jose Schneider Room Ônix

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14:15-14:40 OP143: Dynamics and Quantum Information Transport in Nuclear Spin Chains ∗Paola Cappellaro; Massachusetts Institute of Technology ...... 62

14:40-15:05 OP144: Loschmidt Echoes as Quantifiers of Decoherence, Quantum Phase Tran- sitions and Thermalization in Interacting Spin Systems 1,2∗Horacio M. Pastawski, 1,2Patricia R. Levstein; 1LaNaIS de RMS, Instituto de Física Enrique Gaviol (CONICET-UNC), 2Facultad de Matemática Astronomía y Física, Univer- sidad Nacional de Córdoba ...... 62

15:05-15:20 UP145: Origin of Long-Lived Signals in Dipolar Coupled Spin Systems 1∗Alexej Jerschow, 1Jae-Seung Lee, 2Anatoly Khitrin; 1New York University, 2Kent State University...... 62

15:20-15:35 UP146: High Resolution Para-Hydrogen Induced Polarization (PHIP) in Inho- mogeneous Magnetic Fields 1,2∗Lisandro Buljubasich, 1,2Ignacio Prina, 3María Belén Franzoni, 3Kerstin Münnemann, 3Hans Wolfgang Spiess, 1,2Rodolfo Héctor Acosta; 1FAMAF – Universidad Nacional de Cór- doba, 2IFEG –CONICET, 3Max Plank Institut für Polymerforschung ...... 157

15:35-16:00 OP147: Using single spins for quantum computing and sensing ∗Dieter Suter; TU Dortmund...... 63

16:00-18:30 Poster Session III Rooms Quartzo A/B, Authors with poster numbers starting "TH" present

all day Vendors Exhibit Booths (Foyer)

18:30 Shuttle Buses start leaving to hotels and Brazilian Night 20:00-24:00 Brazilian Night (banquet, show and music) at Fogo de Chão Brazilian Steak House (Av. Repórter Nestor Moreira s/n, Botafogo) – Tickets required. 22:30-24:00 Shuttle Buses will return to hotels

18 Program

Friday – May 24th - Membrane Proteins Chair: Laurent Catoire Room Gávea A

page 8:30-8:55 OP148: NMR studies on the type IV secretion system of Xanthomonas citri Diorge Souza, Luciana Coutinho de Oliveira, Denize C. Favaro, Cristina Alvarez-Martinez, Chuck Farah, ∗Roberto Salinas; Department of Biochemistry, Institute of Chemistry, USP 63 8:55-9:20 OP149: Magic Angle Spinning Solid-State NMR Studies of Membrane Proteins ∗Vladimir Ladizhansky; University of Guelph ...... 63 9:20-9:35 UP150: Comparison of Structure of Cyanobacateria and Spinach PSII Studied by PELDOR 1∗Asako Kawamori, 2Jiang-Ren Shen, 3Hiroyuki Mino; 1AGAPE-Kabutoyama Institute of Medicine, 2Department of Biology, Okayama University, 3Department of Physics, Nagoya University...... 158 9:35-10:00 OP151: Structure Determination of Membrane Proteins in their Native Envi- ronment of Phospholipid Bilayers ∗Stanley Opella; University of California, San Diego ...... 64

Friday – May 24th - Instrumentation Chair: Mario Engelsberg Room Gávea B

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8:30-8:55 OP152: Robust Multi-Phase Flow Measurements Using Magnetic Resonance 1,2∗Daniel José Pusiol, 1Lucas C. Cerioni; 1Instituto de Física Enrique Gaviola IFEG, CONICET, Ciudad Universitaria, Córdoba, Argentina, 2SPINLOCK SRL & CONICET, Córdoba, Argentina ...... 64

8:55-9:20 OP153: Using Time Domain NMR to Study Magnetoelectrolysis phenomenon in Situ 1∗Luiz Alberto Colnago, 1Luis Fernando Cabeça, 2Luiza Maria da Silva Nunes, 2Paulo Falco Cobra, 2Bruna Ferreira Gomes, 1André de Souza Carvalho; 1Embrapa Instrumentação, São Carlos/SP - Brazil, 2Instituto de Química de São Carlos - USP ...... 64

9:20-9:35 UP154: In situ MR: Pore condensation at elevated temperature and pressure 1∗Matthew P. Renshaw, 1S. Tegan Roberts, 2Belinda S. Akpa, 1Mick D. Mantle, 1Andrew J. Sederman, 1Lynn F. Gladden; 1University of Cambridge, 2University of Illinois at Chicago 159

9:35-10:00 OP155: Compact NMR of Materials and Processes ∗Bernhard Blümich; RWTH Aachen University...... 65

Friday – May 24th - Innovative NMR/MRI Methods Chair: Betty Gaffney Rooms Turmalina/Topázio

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8:30-8:55 OP156: Relaxometry and dynamics ∗Claudio Luchinat; Magnetic Resonance Center (CERM), University of Florence ...... 65

8:55-9:20 OP157: The Nitrogen-Vacancy Center In Diamond As A Nanoscale Spin Sensor 1Abdelghani Laraoui, 2Florian Dolde, 2Tobias Staudacher, 2Jörg Wrachtrup, 2Friedemann Reinhard, 1∗Carlos A. Meriles; 1Department of Physics, CUNY - City College of New York, New York, 23rd Physics Institute, University of Stuttgart, Germany ...... 66

9:20-9:35 UP158: Mitochondrial function in diabetes: Novel methodology and new insight ∗Liping Yu, Brian Fink, Judith Herlein, William Sivitz; University of Iowa ...... 66

9:35-10:00 OP159: Using SABRE as a route to hyperpolarization in NMR and MRI 1∗Simon B Duckett, 1Kevin D. Atkinson, 1Alex J. J. Hooper, 1Lyrelle S. Lloyd, 2Gary G. R. Green, 1Marianna Fekete, 1Richard A. Green, 1Ryan E. Mewis, 1Louise A. R. Highton; 1York Centre for Hyperpolarisation In Magentic Resonance, 2York Neuroimaging Centre. . 66

19 Program

Friday – May 24th - Small Molecules and Bioactive Peptides Chair: Marta Bruix Room Ônix

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8:30-8:55 OP160: NMR Studies of the Self-Association and Membrane Binding of Cy- clotides ∗David J Craik, Conan Wang, K Johan Rosengren, Anne Conibear, Sonia T Henriques; Institute for Molecular Bioscience, The University of Queensland, Australia ...... 66

8:55-9:20 OP161: Separative NMR: adapted resolution for complex mixtures of small molecules 1,2∗Stefano Caldarelli; 1Aix Marseille Université,Marseille, France, 2CNRS UPR 2301 Ecole Palaiseau France ...... 67

9:20-9:35 UP162: Structural Basis for the Interaction of Human β-Defensins 1 and 6 and Its Putative Chemokine Receptor CCR2 and Breast Cancer Microvesicles ∗Viviane Silva de Paula, Robson Q. Monteiro, Fabio C. L. Almeida, Ana Paula Valente; Universidade Federal do Rio de Janeiro ...... 155

9:35-10:00 OP163: Breaking the barrier: NMR of membrane-active peptides ∗Frances Separovic; School of Chemistry, Bio21 Institute, University of Melbourne, Aus- tralia...... 67

10:00-10:30 Coffee Break

Friday – May 24th - Plenary Session Chair: Sonia Menezes Room Gávea A

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10:30-11:15 PL164: Nuclear Spins, High Magnetic Fields, and Traveling Waves in Pursuit of Human Brain Function and Connectivity ∗Kamil Ugurbil; CMRR, University of Minnesota, USA...... 68

11:15-12:00 PL165: Synergy between NMR, cryo-EM and large-scale MD simulations - Novel Findings for HIV Capsid Function ∗Angela M. Gronenborn; Department of Structural Biology, University of Pittsburgh . . . 68 Closing Ceremony Room Gávea A

page 12:00-12:30 Closing Remarks by José Daniel Figueroa-Villar and Hans Spiess 12:30 Adjourn

12:30-14:30 Lunch Break

page Free for Excusions 14:30-18:20 Satellite Meeting: Vth Iberoamerican NMR Meeting, Chair: Fabio Almeida (UFRJ, Brazil), Room: 14:30-18:20 Turmalina/Topázio. Requires separate registration.

20 Abstracts of Talks – Dynamics and Catalysis

Abstracts of Talks

Sunday – May 19th PL003: Sensitivity enhancement in High Field NMR Honor and Prizes Session and EPR Presiding: Hans Spiess Vasyl Denysenkov, Petr Neugebauer, Phillip Spindler, Room Gávea A Burkhard Endeward, ∗Thomas F. Prisner Institute of Physical and Theoretical Chemistry and Cen- PL001: Emerging Frontiers in Ultrafast Multidimen- ter of Biomolecular Magnetic Resonance, Goethe University sional NMR and MRI Frankfurt, Germany

Rita Schmidt, Eddy Solomon, Avi Leftin, ∗Lucio Frydman Sensitivity is a major limitation for the application of NMR to samples with low spin concentration or restricted volume. Department of Chemical Physics, Weizmann Institute of Sci- We have explored the use of dynamic nuclear polarization ence, Israel (DNP) to enhance liquid NMR signals at a magnetic field of We have developed a scheme enabling the acquisition of arbi- 9.2 T (400 MHz proton and 260 GHz electron spin resonance trary multidimensional NMR spectra and/or images (MRI), frequency) at ambient temperatures. Enhancements of up within a single scan. This is by contrast to the hundreds or to -80 have been experimentally observed and rationalized thousands of scans that are usually needed to collect this kind with respect to the electron-nuclear spin coupling efficiency, of data. Provided that the target molecule’s signal is suffi- saturation behavior of the electron spin and viscosity of the ciently strong, the acquisition time of NMR/MRI scans can solvent. EPR offers increased sensitivity, compared to NMR, thus be shortened by several orders of magnitude. This new because of the at 660 times larger magnetic moment of the "ultrafast" methodology is compatible with existing multi- electron spin with respect to a proton spin, but is typically dimensional pulse sequences and can be implemented using performed at lower magnetic fields. At high magnetic fields conventional hardware. The manner by which the spatiotem- the EPR line shape becomes typically very broad and thus poral encoding of the NMR interactions –which is the new cannot be efficiently excited by the limited power available principle underlying these new protocols– proceeds in these above 100 GHz microwave frequency. This severely restricts experiments, will be summarized. The new horizons that are the efficiency of pulsed EPR and DNP methods. We will opened by these protocols will also be exemplified with a va- show new approaches to improve the sensitivity by the ap- riety of NMR and MRI projects we are currently involved plication of new pulse schemes for EPR and the use of high in in fields of chemistry, biophysics, biology and medicine. power cw microwave gyrotron sources for DNP and discuss The incorporation into these experiments of nuclear hyper- the potential and limitations of these methods for biomolec- polarization procedures capable of increasing the single-scan ular applications. sensitivity of single-scan liquid state NMR by factors ranging ACKNOLEDGEMENTS: German Research Society and from 103-106, will also be assessed. Center of Excellence Frankfurt Macromolecular Complexes Acknowledgments: : BioNMR EU Grant #261863, EU ERC for financial support Advanced Grant # 246754, Kamin-Yeda Grant (Israel), Is- rael Science Foundation, Fulbright and Natl. Science Foun- Monday – May 20th dations (US) Dynamics and Catalysis Chair: Jarbas Resende th Monday – May 20 Room Gávea A Plenary Session Chair: Daniella Goldfarb OP004: Control of Periplasmic Interdomain Thiol: Room Gávea A Disulfide Exchange in the Transmembrane Oxidore- ductase DsbD PL002: Biomolecular NMR at Very Low Tempera- 1Despoina A.I. Mavridou, 2Emmanuel Saridakis, 1Paraskevi tures Kritsiligkou, 1Lukas Stelzl, 1Erin C. Mozley, 1Alan D. God- 1 1 1∗ ∗Robert Tycko, Kent Thurber, Alexey Potapov, Wai-Ming dard, Julie M. Stevens, Stuart J. Ferguson, Christina Yau, Eric Moore Redfield 1 National Institutes of Health Department of Biochemistry, University of Oxford, 2National Center for Scientific Research Demokritos Our group pursues research on solid state NMR methods and their application to structural problems in biological systems. Oxidative protein folding is essential for stability and func- This lecture will focus on recent efforts to develop and apply tion of numerous proteins. In vivo, thiol:disulfide exchange new technology for NMR measurements on biological systems reactions are catalyzed by a range of oxidoreductases which at temperatures below 30 K. These efforts include the design often contain the thioredoxin fold. The protein DsbD trans- and construction of an apparatus for magic-angle spinning fers electrons from the cytoplasm to the periplasm of Gram- with sample temperatures in the 20-25 K range, in which negative bacteria. This reducing power is required in the the sample is cooled with liquid helium while sample rota- otherwise oxidizing periplasm for the isomerization of in- tion is driven with nitrogen gas, and the development of a correct disulfide bonds (Dsb) and for cytochrome c mat- dynamic nuclear polarization (DNP) system that operates at uration (Ccm). Reductant transfer occurs via a series of temperatures as low as 8 K (without MAS) or 20 K (with thiol:disulfide exchange reactions between pairs of conserved MAS) and uses relatively low microwave powers at 264 GHz cysteines in the three domains of DsbD, tmDsbD (the inte- (the electron spin resonance frequency of nitroxides in a 9.4 gral membrane domain), nDsbD and cDsbD (the N- and C- T magnet). Our latest results from experiments that use terminal periplasmic domains) and their partner proteins on very low temperatures will be described, possibly including both sides of the inner membrane. The flow of electrons starts structural studies of transient intermediate states in a rapid from cytoplasmic thioredoxin and proceeds to tmDsbD, then protein folding process (trapped by rapid freeze-quenching), to cDsbD and nDsbD and ends with the periplasmic proteins structural studies of intermediate states in the self-assembly DsbC and CcmG. NMR spectroscopy has been used to study of beta-amyloid peptides into amyloid fibrils, or attempts to the reactivity and specificity of these processes. cDsbD has a obtain micron-resolution magnetic resonance images with the thioredoxin fold; the N-terminal cysteine, C461, in the char- assistance of low temperatures and DNP. acteristic CXXC motif attacks the disulfide bond of nDsbD. Knowledge of cysteine pKas is important in understanding

21 Abstracts of Talks – General Solids

their reactivity. C461 has an unusually high pKa; this makes constant of the enzyme. Our data firmly underscore that C461, in isolated cDsbD, a poor nucleophile and implies rela- understanding enzyme dynamics in substrate-free states is tive unreactivity towards the target disulfide in nDsbD. NMR required for a full understanding of enzymatic mechanisms. studies of an nDsbD-15N-cDsbD covalent complex allowed specific changes in active-site pKa values due to contact with Reference: Ådèn, J. et. al. (2012), JACS, 134, 16562-16570 its physiological partner to be examined. The observed mod- ulation of pKa values is critical for the specificity and function UP007: Structural and dynamic insights into sub- of cDsbD. In recent studies the interactions of nDsbD and strate binding and catalysis in human Lipocalin cDsbD have been shown to be oxidation-state dependent such Prostaglandin D synthase that the functionally-relevant complex nDsbDox-cDsbDred, 1∗Konstantin Pervushin, 1,2Pär Nordlund, 1,2Sing Mei has a higher affinity than the product complex, nDsbDred- Lim, 1Dan Chen, 1Hsiangling Teo, 2Annette Roos, 2Tomas cDsbDox. The principles established have wider significance 2 for understanding of processes mediated by thioredoxin-like Nyman, Lionel Trésaugues proteins which are critical in prokaryotes and eukaryotes. 1Nanyang Technological University, 2Karolinska Institute Lipocalin-Prostaglandin D synthase converts Prostaglandin- OP005: Hydration of yeast Thioredoxin 1 Modulates H2 (PGH2) to Prostaglandin-D2 (PGD2), an important in- Slow Dynamics flammatory and signaling mediator. It also binds biliverdin, ∗ all-trans retinoic acid and bilirubin, acting as lipophilic com- Fabio C. L. Almeida pound carrier. We employed structural, biophysical and bio- Institute of Medical Biochemistry, National Center of Nu- chemical approaches to address the mechanistic aspects of clear Magnetic Resonance, Federal University substrate entry, catalysis and product exit. The structure of Proteins are dynamic entities that move in a hierarchy of human L-PGDS was solved in a complex with a substrate timescales that goes from picoseconds to seconds. Motions analog and in ligand-free-form. Catalytic Cys 65 thiol group that occur in microseconds to seconds define biologically rel- is found in two different conformations, each making a dis- evant events that are frequently involved in binding, allostery tinct hydrogen bond network to neighboring residues, eluci- and catalysis. We measured relaxation parameter, relaxation dating the mechanism of the cysteine nucleophile activation. dispersion experiments and molecular dynamic simulation in Ligand electron density was observed in the active site de- order to correlate hydration and slow dynamics. We will show marking substrate-binding regions, but did not allow unam- how conformational dynamics of yeast thioredoxin 1 is corre- biguous fitting of the substrate. To further understand lig- lated with the hydration equilibrium of a water cavity near and binding, we used NMR spectroscopy to map the ligand the active site. The residue Asp24 that is well-recognized as binding sites and to show the dynamics of protein-substrate a proton acceptor in the catalytic mechanism also controls and protein-product interactions. A model for ligand bind- dynamic events of the protein that are important for recog- ing at the catalytic site is proposed, showing a second bind- nition and catalysis. ing site involved in ligand exit and entry. NMR chemical shift perturbations and NMR resonance line-widths alter- ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, ations (observed as changes of intensity in 2D cross-peaks 1 15 INBEB-CNPq, Program Science Without Borders-CNPq in [ H, N]-TROSY) for residues at ω helix, E-F loop and G-H loop besides the catalytic sites indicating involvement of these residues in ligand entry/egress. UP006: The magnitude of a pre-existing equilibrium tunes adenylate kinase activity OP008: EPR Studies of Radical SAM Enzymes 1Jörgen Åden, 2Abhinav Verma, 2Alexander Schug, ∗ 1∗Magnus Wolf-Watz R. David Britt 1Umeå University, 2Karlsruhe Institute of Technology University of California, Davis Enzymes are extraordinary biocatalysts that accelerate chem- We are using multifrequency EPR in conjunction with freeze ical reactions by performing several complex tasks simultane- trapping of intermediates and specific isotope labeling to ously. These tasks include; (1) binding of substrates weakly probe the geometrical and electronic structure of such inter- enough to avoid thermodynamic pits; (2) extremely tight mediates formed during catalysis by Radical SAM enzymes. binding to the transition state compound; (3) activation of A number of Radical SAM enzymes have one or more sec- functional groups on the substrate(s); (4) dehydration of ac- ondary FeS clusters in addition to the 4Fe4S cluster that tive sites and (5) conformational changes. binds the SAM cofactors. Two of the enzymes we are study- ing are biotin synthase, which inserts a S-atom in the final The free energy landscape of enzymes is tuned such that step of forming the vitamin biotin, and HydG, which is a ty- functionally important structures are accessed within rele- rosine lyase which produces the CO and CN- ligands for the vant time-scales. Some enzymes have been found to sam- Fe-Fe cluster of [Fe-Fe] hydrogenase. ple "substrate bound-like" structures in the absence of sub- strate and Escherichia coli adenylate kinase (AKeco) belongs th to this class. These enzymes display pre-existing conforma- Monday – May 20 tional equilibria between inactive and active structural states General Solids in their substrate free states. Here, we probe the free energy Chair: Bernhard Blümich landscape of substrate-free AKeco with solution state NMR Room Gávea B spectroscopy. Perturbation of the pre-existing equilibrium constant in the forward and reverse directions was accom- OP009: Structure Determinations of Molecularly plished with mutation or addition of the osmolyte TMAO, Ordered, Non-Crystalline Silicate, Borosilicate, and respectively. Surprisingly, we found that the rate constant for Aluminosilicate Frameworks by Combined NMR, the enzymatic turn-over (kcat) of AKeco is dependent on the Scattering, and DFT Analyses magnitude of the equilibrium constant for exchange between active and inactive structures observed in the substrate-free 1∗Bradley F. Chmelka, 2Sylvian Cadars, 3Darren state. A shift of the equilibrium towards the active structure H. Brouwer, 1Ming-Feng Hsieh, 1Robert J. Messinger, results in a decrease of kcat, and conversely a shift towards 1Matthew T. Aronson, 2Mounesha N. Garaga, 2Zalfa Nour the inactive structure results in an increase of kcat. These 1University of California, Santa Barbara, USA, 2CNRS, seemingly counterintuitive relations depend on that AKeco Université d’Orléans, France, 3Redeemer College, Ancaster, catalysis is rate-limited by a conformational change coupled Ontario, Canada to product release. Thus, for AKeco the magnitude of a pre- existing equilibrium is directly mirrored in the catalytic rate- A general protocol is demonstrated for determining the struc-

22 Abstracts of Talks – General Solids tures of molecularly ordered but non-crystalline solids, which field-effect transistors, and light-emitting diodes, where the combines constraints provided by solid-state NMR, X-ray active semiconducting polymer layer is formed via solution diffraction, and first-principles calculations. The approach processing. This produces a final material that is semi crys- is used to determine the structure(s) of surfactant-directed talline, preventing the direct access to details about the local layered silicates with short-range order in two dimensions, molecular organization from a conventional approach. In this but without long-range 3D atomic periodicity. Structural contribution I will outline a general strategy for determin- constraints are obtained from solid-state 29Si MAS NMR ing structural motifs in π-conjugated systems that combines analyses, including the types and relative populations of dis- X-ray diffraction and solid-state NMR experiments with Nu- tinct 29Si sites, their respective 29Si-O-29Si connectivities cleus Independent Chemical Shift (NICS) maps.[1] This com- and dipolar interactions, and combined with unit cell param- bination provides a useful platform to evaluate specific pack- eters from XRD. A comprehensive search of candidate struc- ing structures and in some cases even allows setting up a tures leads to the identification of frameworks that are each crystal structure, if sufficient constraints can be determined compatible with all of the experimental constraints. Subse- from experiments. The potential of the proposed strategy quent refinements of the candidate structures using density- will be exemplified by recent and ongoing work on poly- functional theory allow their evaluation and the identification thiophenes,[1] donor-acceptor-type polymers,[2,3] and shape- of "best" framework representations, based on their lattice persistent macrocycles, forming empty helical nano chan- energies and quantitative comparisons of experimental and nels.[4] calculated isotropic 29Si chemical shifts and 2J(29Si-O-29Si)- References: scalar couplings. [1] D. Dudenko, A. Kiersnowski, J. Shu, W. Pisula, D. Se- The incorporation of heteroatoms, such as boron or alu- bastiani, H. W. Spiess, M. R. Hansen, Angew. Chem. Int. minum, into otherwise identical silicate frameworks often Ed. 2012, 51, 11068. leads to dramatically improved adsorption or catalytic prop- erties of the materials, as notably exemplified by nanoporous [2] H. N. Tsao, D. M. Cho, I. Park, M. R. Hansen, A. Mavrin- zeolites. Such properties, however, have been difficult to cor- skiy, D. Y. Yoon, R. Graf, W. Pisula, H. W. Spiess, K. relate to specific framework attributes, due to the increased Müllen, J. Am. Chem. Soc., 2011, 133, 2605. complexity of local structural order and disorder, especially near the heteroatom sites. Distributions of heteroatom bond- [3] D. Niedzialek, V. Lemaur, D. Dudenko, J. Shu, M. ing environments, in particular, lead to reduced resolution R. Hansen, J. W. Andreasen, W. Pisula, K. Müllen, J. of 1D 29Si, 11B, or 27Al MAS NMR spectra. By compari- Cornil, D. Beljonne, Adv. Mater., 2013, (in press, DOI: son, 2D Heteronuclear Multiple-Quantum Correlation NMR 10.1002/adma.201201058). measurements are sensitive to through-bond (J) or through- [4] M. Fritzsche, A. Bohle, D. Dudenko, U. Baumeister, D. space (dipole-dipole) interactions that can be used to probe Sebastiani, H. W. Spiess, M. R. Hansen, S. Höger, Angew. the short-range environments of different framework moieties, Chem. Int. Ed., 2011, 50, 3030. including those involving heteroatoms. Intensity correlations 11 29 27 associated with B, Si, or Al atoms provide detailed UP012: NMR Crystallography, from “Pas de deux” information on the bonding connectivities and distributions to Spin Choreography. Nanoporous Crystal Struc- of heteroatom sites in boro- and aluminosilicate frameworks, tures of Powders : Methods and Hardware. yielding new understanding of the compositions, structures, and properties of these technologically important materials. 1∗Francis Taulelle, 2Frank Engelke, 2Frank Decker, 1Boris Bouchevreau, 1Charlotte Matineau Acknowledgements: U.S. National Science Foundation, 1 2 Agence National de Recherche de France, Chevron University of Versailles, Bruker-Biopspin GmbH Inorganic-organic hybrid nanoporous solids exhibits many OP010: Zero- and low-field NMR spectroscopy challenges for their structure determination. They contain 1,2∗ a framework, that is usually periodic or at least topologi- Dmitry Budker cally periodic. However, these materials are hard to form 1Department of Physics, University of California, Berkeley, single crystals, most of the time one has to resolve their 2Nuclear Science Division, E. O. Lawrence Berkeley Na- crystal structure from powder. Additionally, they contain tional Laboratory, Berkeley either additional templating agents, of the same nature than We will present an overview of the recent work on NMR the organic linkers, making the activation (emptying the spectroscopy based on measuring J-couplings at zero and low nanopores) of the materials difficult, if not impossible, and magnetic fields, including experiments on NMR entirely with- by and large, partial. Therefore, at synthesis or later when out magnets (not counting a low-field-pulse coil), where sig- exchanged with gases or molecules, these exchanged crystals nal detection is accomplished with atomic magnetometers. exhibit a periodic network and a non-periodic sub-lattice. The experiments have been performed by our group in col- It will be shown on an alumina-phosphate lamellar laboration with the group of Prof. Alexander Pines at the nanoporous crystal [1] that X-ray diffraction has intrinsic lim- Department of Chemistry at UC Berkeley and the Materi- its for structure computability from a powder and this limit als Science Division of LBNL, and our various American and can be overcome by using NMR data. With this combined international collaborators. Up-to-date bibliography of this usage of XRD and NMR the average periodic structure can work can be found at the following web sites: be determined. At a second level of structure determina- http://budker.berkeley.edu/PubList.html tion NMR can resolve the structural non-periodic part of the crystal. http://waugh.cchem.berkeley.edu/publications.php . In a second part, it will be shown that the large number of ACKNOLEDGEMENTS: NSF, DOE NMR experiments that must be acquired to solve structures can be accelerated by using multiple channels probe (4 and 5 UP011: Use of 1H Chemical Shifts to Determine channels) [2] with multiple receivers acquisition. Multi- de- Structural Motifs in π-Conjugated Polymers coupling enhances the resolution that reaches about five times higher resolution power than the best synchrotron resolution ∗ Michael Ryan Hansen power. Max Planck Institute for Polymer Research NMR Crystallography encompasses usage of prior data from Polymers with extended π-conjugation and narrow band gaps XRD as cost function for the average structure determina- are of broad technological importance due to their promising tion as well as using new NMR hardware and software [3,4] applications as semiconductors in organic electronic devices. with additional pulse sequences [5] to solve all the structure Examples of such devices include organic-based solar cells, features of nanoporous crystals.

23 Abstracts of Talks – NMR in Medicinal Chemistry

With this instrumental developments, one can devise for of large collection of compounds (>100,000). A key advan- each structure resolution an original combination of pulse se- tage of FBLD approaches is that these often rely on robust quence manipulating the resonances between spins in a com- biophysical methods such as NMR spectroscopy for detection plex choreography of spins, grouped in a variety of motives of ligand binding, hence are less prone to artifacts that too that would progressively address all the aspects of the crystal often plague the results from HTS campaigns. However, the complexity. challenge of FBLD approaches is that the evolution of initial weakly interacting fragments into more mature compounds References with low micromolar is not trivial. In this presentation I will [1] B. Bouchevreau, C. Martineau, C. Mellot-Draznieks, A. introduce a new fragment screening strategy, that takes ad- Tuel, M. R. Suchomel, O. Lafon, J. Trébosc, J. P. Amoureux, vantage of both, the robustness of protein NMR spectroscopy F.; Taulelle, Chem. Eur. J. in press. as the detection method, and the basic principles of combi- natorial chemistry to enable the screening of large libraries of [2] C. Martineau, F. Engelke, F. Taulelle, J. Magn. Reson. fragments pre-assembled on a common backbone (>100,000 212 (2011) 311-319. compounds). Hence we term the approach HTS by NMR. The approach seems particularly suited to target protein- [3] E. Kupce, R. Freeman, J. Magn. Reson. 206 (2010) 147- protein interactions and three examples of such successful 153, J. Magn. Reson. 213 (2011) 1-13. implementations will be discussed. Identification of simple [4] E. Kupce, L. E. Kay, J. Biomol. NMR 54 (2012) 1-7. peptides or even small molecules with this approach does not require expensive resources or medicinal chemistry expertise, [5] Herbst, C.; Riedel, K.; Ihle, Y.; Leppert, J.; Ohlen- hence we believe the method could become routinely used schläger, O.; Görlach, M.; Ramachandran, R. MAS solid in NMR based structural biology laboratories that are in- state NMR of RNAs with multiple receivers. J Biomol NMR terested in making a significant contribution to translational 2008, 41, 121–125. research. [6] C. Martineau, F. Engelke, F. Decker, F. Taulelle. J. Magn. Wu et al. Chemistry & Biology, 2013, 20, 19-33. Reson. Submitted. OP015: Enzyme Kinetics and Inhibition by NMR [7] V. Munch, F. Taulelle, T. Loiseau, G. Férey, A. Cheetham, S. Weigel, G.D. Stucky, Magn. Reson. Chem. 1999, 37, ∗José Daniel Figueroa-Villar S100–S107. Mititary Institute of Engineering Enzyme inhibitors are important bioactive compounds for OP013: High Frequency Dynamic Nuclear Polariza- treatment of diseases. However, some available methods tion for evaluation of enzyme inhibition display important prob- ∗Robert G. Griffin lems. For example, the Ellman test, applied to inhibition of acetilcolinesterase (AChE) requires the use of thioacetyl- Francis Bitter Magnet Laboratory and Department of Chem- choline instead of acetylcholine (ACh), leading to different istry, MIT results. Also, with ultraviolet-visible spectroscopy methods, Over the last few years we have developed cyclotron reso- when the initial enzyme product displays lmax similar to the nance maser (a.k.a. gyrotron) microwave sources that oper- substrate, is necessary the use of a second enzyme in order ate at frequencies of 140-527 GHz that permit DNP enhanced to transform the product to a very different compound. The NMR (DNP/NMR) experiments in magnetic fields of 5-18.8 problem using a second enzyme is that some inhibitors of the 1 T( H NMR frequencies of 211-800 MHz, respectively). We first enzyme may also have effects on the second one, leading review the instrumentation used for these experiments, which to unacceptable results. The use of NMR methods for eval- include new NMR probe designs and tunable gyrotron oscilla- uation of enzyme inhibition is a very acceptable procedure tors and amplifiers. In addition, we discuss two mechanisms because this technique is relatively easy for monitoring sub- used for DNP experiments in solids at high fields – the solid strates and products concentration, without the use of other effect and cross effect – and the polarizing agents appropri- enzymes and/or different substrates, even allowing the ob- ate for each. These include metals, biradicals and mixtures tention of Lineweaver-Burk graphics to determine the type of narrow line radicals that enable increased enhancements at of inhibition of each tested compound. The problem with reduced concentrations of the paramagnetic center. In addi- NMR is its relatively NMR slowness in comparison with the tion, we discuss applications of DNP/NMR that illustrate its enzyme properties, a process that was resolved using very low utility in enhancing signal-to-noise in MAS NMR spectra of enzyme concentrations on the tests and simple 1H NMR pro- a variety of biological systems including membrane and amy- cedures. This method was successfully used for inhibition of loid proteins whose structures are of considerable scientific AChE, nucleoside hydrolase (NH) of Leishmania infantum, interest. Presently, enhancements that are routinely available dihydrofolate reductase (DHFR) of Plasmodium falciparum, and range from 40-250 depending on experimental variables human tyrosinase and reactivation of phosphorylared AChE. such as temperature, magnetic field, microwave B1, polariz- ing agent, etc. Interaction of inhibitors with enzymes were monitored by re- laxation times (T1 and T2), modification of chemical shifts and DOSY and kinetics by 1H NMR. Monday – May 20th NMR in Medicinal Chemistry ACKNOLEDGEMENTS: CAPES/Pro-defesa, INBEB, FAPERJ. Chair: Anita Marsaioli Rooms Turmalina/Topázio UP016: Integrated structural biology shows how scarce sequence elements control the function of sin- OP014: HTS by NMR of combinatorial libraries: a gle ß-thymosin/WH2 domains in actin assembly new approach to peptide and ligand discovery 1François-Xavier Cantrelle, 2Dominique Didry, 1Célia Dev- ∗Maurizio Pellecchia ille, 1Jean-Pierre Placial, 2Clotilde Husson, 3Javier Perez, 2Marie-France Carlier, 2Louis Renault, 1Carine van Hei- Chemical Biology, Sanford-Burnham Medical Research Insti- 1∗ tute, La Jolla, CA jenoort, Eric Guittet 1CNRS-ICSN, 2CNRS-LEBS, 3Synchrotron SOLEIL Recently, fragment based ligand design (FBLD) approaches have become more widely used in drug discovery projects β-thymosin (βT) and WH2 domains are found as single or from both academia and industry, and are even often pre- repeated units in a large number of multi-domain actin- ferred to traditional ultra-high-throughput screening (HTS) binding proteins involved multiple cellular processes. They

24 Abstracts of Talks – EPR and New Materials are archetypes of small intrinsically disordered actin-binding means of 1H and 19F NMR studies applying saturation trans- modules with highly variable sequences of 25-55 residues that fer difference (STD) and T2 perturbation strategies comple- fold upon binding. Understanding the structure-function re- mented by molecular modeling and docking studies[2]. The lationship governing the versatile or multiple functions of importance of all hydroxyl groups of the galactose-derived these domains remains extremely challenging. monosaccharide ligands and the dependency of calcium for carbohydrate recognition by this c-type lectin has been de- Here, we reveal the structural and dynamics determinants, duced from those studies. which govern the assembly inhibition (Thymosin-β4) or the motility enhancement by directing polarized filament assem- [1] "Sweet preferences of MGL: carbohydrate specificity and bly (Ciboulot βT or WASP/WAVE WH2 domains). To reach function", van Vliet SJ, Saeland E, van Kooyk Y. Trends this goal, we combined mutational, functional and structural Immunol. 2008 29(2):83-90. analyses by X-ray crystallography, SAXS and NMR in a typ- [2] "Carbohydrate-protein interactions: a 3D view by NMR", ical integrated structural biology approach. We show that Roldós V, Cañada FJ, Jiménez-Barbero J., Chembiochem. functionally different βT/WH2 domains do not target al- 2011 12(7):990-1005. ternative actin binding sites but rather differ by alternative dynamics of their C-terminal half interactions with G-actin Acknowledgments: This work has been carried out with pointed face [1-3]. financial aid of the MINECO, Spain (Project number CTQ2009-08536) The detailed NMR analysis of the dynamics of the free chimeras using 15N relaxation, residual dipolar couplings and relaxation dispersion experiments demonstrates specific OP018: High-yield expression and NMR structural individual behaviors, which bring insight into the folding studies of Antimicorbial Peptides upon binding mechanisms of these peptides. Isotope-filtered Ji-Ho Jung, Ji-Sun Kim, ∗Yongae Kim NOESY experiments allow the precise characterization of the structure of the complexes. The structural plasticity of WH2 Depart of Chemistry and Protein Research Center for Bio- domains is partially conserved and functional in the G-actin Industry, Hankuk University of Foreign Studies, Korea bound state. The distribution of static and dynamic interac- Lactophoricin (LPcin), a cationic amphipathic peptide con- tions induces their ability to control the function in an ionic sists of 23-mer peptide, corresponds to the carboxy terminal strength dependent manner. A single residue controls the lo- 113–135 region of component-3 of proteose-peptone. LPcin is cal interaction dynamics by strong electrostatic interactions a good candidate as a peptide antibiotic because it has an an- [3]. tibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, The results open perspectives for elucidating, including by LPcin-yk1 and LPcin-yk3 that has shorter mutant amino NMR, the functions of βT/WH2 domains in other modular acids are recently developed by using peptide engineering in proteins and enlighten how intrinsic structural disorder can our laboratory. These three LPcin analogs show better an- lead to a novel mode of functional versatility. tibioitic activities than wild-type LPcin and no toxicity at [1] Hertzog, M.; van Heijenoort, C.; Didry, D.; Gaudier, M.; all. Coutant, J.; Gigant, B.; Didelot, G.; Preat, T.; Knossow, M.; In order to understand the structural correlation between Guittet, E.; Carlier, MF Cell 2004, 117, 611-623. LPcin analogs structure and antimicrobial activity under the [2] Domanski, M.; Hertzog, M.; Coutant, J.; Gutsche- membrane environments, we tried to express and purify as Perelroizen, I.; Bontems, F.; Carlier, MF.; Guittet, E.; van large as amounts of LPcin and three different LPcin analogs. Heijenoort, C. J Biol Chem 2004, 279, 23637-45. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with bio- [3] Didry D, Cantrelle FX, Husson C, Roblin P, Moorthy AM, physical techniques like Ni-affinity chromatography, dialysis, Perez J, Le Clainche C, Hertzog M, Guittet E, Carlier MF, centrifuge, chemical cleavage, and reversed-phase semiprep van Heijenoort C, Renault L. EMBO J. 2011, 31, 1000-13. HPLC. In here, we will present the optimizing processes for high-yield expression and purification and solution NMR UP017: Human Macrophage Lectin (CLEC10A) spectra and solid state NMR spectra for antimicrobial mech- recognition of monosaccharides related to tumor anisms. 1 marker Tn-antigen studied by H and 19F NMR. Reference: 1) Tae-Joon Park, Sung-Sub Choi, Ji-Sun Kim, 1∗ 1 and Yongae Kim (2008) "Expression, Purification, and Francisco Javier Cañada, Anneloes Oude Vrielink, 1,2Filipa Marcelo, 1Ana Manzano, 1Pilar Blasco, 1Jesús NMR structural studies of antibacterial peptide, bovine lac- Jiménez-Barbero, 3Sabine Andre, 3Hans-Joachim Gabius tophoricin" The 1st Italy-Korea Symposium on Antimicro- bial Peptides Proceedings 1, P14 2) Tae-Joon Park, Ji-Sun 1 Centro de Investigaciones Biológicas, CIB-CSIC Chem- Kim, Sung-Sub Choi, and Yongae Kim (2009) "Cloning, Ex- ical and Physical Biology Department. Ramiro de, pression, Isotope Labeling, Purification, and Characteriza- 2 REQUIMTE, CQFB, DQ, FCT-UNL, 2829-516 Caparica, tion of Bovine Antimicrobial Peptide, Lactophoricin in E. 3 Portugal, Institute of Physiological Chemistry, Faculty of Coli" Prot. Exp. Purification 65, 1, 23 3) Tae-Joon Park, Veterinary Medicine, Ludwig-Maximilians-University, Ji-Sun Kim, Hee-Chul Ahn, and Yongae Kim (2011) "Solu- In pathological tissues mucins, heavily O-glycosilated pro- tion and Solid-state NMR Structural Studies of Antimicrobial teins of cellular surfaces, present perturbed glycosylation pat- Peptides, LPcin-I and LPcin-II." Biophy. J. 101, 5, 1193 terns that are being consider as tumor markers and are at- tracting a growing interest for the design and developing of Monday – May 20th antitumor vaccines. Interestingly, some of these carbohydrate EPR and New Materials related tumor markers are known carbohydrate antigens rec- ognized by C-type (calcium dependent) lectins expressed in Chair: Eduardo Di Mauro cells of the immune system envisioning an important role Room Ônix for these interactions in both innate and adaptive immune responses[1]. Human macrophages express in their surface OP019: EPR of thermo- and photoswitchable the so call Human Macrophage Lectin (HML-2, c-type lectin copper-nitroxide based molecular magnets from family 10, CLec10A) that recognizes carbohydrartes re- lated to Tn antigen, (O-glicosides of N-Acetyl–Galactosamine 1∗Matvey Fedin, 1Sergey Veber, 1Irina Drozdyuk, 1Ksenia with serine or threonine). In the presented work the binding Maryunina, 1Evgeny Tretyakov, 2Hideto Matsuoka, 2Seigo mode of Galactose and Galactosamine and fluorogalactoses Yamauchi, 1Renad Sagdeev, 1Victor Ovcharenko, 1,3Elena monosaccharides will be shown as it has been characterize by Bagryanskaya

25 Abstracts of Talks – EPR and New Materials

1International Tomography Center SB RAS, Novosi- UP021: Multifunctional in vivo EPR-based spec- birsk, Russia, 2Tohoku University, Sendai, Japan, 3N.N. troscopy and imaging of paramagnetic probes Vorozhtsov Novosibirsk Institute of Organic Chemistry SB ∗ RAS, Novosibirsk, Russia Valery V. Khramtsov R The Ohio State University Medical Center, Columbus, OH, Copper-nitroxide based molecular magnets Cu(hfac)2L ("breathing crystals") represent the new and interesting USA type of thermo- and photoswitchable materials. The un- Biological effects of various drugs and treatments observed in usual type of reversible magnetic switching between weakly- vitro are often not reproduced in vivo, therefore significantly and strongly exchange-coupled states occurs in spin triads decreasing their potential for translation to medical practice. nitroxide-copper(II)-nitroxide and can effectively be stud- In recent years it came to realization that tissue microen- ied using EPR. In this report we overview experimental ap- vironment (TME) plays a key role in this discrepancy. For proaches developed for these systems, discuss general trends two leading causes of mortality in the United States, cancer and focus on the characteristics and mechanism of light- and ischemic heart disease, tissue hypoxia is well documented induced spin state switching and following relaxation to the and is accompanied by changes in glycolysis resulting in tissue ground state using continuous wave X/Q-band and time- acidosis and tissue redox changes. It makes of primary impor- resolved (TR) W-band EPR. We found that the light-induced tance the development of approaches for noninvasive moni- spin dynamics leading to a formation of metastable state oc- toring of these TME parameters in vivo. Magnetic resonance curs on a nanosecond timescale and can be studied by TR techniques have the advantage of in vivo applications in ani- EPR. The first application of TR EPR to switchable molec- mals and humans due to sufficient depth of microwave pene- ular magnets has shown its potential for estimation of the tration in living tissues. The unique advantage of EPR over switching times and study of the fast electron relaxation NMR is the functional specificity due to the absence of over- rates. After being formed, the light-induced metastable state lap with endogenous signals. However EPR-based techniques slowly relaxes back to the ground spin state on a timescale of are far from attaining their maximum potential because of hours at cryogenic temperatures (T<20 K). This relaxation a lack of stable in vivo paramagnetic probes, and technical shows pronounced self-decelerating character for all studied limitation of EPR and EPR imaging. Here we discussed the compounds, which is explained by the broad distribution of recent advances in chemistry of paramagnetic probes[1, 2] activation energies in exchange-coupled clusters. Since light- and EPR-based techniques[3-5] which make multifunctional induced phenomena in "breathing crystals" are promising monitoring of TME feasible. The exemplified applications in- for spintronics, our recent efforts have been focused on opti- clude concurrent monitoring of ischemia-induced myocardial mization of their optical and relaxation properties by chem- oxygen depletion and acidosis in isolated rat hearts[6], and ical modification of nitroxide ligands. In particular, com- multifunctional (pH, redox, oxygen and glutathione content) plexes with tert-butylpyrazolylnitroxides show superior op- monitoring of tumor TME[3], including pH mapping of living tical properties and slower relaxation rates, allowing photo- tissues using low-field EPR imaging and functional proton- switching at up to 65 K. Another interesting direction on electron-double-resonance imaging (PEDRI)[4, 7]. Supported the way to applications is the study of light-induced phe- by NIH grant EB014542. nomena in thin polymer films and layers based on "breath- ing crystals", where the high sensitivity of EPR compared to [1] Khramstov VV, 2012. In Nitroxides - Theory, Experiment SQUID-magnetometry is crucial. We discuss recent results and Applications, Kokorin AI, Ed. InTech: 317-346. and future research in this field. [2] Bobko et al. 2012. Anal Chem, 84: 6054-6060.

ACKNOWLEDGEMENTS: RFBR (11-03-00158, 12-03- [3] Bobko AA et al. 2012. Magn Reson Med 67, 1827-1836. 33010), Russian Ministry for Education and Science [4] Khramtsov VV et al. 2010. J Magn Reson 202: 267-73. (8436, 8456), RF President grants (MK-1662.2012.3, MK- 1165.2012.3). [5] Koda S et al. 2012. Anal Chem 84: 3833-7.

[6] Komarov DA et al. 2012. Magn Reson Med 68: 649-55. OP020: Theoretical EPR Spectroscopy of Open- [7] Efimova OV et al. 2011. J Magn Reson 209: 227-232. Shell Transition Metal Complexes with Strong Spin Orbit Coupling UP022: New Kind of Mesoscopic EPR In Some Itin- ∗Frank Neese, Mihail Atanasov, Michael Römelt, Kan- erant Undoped Ferromagnets tharuban Sivalingam ∗Vladimir A. Ivanshin, Eduard M. Gataullin Max-Planck-Institut für Chemische Energiekonversion Kazan (Volga region) Federal University The majority of molecules have orbitally nondegenerate ground states. The molecular electric and magnetic prop- New Kind of Mesoscopic EPR In Some Itinerant Undoped erties are well described by standard models of electronic Ferromagnets Hybridization between local f or d states and structure theory, e.g. perturbation or linear response the- conduction electrons (CE) can lead to a dichotomy of local- ory. These methods have found very widespread use in the ized/itinerant characters of electrons in some strongly corre- community of quantum chemistry users. The ORCA program lated electron systems (SCES). Electron paramagnetic reso- developed in our group features many such methods for the nance (EPR) probes microscopically both the local moment calculation of optical and magnetic properties of transition (LM) spins and CE and could be observed in some dense metal complexes. There is, however, a significant class of SCES even without any kind of paramagnetic doping [1, 2]. molecules with orbitally (nearly) degenerate ground states According to very recent different theories [3-5], the key ori- where the established methods do not work. Here, a more gin of the unexpected narrow EPR signals observed in some careful treatment of the leading relativistic effects is neces- undoped intermetallics can be related to a strong hybridiza- sary in order to correctly predict their spectroscopic proper- tion effects between localized electrons and conduction bands ties and obtain molecular level electronic structure insight. in conjunction with ferromagnetic (FM) correlations. We re- This is not possible on the basis of density functional theory view the EPR and inelastic neutron scattering experiments in (DFT). Recently efficient methods based on multireference several undoped Yb, Ce, and Eu-based intermetallics [2, 6-8] wavefunction theory have been implemented into ORCA that and discuss the role of chemical composition, Kondo effect, allow such calculations on large molecules. Their use will be anisotropy, and RKKY interactions for the EPR linewidth. demonstrated by a recent study that deals with the electronic It is shown that an increase of the Kondo interaction and FM structure of the only low-molecular weight catalyst known to correlations leads to a stronger hybridization and an essential be capable of reducing dinitrogen to ammonia. EPR line narrowing. Possible hybridization mechanisms and the mesoscopic character of EPR are discussed.

26 Abstracts of Talks – Membrane Proteins

[1] J. Sichelscmidt, V.A. Ivanshin et al., Phys. Rev. Lett. inter-dependent, structure determination of membrane pro- 91, 156401(2003) teins needs to include the membrane environment. This also eliminates the potential for distorting protein structure, dy- [2] V.A. Ivanshin et al., Difusion and Defect Data Part B: namics and function due to crystal contacts or detergent Solid State Phenomena 170, 170 (2011) molecules, and enables functional and ligand binding stud- [3] P. Wölfle, E. Abrahams, Ann. Phys. (Berlin) 523, 598 ies to be performed in a natural setting. Solid-state NMR (2011) spectroscopy is compatible with structure determination of membrane proteins in membranes at physiological conditions. [4] P. Schlottmann, Phys. Rev. B 79, 045104 (2009) Advances in sample preparation, instrumentation, NMR ex- periments and effective computational methods, enable very [5] D. Huber, Mod. Phys. Lett. B 26, 1230021 (2012) high-resolution solid-state NMR spectra to be obtained from [6] L.M. Holanda et al., Phys. Rev. Lett. 107, 026402 (2011) isotopically labeled membrane proteins in lipid bilayers and high quality structures of membrane proteins to be deter- [7] T. Gruner et al., Phys. Rev. B 85, 035119 (2012) mined in membranes. Orientation restraints are particularly [8] C. Stock et al., Phys. Rev. Lett. 109, 127201(2012) useful for solid-state NMR structural studies of membrane proteins since they provide not only information about three- dimensional structure, but also information about protein OP023: Elucidating Smart Polymeric Materials Us- orientation in the membrane. We describe recent results ob- ing Electron Paramagnetic Resonance (EPR) Spec- tained for a family of bacterial outer membrane proteins im- troscopy plicated in cell adhesion and pathogenesis. ∗ Dariush Hinderberger ACKNOWLEDGEMENTS: This research was supported by Max Planck Institute for Polymer Research the National Institutes of Health. Magnetic resonance methods such as nuclear magnetic reso- nance (NMR) or electron paramagnetic resonance (EPR) can OP025: Brain Fatty-acid Binding Protein and its In- give valuable insights into complex systems that show long teractions with Membrane Model Systems range disorder and some short range or intermediate range 1 1 1 order, e.g. synthetic and biological soft matter. In particular, Fábio H. Dyszy, Ítalo A. Calvini, Daniel F. Silva, 1Andressa P. A. Pinto, 1Ana P.U. Araújo, 2Hans Robert EPR spectroscopy with its high sensitivity and selectivity is 1 1,3∗ complementary to the well-established methods of soft matter Kalbitzer, Claudia Elisabeth Munte, Antonio José characterization (e.g. light-, X-ray, neutron scattering) and Costa-Filho delivers information that is otherwise not accessible. While 1Grupo de Biofísica Molecular Sérgio Mascarenhas, Insti- conventional continuous-wave (CW) EPR allows studying ro- tuto de Física de São Carlos, USP, 2Department of Bio- tational dynamics in the microsecond to picosecond regime, physics and Physical Biochemistry, University of Regens- EPR methods that measure the dipolar (through-space) in- burg, 3Laboratório de Biofísica Molecular, Faculdade de teraction of individual electron spins can be used to gain in- Filosofia, Ciências e Letras de Ribeirão Preto, USP formation on the spatial distribution of paramagnetic probe Fatty acid-binding proteins (FABP) constitute a family of molecules up to 8 nm. low molecular weight proteins that share structural homol- Here, I will show that methods of EPR spectroscopy on small ogy and the ability to bind fatty acids. The common struc- radicals as probe molecules can help illuminating the molec- tural feature is a β-barrel of 10 antiparallel strands forming ular scale processes in smart, responsive polymeric materials. a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short α-helices. This will be highlighted through examples from my lab that Here we used magnetic resonance methods (ESR and NMR) span a broad range of thermoresponsive synthetic and bio- to correlate structure and function of B- FABP in the pres- logical macromolecules, such as poly(alkylene oxide)-based ence of membrane mimetics. Spin probes selectively placed in copolymers and dendronized polymers or genetically engi- the portal region and fatty acid spin labels (n-SASL) allowed neered elastin-like polypeptides (ELPs). These polymers are us to monitor structural changes both in the protein and a very interesting class of materials envisioned for a multitude in the membrane models. ESR spectra of B-FABP labeled of applications such as molecular transport or sensing. I will mutants were overall divided in two groups that responded show that using simple CW EPR on amphiphilic spin probes, differently to the presence of membranes. Group I residues, mainly simple TEMPO, - performed in a benchtop EPR spec- whose side chains point outwards from the contact region trometer - it is possible to obtain fundamental insight into between the helices, had their mobility decreased upon mem- molecular scale processes during thermal responses of these brane addition. Group II residues, composed by residues with polymers. This insight can finally be correlated with their side chains situated at the interface between the a-helices, macroscopically observable function on much longer length showed ESR spectra associated to an increase in mobility in scales to derive structure-function relationships as they are the presence of the model membranes. These modifications known from proteins and other biomacromolecules. in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues th (group II) that is likely to facilitate the delivery of FAs to Monday – May 20 target membranes. On the other hand, residues in group Membrane Proteins I and probes in the membrane mimetic presented decreased Chair: Ana Carolina Zeri mobilities probably as a result of the formation of a collisional Room Gávea A complex. NMR HSQC of isotopically labeled B-FABP in the presence of bicelles was also used to identify residues that OP024: Structural studies of membrane proteins in participate in the binding/gating and allowed us to find a membrana new series of residues that have not been implicated in those processes before. Our results bring new insights for the un- ∗ Francesca M. Marassi derstanding of the binding, gating and delivery mechanisms Sanford-Burnham Medical Research Institute, La Jolla CA, in FABPs. USA ACKNOLEDGEMENTS: FAPESP, CNPq, CAPES. The two principal components of biological membranes - the lipid bilayer and the proteins integrated within it - have co- UP026: Defining the Flexible and Fixed Sides of a evolved for specific functionalities that mediate all interac- Protein Channel by EPR tions of cells with their environment. Since the physical and chemical properties of lipids and proteins are highly ∗Betty J. Gaffney

27 Abstracts of Talks – Dynamics and Recognition

Florida State University [4] Catoire et al. (2010) Structure of a GPCR ligand in its Lipoxygenases are ubiquitous proteins that oxidize polyun- receptor-bound state: leukotriene B4 adopts a highly con- saturated chains at different positions, but do so with a highly strained conformation when associated to human BLT2. J conserved structural motif of 20 helices. Because mechanistic Am Chem Soc, 132, 9049-9057 details have been difficult to obtain by crystallography, EPR [5] Catoire et al. (2011) Electrostatically-driven fast associa- spectroscopy approaches have been implemented. Lipoxy- tion and perdeuteration allow detection of transferred cross- genases are naturally paramagnetic due to an iron involved relaxation for G protein-coupled receptor ligands with equi- in catalysis. We have made them additionally paramagnetic librium dissociation constants in the high-to-low nanomolar by adding site-directed spin labels at selected sites. Using range. J Biomol NMR, 50, 191-195 several EPR approaches, we have found the entrance to the active site and have examined an α-helix that must move to allow substrate to enter the deep cavity. The non-heme iron OP028: Structural Biology by DNP MAS NMR and is bound to five protein side chains and water and is located Investigations on the Transport Cycle of an ABC centrally in the structure, 25-35 Å from the surfaces. The po- Transporter lar end of a lipid substrate analog was found on the surface ∗Hartmut Oschkinat at one end the internal cavity in a lipoxygenase with pH- gating of substrate access (Biophys J 103: 2134 (2012)). A Leibniz-Institut für Molekulare Pharmakologie, Berlin, Ger- new bacterial lipoxygenase structure, with a lipid bound also many seems to have a helical "lid" protecting a similar entrance to Solid-state NMR enables the investigation of heterogeneous, the active site. To examine how lipid binding "opens" the complex biological samples at high reso-lution. A major fac- way to catalytic iron, we have scanned the gating helix with tor enabling such investigations is dynamic nuclear polari- spin labels and examined, by power saturation, relaxation of sation (DNP), which was introduced to increase signal-to- the spin at many helix residues when a lysolipid is present. noise by one or two orders of magnitude. The application In comparison to substrate insensitive positions, measured of dynamic nuclear polarisation (DNP) requires further opti- in the reference above, the gating helix distances to iron in- mization of samples, experimental parameters and concepts. crease in the presence of substrate analog, indicating a shift In this context, the possibility of ’high-temperature’ DNP in of the helix backbone. The EPR spectra of ferric lipoxyge- solid-state NMR is discussed. A major application of DNP nases with and without lipids in the active site also confirm involves investigations of the nascent chain within and di- remarkable conservation of active site structure in lipoxyge- rectly after leaving the ribosome. The initial folding events nases from bacteria and eukaryotes. are not yet well understood, and this investigation provides an NMR view onto this process. Of particular interest is the UP027: Conformational selection of GPCR ligands occurence of secondary structure within the tunnel. upon binding to their receptors as observed by NMR Investigations of membrane proteins may be facilitated by 1∗Laurent Catoire, 2Marc Baaden, 1Jean-Luc Popot, 3Eric extensive deuteration, and subsequent de-tection of protons Guittet, 4Jean-Louis Banères under magic-angle spinning conditions. Various schemes for 1Laboratory of Biology and Physico-Chemistry of Mem- the usage of proton chemical shifts for achieving sequence- brane Proteins - UMR 7099 CNRS & University Paris, specific assignments will be discussed, including a substitute 2Laboratory of theoretical Biochemistry - UPR 9080 CNRS, of it, 2H-double quantum chemical shifts. An ABC trans- 3ICSN- UPR 2301 CNRS, 4IBMM - UMR 5247 CNRS, Uni- porter reconstituted into native lipid bilayers is investigated versite Montpellier 1 & 2 in variously labelled forms, and spectra of different functional states will be presented to-gether with initial assignments and The mechanism of signal transduction mediated by G functional considerations. In essence, the solid-state NMR protein-coupled receptors (GPCRs) is a subject of intense data suggest a new transport cycle. research in pharmacological and structural biology. An in- creasing amount of high-resolution GPCR crystal structures have been established these past few years, in parallel to Monday – May 20th a large panel of pharmacological, biophysical and biological Dynamics and Recognition studies. In spite of these structures have provided a ma- Chair: Robert Kaptein jor breakthrough in the field of structural biology of these Room Gávea B receptors, and although these structures reveal several com- mon features between all these receptors, the mechanism of GPCR activation remains to be understood at the molecu- OP029: Dynamic Aspects of Indirect DNA Readout lar level. Beside technical issues, like obtaining a sufficient by Nucleoid Associated Proteins amount of functional receptor [1-3], this difficulty to clearly 2 2 3 1∗ propose a mechanism for a given receptor is due to the confor- Tiago Cordeiro, Jesús García, Oscar Millet, Miquel mational complexity and variability displayed by these mem- Pons brane proteins, making GPCR signaling a rather sophisti- 1University of Barcelona, 2Institute for Research in cated and subtle biological process. Ligand association to Biomedicine, 3CIC-bioGUNE the receptor constitutes a critical event in the activation pro- The capacity of Enterobacteriaceae to regulate horizontally cess. In this context, NMR, with the support of molecular transferred genes independently from the core genome is modeling can provide dynamics pictures of receptor-ligand probably relevant for their success as pathogens. Escherichia, interactions, and has therefore the potential of becoming a Yersinia, and Salmonella are among the best-known genus of leading technique in the study of the structure-function rela- pathogenic bacteria of the enteric group. tionships of GPCRs. This will be illustrated here with two fatty acid molecules in interaction with the leukotriene BLT2 At least two mechanisms allow the selective recognition of receptor [4,5]. horizontally transferred genes: i) the use of specialized forms of the nucleoid-associated protein H-NS and, ii) the use of a [1] Dahmane et al. (2009) Amphipol-assisted in vitro folding co-regulator of the Hha family that converts standard H-NS of G protein-coupled receptors. Biochemistry, 48, 6516-6521 into a selectively regulating complex [1].In the two mecha- [2] Banères et al. (2011) New advances in production and nisms, recognition of DNA takes place through an indirect functional folding of G-protein-coupled receptors. Trends readout mechanism [2]. Biotechnol, 29, 314-322 We shall present unpublished results on the structure of the [3] Popot et al. (2011) Amphipols from A to Z. Annu Rev complex between Hha and H-NS obtained from NMR, includ- Biophys, 40, 379-408 ing paramagnetic relaxation enhancement experiments.

28 Abstracts of Talks – Dynamics and Recognition

Proteins of the Hha family (Hha and YmoA) present intrin- ing along DNA have caused a revival in this field. Our NMR sic pervasive dynamics in the millisecond timescale [3]. Using approach shines new light on this matter and has the advan- CPMG-relaxation dispersion experiments we have character- tage that it combines kinetics with the possibility to observe ize the collective motion of YmoA and the effect of mutations the process involved at an atomic level. known to change its capacity to bind H-NS. We have iden- In non-specific lac headpiece-DNA complexes selective NMR tified residues that play a dual role in stabilizing the H-NS line broadening is observed that strongly depends on length complex and in modulating the dynamics of the apo-form. and composition of the DNA fragments. This broadening We could also show that a mutation of a highly evolutionarily involves amide protons found in the non-specific lac-DNA conserved residue, which had no apparent effect on in-vitro structure to be interacting with the DNA phosphate back- H-NS binding, had a strong effect on the dynamics of the bone, and can be ascribed to DNA sliding of the protein apo-form of YmoA. along the DNA. This NMR exchange broadening has been These results suggest a model in which core and horizon- used to estimate the 1D diffusion constant for sliding along tally acquired DNA forms interact with the same protein (H- non-specific DNA. The observed slow sliding rate indicates NS) alone or in a complex with Hha/YmoA, respectively. that other processes such as hopping and intersegment trans- Hha/YmoA dynamics plays a key role disconnecting the two fer contribute to facilitate the DNA recognition process. pools of H-NS and therefore, enables selective regulation. ACKNOLEDGEMENTS: MINECO, Spain (BIO2010- UP032: RNA regulation - A new Twist th 15683), Generalitat de Catalunya (2009SGR1352), EC 7 ∗Harald Schwalbe FP (BioNMR contract 261863). Goethe-University Frankfurt References: In the recent past, the importance of RNA-based regula- [1] RC Baños et al. PLoS Genet 5(6): e1000513. tion has become increasingly recognized. Regulation is based doi:10.1371/journal.pgen.1000513 (2009). on structural transitions of RNA modules located in the 5’ untranslated region of mRNA. The regulation mecha- [2] T Cordeiro, et al. PLoS Pathog 7(11): e1002380. nism act on the level of transcription as well as translation. doi:10.1371/journal.ppat.1002380 (2011). Riboswitches sense changes in the concentration of small [3] J García, et al. Biochem. J. 388, 755–762 (2005). molecule metabolites while RNA thermometers respond to temperature changes. OP030: Protein Kinase A and Conformational Dy- In this contribution, we will describe novel novel NMR meth- namics: ‘Not too fast. . . not too slow. . . but just ods to monitor such structural transitions at atomic resolu- right’ tion in vitro and in cell. In addition, we have developed a ∗ new T-jump probe to monitor temperature-induced regula- Gianluigi Veglia tion events in RNA. University of Minnesota Protein kinases mediate a myriad of cell signaling events. OP033: Protein interactions and function studied by Protein kinase A (PKA) is considered the prototypical kinase, novel NMR methods sharing its catalytic core (PKA-C) with many kinases. We ∗ have analyzed PKA-C’s conformational dynamics in the slow S. Grzesiek, M. Gentner, J.-r. Huang, S. Morin, L. Nisius, and fast scale of NMR spectroscopy and found that the slow H.-.J. Sass, L. Skora, N. Vajpai, M. Wiktor time scale dynamics are linked with the enzyme turnover. Biozentrum, University of Basel, Switzerland Motions synchronous with the kcat are initiated upon nu- We extensively use residual dipolar coupling (RDC), para- cleotide binding and pervade the entire enzyme, defining a dy- magnetic labeling (PRE, PCS), scalar couplings and other namically committed state. The binding of inhibitors quench NMR parameters to characterize folded and unfolded pro- the enzyme’s conformational dynamics, creating a dynam- tein states in a quantitative manner [1-11]. We will discuss ically quenched state. We also found a mutant of PKA-C several recent examples. that enhances the motions in the fast time scale. However, these enhanced motions reduce the catalytic efficiency of the Scalar couplings across hydrogen bonds (H-bonds), which re- kinase. We conclude that the conformational dynamics of the port on the electronic overlap between donor and acceptor enzyme is linked to the overall turnover, and an increase or orbitals, present a highly sensitive measure of H-bond geom- decrease of these motions has negative effects on the catalytic etry [1,2]. Using H-bond scalar couplings, we have recently cycle. mapped the pressure and temperature dependent deforma- tion of ubiquitin’s H-bond network at unprecedented resolu- UP031: Sliding and target location of DNA-binding tion [3]. proteins: an NMR view of the lac repressor system We recently also obtained atomic details of the pressure- 1∗Rolf Boelens, 2Karine Loth, 4Manuel Gnida, 5Julija Ro- assisted, cold-denatured state of ubiquitin at 2500 bar and manuka, 1,3Robert Kaptein 258 K by high-resolution NMR techniques [4]. This state has structural propensities for a native-like N-terminal β-hairpin 1Utrecht University, 2University of Orleans, 3Novosibirsk and α-helix and a non-native C-terminal α-helix, which State University, 4Paderborn University, 5Shell Research are very similar to ubiquitin’s alcohol-denatured (A-) state. Laboratories The close similarity of pressure-assisted, cold-denatured and Gene regulatory proteins exert their function through binding alcohol-denatured state supports a hierarchical mechanism to specific DNA sequences. Much progress has been made in of folding and the notion that similar to alcohol, pressure understanding specific protein-DNA interactions, primarily and cold reduce the hydrophobic effect. Indeed, at non- from X-ray or NMR structures of the involved complexes. denaturing concentrations of methanol, a complete transi- Another important question is how these proteins can ef- tion from the native to the A-state can be achieved at ambi- ficiently find their targets in the presence of the abundant ent temperature by varying the pressure from 1 to 2500 bar. non-specific DNA in the nucleus. This method should allow highly detailed studies of protein folding transitions in a continuous and reversible manner. We present a new approach based on NMR exchange mea- surements to analyze 1D diffusion of DNA-binding proteins Abelson kinase (Abl) is an important drug target in the treat- along DNA sequences. Although this problem has been dis- ment of chronic myelogenous leukemia. We have recently cussed since the pioneering studies of Berg and von Hippel characterized the solution conformations of the kinase do- in the early 80’s, results of recent single molecule fluores- main in complex with different inhibitors by RDCs and other cence measurements of lac repressor and other proteins slid- parameters [5]. The NMR analysis has also helped to locate

29 Abstracts of Talks – Methods for Inorganic Materials the interaction side of a new class of allosteric inhibitors that glasses. We have also found that 29Si nuclear shielding ten- can overcome resistance to ATP-binding-site inhibitors [6]. sor parameters of SiO4 tetrahedra not only reveal tetrahedral Time permitting we will show recent results on these inter- connectivity but can also be related to the nature and coor- actions. dination number of network modifier cations. These studies have direct implications for the continuous random network [1] A. J. Dingley, L. Nisius, F. Cordier, and S. Grzesiek, Na- and modified random network structural models of silicate ture Protocols 3, 242 (2008). glasses. They can provide insight for researchers needing to [2] F. Cordier, L. Nisius, A. J. Dingley, and S. Grzesiek, Na- refine potentials for MD simulations and to connect struc- ture Protocols 3, 235 (2008). ture to viscosity, density, strength, transport properties, and thermodynamic properties, impacting fields such as inorganic [3] L. Nisius and S. Grzesiek, Nat Chem 4, 711 (2012). geochemistry, waste containment technology, and ionic con- ductors. [4] N. Vajpai, L. Nisius, M. Wiktor, and S. Grzesiek, Proc Natl Acad Sci USA (2013). [5] N. Vajpai, A. Strauss, G. Fendrich, S. W. Cowan-Jacob, OP035: Hunting for Hydrogen in Wadsleyite: Mult- P. W. Manley, S. Grzesiek, and W. Jahnke, J Biol Chem 283, inuclear Solid-State NMR and First-Principles Cal- 18292 (2008). culations 1∗ 1 2 [6] J. Zhang, F. J. Adrián, W. Jahnke, S. W. Cowan-Jacob, A. Sharon E. Ashbrook, John M. Griffin, Andrew J. 3 G. Li, R. E. Iacob, T. Sim, J. Powers, C. Dierks, F. Sun, G.- Berry, Stephen Wimperis R. Guo, Q. Ding, B. Okram, Y. Choi, A. Wojciechowski, X. 1School of Chemistry and EaStCHEM, University of St An- Deng, G. Liu, G. Fendrich, A. Strauss, N. Vajpai, S. Grze- drews, UK, 2Research School of Earth Sciences, Australia siek, T. Tuntland, Y. Liu, B. Bursulaya, M. Azam, P. W. National University, Canberra, Australia, 3School of Chem- Manley, J. R. Engen, G. Q. Daley, M. Warmuth, and N. S. istry and WestCHEM, University of Glasgow, UK Gray, Nature 463, 501 (2010). It is thought that the inner Earth contains a vast amount of [7] M. Gentner, M. G. Allan, F. Zaehringer, T. Schirmer, and water in the form of hydrogen bound at defect sites within S. Grzesiek, J Am Chem Soc 134, 1019 (2012). the nominally anhydrous silicate minerals present in the man- tle. Structural studies of silicates, therefore, play an impor- [8] J. Habazettl, M. G. Allan, U. Jenal, and S. Grzesiek, J tant role in our understanding of the physical and chemical Biol Chem 286, 14304 (2011). properties of the Earth’s interior. However, the high-pressure [9] J.-R. Huang and S. Grzesiek, J Am Chem Soc 132, 694 synthesis conditions typically result in small sample volumes (2010). ( 1-10 mg), compromising sensitivity. Here we present exper- imental solid-state NMR results and first-principles calcula- [10] D. Häussinger, J.-R. Huang, and S. Grzesiek, J Am Chem tions that provide insight into local structure and disorder in Soc 131, 14761 (2009). hydrous wadsleyite (β-Mg2SiO4). This deep-Earth mineral has received widespread attention due to its high capacity for [11] S. Meier, M. Blackledge, and S. Grzesiek, The Journal the incorporation of water. 17O MAS and STMAS spectra of Chemical Physics 128, 052204 (2008). recorded at 20.0 T enable the identification of hydroxyl oxy- gen sites in samples with different hydration levels. This is Monday – May 20th consistent with a model structure previously proposed in the Methods for Inorganic Materials literature; however, considerable broadening is also observed, indicating that the structure is not fully ordered. 1H/2H Chair: Bradley Chmelka MAS and two-dimensional 1H dipolar correlation spectra re- Rooms Turmalina/Topázio veal multiple proton environments in both samples, including some with chemical shifts that are higher than expected for OP034: NMR Non-Crystallography Mg-OH environments. Indeed, DFT calculations carried out for different model structures derived from disordered super- 1∗ 1 1 Philip J. Grandinetti, Kevin Sanders, Eric Keeler, cells suggest that some of the observed resonances correspond 2 Jay H. Baltisberger Si-OH protons. This is supported by 29Si CPMAS and het- 1Ohio State University, Columbus, USA, 2Berea College, eronuclear correlation spectra, while 1H-17O CPMAS and Berea, KY, USA two-dimensional spectra also indicate the presence of both Mg-OH and Si-OH groups in the structure. The DFT cal- A great advantage of solid-state NMR spectroscopy is its abil- culations additionally reveal it is an Mg3, not an Mg2, va- ity to reveal atomic-level structure in materials where diffrac- cancy that charge balances the structure. The combination of tion techniques fail. This is generally true in non-crystalline multinuclear solid-state NMR and DFT calculations enables solids, where diffraction methods rarely reveal structural de- the most complete study to date of this extremely important tails beyond the first-coordination sphere except in the sim- mantle mineral, providing new insight into the mechanism of plest of compositions. While it also true that the spectra water storage. of non-crystalline solids in many spectroscopies are often broad and featureless, magnetic resonance spectroscopy has a unique advantage in that the different NMR frequency con- UP036: Uniform excitation and inversion of broad tributions leading to these broadenings can be separated and NMR spectra in rotating solids using DANTE correlated in multi-dimensional experiments. This talk will describe efforts in our laboratory over the last few years de- 1Veronika Vizthum, 1Marc Caporini, 1Diego Carnevale, veloping new and improved multi-dimensional NMR methods 1Simone Ulzega, 2Julien Trébosc, 2Olivier Lafon, 1Geoffrey for structure determination in non-crystalline solids. This Bodenhausen, 2∗Jean Paul Amoureux work has focused on silicates and involves nuclei, such as 29Si 1Ecole Polytechnique Fédérale de Lausanne, Switzerland, and 17O, where we have found strong relationships between 2Lille University, France NMR couplings and local structure and used these relation- ships to convert multi-dimensional NMR spectra into multi- Many solid-state NMR powder patterns span several MHz. dimensional structural correlations. For example, we have These broad patterns arise from large anisotropic interactions shown that 17O quadrupole coupling constant and asymme- (e.g. quadrupolar interaction or electron-nuclei interactions try parameters can be directly related to Si-O bond lengths, in paramagnetic solids). These ultra-wide-line NMR spectra Si-O-Si angles, and the presence of coordinating alkali or al- can provide detailed information on the structure and dynam- kaline earth cations, and exploit these relationships to fol- ics at molecular level but high-power rectangular pulses are low structural changes in various silica and modified silicate usually insufficient to excite uniformly these broad patterns.

30 Abstracts of Talks – Hyperpolarization/ Instrumentation

Herein, we show how trains of rotor-synchronized short pulses can be prepared with isotopic enrichment (13C, 15N), making in the manner of Delays Alternating with Nutation for Tai- it also applicable to biological solids. lored Excitation (DANTE) can provide uniform excitation, inversion or other coherence manipulation for broad spec- ACKNOWLEDGEMENTS: DFG, FAPESP, CNPq tra of rotating solids, spanning several MHz. In that case, DANTE overcome many limitations of rectangular pulses. OP038: Topologic, Geometric, and Chemical Order Furthermore, interleaved DANTE trains can achieve shorter in Materials: Insights from Solid-State NMR excitation and hence are less affected by coherent or inco- herent decays, and can facilitate the excitation of different Dominique Massiot, Sylvian Cadars, Michael Deschamps, chemical environments. Emmanuel Véron, Mounesha N.Garaga, Robert J. Messinger, Mathieu Allix, ∗Pierre Florian, Franck Fayon We first demonstrate the efficiency of DANTE for the 14N CEMHTI UPR3079 CNRS, France observation.[1-3] We show applications to direct detection of 14N nuclei subject to large quadrupole interactions, using Local order, as opposed to the long-range order of the ideal fast MAS, backed up by simulations that provide insight into crystalline structures, can be considered as an intrinsic char- the properties of DANTE. When used for indirect detection, acteristic of real materials, being often the clue to the tuning we show that DANTE can lead to large boost of efficiency of their properties and their final applications. While order- compared to rectangular pulses. ing can be easily assessed in two-dimensional imaging tech- niques with resolution approaching the atomic level, the di- We also demonstrate that DANTE can manipulate in para- agnostic, description, and qualification of local order in three magnetic samples a large number of spinning sidebands of dimensional systems is much more challenging. spin-1/2 nuclei (19F) that extend over more than 1.3 MHz, with rf-fields as low as 20-30 kHz.[4] Solid-state nuclear magnetic resonance [NMR] and its panel of constantly developed new instruments and methods en- [1] Vitzthum, Caporini, Ulzega, Bodenhausen, J. Magn. Re- able local, atom selective characterization of structures and son. 212 (2011) 234. assemblies ranging from atomic to nanometer length scales. [2] Vitzthum, Caporini, Ulzega, Trébosc, Lafon, Amoureux, This opens unique opportunities for characterizing a variety Bodenhausen, J. Magn.Reson. 223 (2012) 228 of materials, ranging from crystalline compounds to amor- phous or glassy materials, for which we show that it becomes [3] Lu, Trébosc, Lafon, Carnevale, Bodenhausen, Amoureux, possible to separate topologic, geometric and chemical con- submitted. tributions to the order or disorder, in cooperation with other experimental techniques and in-silico approaches. [4] Carnevale, Vitzthum, Lafon, Trébosc, Amoureux, Boden- hausen, Chem. Phys. Lett. 553 (2012) 68. As identified by solid state NMR, the local structure of amor- phous materials or glasses consists of well-identified struc- UP037: A homonuclear version of REDOR for recou- tural entities up to at least the nanometer scale. Instead of pling dipolar interactions in multi-spin systems: Ap- speaking of disorder, we propose a new paradigm to describe plications to inorganic phosphorus clusters and net- their structures in terms of a continuous assembly of locally works defined structures, reminiscent of locally favored structures (LFS). This draws a comprehensive picture of amorphous 1,2∗Hellmut Eckert, 2Jinjun Ren structures based on fluctuations of chemical composition and 1University of Sao Paulo, 2WWU Münster structure over different length scales. We hope that these new local or molecular insights could open new possibilities A new solid state NMR technique is presented to re-couple for considering key questions related to nucleation and crys- the homonuclear dipole-dipole interactions within spin pairs, tallization, as well as chemically (spinodal decomposition) 1 clusters and multi-spin distributions of coupled I = ⁄2 sys- or density driven (polyamorphism) phase separation, which tems under MAS conditions. In contrast to the usual double- could enable future applications. quantum coherence intensiy buildup curves, this method measures the amplitude of Iz, both in the presence and in the References absence of the dipolar coupling. Recoupling is accomplished in the form of an effective double quantum Hamiltonian cre- [1] D.Massiot, S.Cadars, M.Deschamps, V.Montouillout, ated by a symmetry-based POST-C7 sequence consisting of N.Pellerin, E.Véron, R.J.Messinger, M.Allix, P.Florian, two excitation blocks, attenuating the signal (intensity S’). F.Fayon, Topologic, Geometric, and Chemical order in Mate- For comparison, a reference signal S0 with the dipolar recou- rials : Insights from Solid-State NMR, Accounts Chem. Res. pling absent is generated by shifting the phase of the second (submitted) block by 90◦ relative to the first block. As in rotational echo double resonance (REDOR), the homonuclear dipole-dipole [2] E.Véron, M.N.Garaga, D.Pelloquin, S.Cadars, coupling constant can then be extracted from a plot of the M.Suchomel, E.Suard, D.Massiot, V.Montouillout, G.Matzen, M.Allix, Synthesis and structure determination of normalized difference signal (S0-S’)/S0 versus dipolar evo- lution time. Detailed simulations indicate that within the CaSi1/3B2/3O8/3, a new calcium borosilicate, Inorg. Chem. ASAP (2013) range of (S0-S’)/S0 0.5 such "homonuclear REDOR curves" can be approximated by simple parabolae, yielding effective [3] F.Fayon, C.Duée, T.Poumeyrol, M.Allix, D.Massiot, Ev- squared dipole-dipole coupling constants summed over all the idence of Nanometric-Sized Phosphate Clusters in Bioactive pairwise interactions present. The method has been success- 31 31 Glasses as Revealed by Solid-State P NMR, J. Phys. Chem. fully validated for P in a large number of inorganic crys- ASAP (2013) talline model compounds and successfully applied to various structural issues in inorganic phosphate glasses. It combines [4] P.Florian, E.Véron, T.Green, J.R.Yates, D.Massiot, Elu- efficiency, facile data analysis, and easy extension to multi- cidation of the Al/Si ordering in Gehlenite Ca2Al2SiO7 by ple spin systems covering a wide range of dipolar coupling combined 29Si and 27Al NMR spectroscopy / quantum chem- strengths. The method is not affected by dipolar truncation ical calculations, Chem. Mater. 24 4068–4079 (2012) and allows CSA effects to be accounted for by simple cali- bration. All of these advantages will help to trigger novel [5] R.N.Kerber, A.Kermagoret, E.Callens, P.Florian, structural applications elucidating short-, medium-range and D.Massiot, A.Lesage, C.Copéret, F.Delbecq, X Rozanska, nanoscale ordering phenomena disordered state of matter. P.Sautet, Nature and structure of aluminum surface sites While the present contribution focuses on 31P NMR studies grafted on silica from a combination of high field aluminum- of inorganic materials, the method can be applied analogously 27 solidstate NMR spectroscopy and first principle calcula- to other spin-1/2 nuclei that are either abundant (19F, 1H) or tions, J. Am. Chem. Soc. 134 6767–6775 (2012)

31 Abstracts of Talks – Hyperpolarization/ Instrumentation

13 13 Monday – May 20th (ALT) pathway; and CO2 & C-bicarbonate through the Hyperpolarization/ Instrumentation pyruvate dehydrogenase (PDH) catalyzed metabolic path- way. The value of this powerful metabolic imaging technique Chair: Dmitry Budker for cancer imaging was shown first by Golman et al 2006 and Room Ônix we have applied it in a number of preclinical animal stud- ies for detecting cancer presence, progression and response OP039: Imaging Pulmonary Gas Exchange from to therapy and in a first-ever human Phase 1 clinical trial in Mouse to Human with Hyperpolarized 129Xe MRI prostate cancer patients. This study demonstrated the feasi- bility to characterize prostate tumors based on dramatically ∗ Bastiaan Driehuys increased conversion of 13C-pyruvate to 13C-lactate through Department of Radiology, Duke University Medical Center, LDH. Recent studies have demonstrated significant correla- Durham tions of gene expression and enzyme activity with new MR techniques to measure kinetic rates in vivo. Also a variety Among the most interesting properties of hyperpolarized of new HP agents to investigate physiological parameters in- (HP) 129Xe, are its high solubility and large chemical shift. cluding perfusion, pH, and membrane transport as well as These have long been prized by NMR spectroscopists, but metabolism have been applied in disease models. are only now beginning to be exploited in biomedical imag- ing. Here we describe progress in using the dissolved phase of HP 129Xe to image the lung’s most fundamental function UP041: Universal tags to sustain long-lived hyper- – gas exchange. When inhaled, HP 129Xe diffuses into the polarized signal: Extension of hyperpolarization to pulmonary blood and tissues where in most mammals, in- broad range of biomarkers cluding humans, it exhibits distinct resonances in each com- 129 Yesu Feng, Thomas Theis, Ryan M. Davis, Kevin Claytor, partment. Here we explain the techniques used to HP Xe ∗ as it transfers from airspace to blood. Particularly in healthy Qiu Wang, Pei Zhou, Warren S. Warren human subjects, these 129Xe gas transfer images exhibit in- Duke University trinsic heterogeneity and are strongly influence by gravity. In vivo applications of hyperpolarization are largely hin- This heterogeneity reveals the extraordinary reserve capacity dered due to the short T1 relaxation times of molecules with of the healthy lung, and may explain why lung disease has metabolic relevance. Up till now, very few molecules have historically been is so difficult to detect early. We will dis- been hyperpolarized that have long enough T1 to acquire cuss not only the basic lung physiology we are learning from in vivo metabolic images. By accessing the singlet state HP 129Xe MRI, but also introduce new techniques to simul- between chemically equivalent or slightly inequivalent spin taneously acquire gas- and dissolved phase 129Xe images by pairs, lifetime extensions above an order of magnitude have efficient radial acquisition. This approach enables short echo been achieved. However, most of the molecules supporting times and improves SNR, but also opens the door to sep- long-lived singlet states have no direct biological function arating the dissolved-phase 129Xe image into its blood and and using these molecules as a tag to label interesting tar- tissue components. Such separation is critically important as get molecules is not practical given the molecule’s size and we show in our recent work in patients with interstitial lung symmetry requirements. As a result, universal tags to gen- disease. To complement these human trials, it is equally im- eralize the implementation of long-lived singlet state are still portant to exploit hyperpolarized 129Xe MRI to image mouse awaited. Here, we present two tags that sustain a long-lived models of lung disease. We now routinely acquire isotropic 13 13 129 singlet state between either a pair of C spins in C2- images of Xe in the mouse with a resolution of <250µm 15 15 diphenylethyne or a pair of N spins in N2-diazirine. We and demonstrate novel ways to extend gas exchange imag- 13 will also demonstrate two immediate applications of C2- ing to the mouse by taking advantage of genetically modified diphenylethyne used as a tag. The first one is a potential strains. Together, these findings are positioning HP 129Xe antibiotic drug which contains diphenylethyne as a substruc- MRI as a powerful platform to enable progress in developing ture. This molecule has been shown to function as an LpxC new therapies for chronic lung diseases. inhibitor to suppress the lipid biosynthesis of gram-negative 13 ACKNOLEDGEMENTS: NIH R01HL105643 and P41 bacteria. The second is to use C2-diphenylethyne to label EB015897 suberoylanilide hydroxamic acid (SAHA), which is an FDA approved anti-cancer drug serving as a histone deacetylases 15 inhibitor. As to N2-diazirine, which is widely used as a OP040: Hyperpolarized Carbon-13 MR Pre-Clinical tag for photoaffinity labeling, it is also one of the smallest and Human Research Studies tags that can support a long-lived singlet state. Studies us- 1∗Daniel B. Vigneron, 1Sarah Nelson, 2Andrea Harzstark, ing diazirine as a tag cover almost every category of drugs 3Marcus Ferrone, 1Robert Bok, 1John Kurhanewicz (antibiotics, anti-cancer, anesthetics etc.) and endogenous biomolecules (amino acids and their derivatives, nucleic acids 1Department of Radiology & Biomedical Imaging, Univer- and fatty acids etc.). We will show the very first case of ex- sity of California, San Francisco, 2Investigational Thera- 3 tending the hyperpolarized signal through the singlet state on peutics, University of California, San Francisco, Clinical 15 a N2-diazirine compound. This might significantly extend Pharmacy, University of California, San Francisco the application of hyperpolarization in metabolic imaging. Hyperpolarized Carbon-13 MR has the potential to become an important new radiological tool for cancer metabolic imag- UP042: Avalanching Amplification of NMR/MRI ing by directly investigating key cellular enzymatic pathways Sensitivity by Solvent-Generated Feedback Fields in vivo as described in a Feb. 2011 "White Paper" in Neo- plasia titled "Analysis of Cancer Metabolism by Imaging Zhao Li, Susie Y. Huang, Jamie D. Walls, ∗Yung-Ya Lin Hyperpolarized Nuclei: Prospects for Translation to Clini- Department of Chemistry and Biochemistry, UCLA cal Research". Hyperpolarized 13C imaging using the dis- solution DNP (dynamic nuclear polarization) method pro- Introduction. Low sensitivity is particularly problematic in vides a >10,000 fold signal enhancement for detecting 13C biomolecular NMR and heteronuclear MRI, where insensitive probes of endogenous, nontoxic, nonradioactive substances and/or dilute spins are detected. A general spin amplifica- such as pyruvate to monitor metabolic fluxes through mul- tion scheme has now been developed to enhance sensitivity tiple key biochemical pathways (glycolysis, citric acid cy- in NMR/MRI based on dynamic instability of the solvent cle and alanine transaminase). The hyperpolarization of [1- magnetization under collective feedback fields. The solute 13C]pyruvate has demonstrated the ability to not only de- spins are first detected by the solvent spins through various tect pyruvate uptake but also the in vivo enzymatic conver- magnetization transfer mechanisms and serve as small "in- sion to 13C-lactate through the enzyme lactate dehydroge- put" signals to perturb the solvent magnetization, which is nase (LDH), 13C-alanine through the alanine transaminase prepared in an unstable state. The weakly detected signal is

32 Abstracts of Talks – Dynamics and Recognition then amplified through subsequent nonlinear evolution of the an initial demonstration of this detection technology, a mil- solvent magnetization. By manipulating bulk solvent spins limeter scale WAND was chronically implanted on the medial near the threshold of instability to detect dilute solute spins, surface of a rat kidney. High resolution images were obtained sensitivity and signal-to-noise ratios (SNR) can be markedly enabling in vivo observation of kidney microstructures, such improved. as individual glomeruli. This wireless detector may find ap- plications for high-resolution imaging of other body parts and Methods and Results. This general spin amplification hold promise to enable integration with tissue engineered con- scheme is shown here to amplify indirectly detected heteronu- structs for monitoring after implantation. clear solute signals. Low-gyromagnetic ratio nuclei can be de- tected through the large 1H solvent magnetization by the dis- 1 tant dipolar field (DDF) [1,2]. The modulated H transverse Tuesday – May 21st magnetization precesses under the DDF created by the spa- Plenary Session tially modulated 13C longitudinal magnetization, generating Chair: Ad Bax an echo in the 1H solvent magnetization that carries informa- Room Gávea A tion about the 13C spins. Recently discovered self-refocusing of dephased solvent magnetization due to the joint action of radiation damping and the DDF [2] is exploited to enhance PL044: Singlet Nuclear Magnetic Resonance the indirectly detected echo signal. The extreme sensitivity of ∗ the first and largest self-refocused echo’s phase and amplitude Malcolm H. Levitt to the phase and amplitude of the initial triggering magne- School of Chemistry, University of Southampton, UK tization (here, the indirectly detected signal) suggests that Singlet nuclear spin states are quantum states of a nuclear the nonlinear spin dynamics can serve as a high-gain spin spin-1/2 pair that are antisymmetric with respect to spin ex- amplifier to enhance the small initial magnetization trans- change, and which have total spin zero. They are therefore ferred to the solvent from the dilute 13C solute spins. The non-magnetic and protected against many important relax- resulting SNR of the amplified indirectly detected echo sig- ation mechanisms. These states often exhibit long relaxation nal, e.g., 10% 2-13C acetone solution, is improved by 3-4x times which may exceed the normal relaxation time T1 by compared to without amplification. The amplification fac- an order of magnitude or even more. tor can be further improved by controlling the nonlinear spin evolution under the feedback fields. For example, if the 1H ◦ 1 I will discuss the phenomenon of singlet nuclear spin order pulse flip angle q>90 , the instability of the inverted net H in a variety of contexts - including gas-phase para-hydrogen, longitudinal magnetization under radiation damping aids in 1 small molecules in solution, and some examples in solids. I refocusing more H transverse magnetization. The result- will discuss how nuclear singlet spin order may be generated ing SNR is enhanced by an additional 4x, as demonstrated 13 from magnetization, how it is maintained, and how it is con- on U- C glucose. Moreover, field inhomogeneity or weak verted back into observable magnetization. continuous gradients may also be exploited to accelerate the self-refocusing process [3] and increase SNR by more than Some recent developments in our group include: 10x overall. Application of this approach to 13C MRI will also be shown. • direct generation of hyperpolarized nuclear singlet order using dynamic nuclear polarization Conclusion. Sensitivity enhancement by the dynamic in- stability of solvent magnetization represents a new direction • storage and repeated detection of hyperpolarized 13C nu- for surmounting a long-standing weakness of MR through clear singlet order in solution, over a total time of 30 minutes nonlinear feedback. • the use of 18O substitution to access long-lived nuclear sin- Reference. [1] R. Bowtell, J. Magn. Reson. 100, 1 (1992) [2] glet states Warren et al. J. Chem. Phys. 108, 1313 (1998) [3] Y.-Y. Lin et al. Science 290, 118 (2000). • demonstration MRI experiments involving hyperpolarized nuclear singlet order OP043: An Implantable Wireless Amplified NMR 13 Detector for High Resolution MRI • detection of hyperpolarized C singlet order in vivo

Chunqi Qian, Xin Yu, Der-Yow Chen, Stephen Dodd, Joseph • modelling of nuclear singlet relaxation in solution using Murphy-Boesch, ∗Alan P. Koretsky molecular dynamics and quantum chemistry Laboratory of Functional and Molecular Imaging, NINDS, • NMR detection of singlet-triplet conversion between ortho- NIH, Bethesda, MD water and para-water in the solid state. Optical microscopy is sensitive enough to observe tissue mi- crostructures at sub-cellular resolution but application to PL045: Visualizing Transient Structures of DNA and deep lying structures in vivo requires a light path in and out RNA using NMR of the sample being imaged. Magnetic Resonance Imaging (MRI) has the unique advantage that radiofrequency signals ∗Hashim M. Al-Hashimi have a large penetration depth and the "near-field" imaging Department of Chemistry and Biophysics, University of properties of MRI can, in principle generate very high res- Michigan olution. Indeed, resolution in deep-lying internal organs in vivo is presently limited by sensitivity. Conventional MRI We will describe the development and application of relax- utilizes external detectors to receive signals from inside the ation dispersion NMR methods to visualize transient state body, and thus sensitivity is often limited, especially for re- structures of DNA and RNA that exist in low abundance gions deep inside the body. In this work, a Wireless Am- (<13%) and for small periods of time (lifetimes < 1 millisec- plified NMR Detector (WAND) is developed to enhance the onds). We will provide evidence that base-pairs in canoni- MRI sensitivity of deep-lying internal tissues. This detec- cal duplex DNA transiently form Hoogsteen base-pairs, and tor has a small dimension to observe the region of interest in describe basic aspects of these transitions, their occurrence close proximity to the WAND. Unlike the MRI detectors that across a variety of DNA sequence contexts, and possible bio- use a wired connection, the WAND utilizes a wireless power logical functions. We will also report transient structures for source to amplify the MR signal in situ before transmitting three distinct RNAs and describe their potential roles in HIV the signal to an external receiver. A parametric amplifier replication and ribosome translation. The possibility of tar- is integrated with the detection coil to enable wireless opera- geting these transient state structures with small molecules tion and amplification before transmission to the receiver. As will also be discussed.

33 Abstracts of Talks – Dynamics and Recognition

Tuesday – May 21st Bacteria live in complex and changing environment and must Dynamics and Recognition adapt to extracellular medium variations including light, ox- idative stress, pressure, temperature and presence of some Chair: Jose Pires nutrients. They thus need external probes and signalling sys- Room Gávea A tems that enable them to sense these extracellular changes and respond by differential gene expression. OP046: Protein dynamics and interaction mediated by local water dynamics at the ps timescale In our model system, the availability of heme, the major iron source in mammals, is detected by some Gram-negative bac- ∗Songi Han teria via an outer membrane receptor, HasR. A cascade of Departement of Chemistry and Biochemistry, University of molecular interactions between five proteins, located in three California Santa Barbara subcellular compartments, is then used to send this signal from outside to inside the bacteria. An emerging NMR-ESR double-resonance technique dubbed Overhauser-effect Dynamic Nuclear Polarization (ODNP) Using structural and biophysical approaches (NMR, Xray, has demonstrated the ability to probe the diffusion dynamics SAXS, microscopy, SPR and ITC), we have studied the of "soft", fast moving, hydration water, surrounding the pro- molecular interactions between HasR and its various part- tein surface and interfacing bulk water. Specifically, ODNP ners in order to define the structural basis of this signalling probes the dynamics within 2-4 water layers (i.e. 5-10 Å) pathway. Furthermore, we have recently shown, for the first of an ESR-active stable nitroxide radical - i.e. "spin-label" time, that a partially folded protein is involved in this sig- - attached to the surface of a bio-macromolecule. We show nalling process. Our current data represent the first detailed that changes to the protein surface induced by folding, bind- characterization of this type of bacterial signalling. ing, or aggregation can cause these local hydration dynamics to vary by factors of up to 30, offering a dramatic dynamic UP049: NMR Mapping of PCNA Interaction with range for probing, and possibly predicting, interactions in- Translesion Synthesis DNA Polymerase Rev1 Medi- volving proteins. Studies of monitoring early protein aggre- ated by Rev1-BRCT Domain gation events and the adsorption and insertion of proteins into lipid membrane bilayers will be presented. Interestingly, Yulia Pustovalova, Mark W. Maciejewski, ∗Dmitry M. Ko- we show a very weak, nonlinear, scaling of the surface hydra- rzhnev tion diffusion with bulk water viscosity that is increased by University of Connecticut Health Center factors of 10-20 by the addition of various molecular crowd- ing agents. Specifically, both a >10-fold increase in bulk Rev1 is a Y-family translesion synthesis (TLS) DNA poly- viscosity and the restriction of diffusion by confinement on merase involved in bypass replication across sites of DNA a multiple-nm length-scale change the local translational dif- damage and postreplicational gap filling. In the process of fusion coefficient of the surface water by less than 2.5-fold, TLS high-fidelity replicative DNA polymerases stalled by which is in contrast to the vast modulation experienced by DNA damage are replaced by error-prone TLS enzymes re- water dynamics on protein surfaces. We begin to suspect that sponsible for the majority of mutagenesis in eukaryotic cells. the surface topology and chemistry at the ≤ 1.5 nm scale, The polymerase exchange that gains low-fidelity TLS poly- rather than the characteristics of the bulk fluid, nearly exclu- merases an access to DNA is mediated by their interactions sively determine the surface hydration dynamics of aqueous with proliferating cell nuclear antigen (PCNA). Rev1 stands macromolecular solutes, which presents an opportunity for alone from other Y-family TLS enzymes since it lacks the hydration dynamics-based study of protein interaction. consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. ACKNOLEDGEMENTS: U.S. National Science Foundation, Here we report solution NMR structure of an 11 kDa BRCA1 National Institute of Health C-terminus (BRCT) domain from S. cerevisiae Rev1, and demonstrate with the use of TROSY NMR methods that OP047: Molecular Recognition and Dynamics: NMR Rev1-BRCT domain directly interacts with an 87 kDa PCNA Studies of Defensins and Epitopes in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2- β4-α3-α4) typical for BRCT domain superfamily. PCNA- ∗Ana Paula Valente binding interface of the Rev1-BRCT domain comprises con- Federal University of Rio de Janeiro served residues of the outer surface of the α1 helix, α1-β1, β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT Conformational dynamics play a key role in protein recog- binds to the inter-domain region of PCNA that overlaps with nition as well as in many biological processes. In order to the binding site for the PIP-box motif. Furthermore, Rev1- interfere with these processes, we must characterize in detail BRCT domain bound to PCNA can be displaced by increas- the nature of motion and its time scale. In the talk I will ing amounts of the PIP-box peptide from TLS DNA poly- show the importance of protein conformation exchange pro- merase polη, suggesting that Rev1-BRCT and polη PIP-box cess both in defensins-membrane and antibody-epitope recog- interactions with a given PCNA subunit are mutually exclu- nition. We have used chemical shift perturbation and relax- sive. These results provide structural insights into PCNA ation parameters measurements to evaluate the importance recognition by TLS DNA polymerases that help better un- of motions in the ps-ns and µs-ms timescales. In the case of derstand TLS regulation in eukaryotes. defensins, multiple motions seem to be correlated with the stages of membrane recognition and disruption over a large timescale range. I will also discuss the modulation observed OP050: Structural basis of epigenetic regulation in the antibody-epitope affinity due to changes in dynamic. ∗Yunyu Shi We observed that the decrease in conformational exchange and no change in the structure of epitope, lead to the higher School of Life Sciences, University of Science and Technol- affinity, inducing higher cross-linking to fc receptors. ogy of China, Hefei, China PHD (plant homeodomain) zinc fingers that specifically rec- ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, INCT- ognize different histone modification are structurally con- INBEB. served modules found in proteins. Acetyltransferase MOZ is important for HOX gene expression as well as embryo and UP048: Bacterial transmembrane signalling through postnatal development. In vivo, it forms a tetrameric com- a heme/hemophore receptor plex with other subunits including several chromatin binding 1,2∗ modules with regulatory functions. Here we report the solu- Nadia Izadi-Pruneyre tion structure of the first and second PHD fingers in BRPF2, 1Institut Pasteur, Paris, France, 2CNRS UMR 3528 a component of MOZ complex. We also report the solution

34 Abstracts of Talks – IDPs, Folding and Misfolding structure of tandem PHD finger (PHD12) of human MOZ PLanck Laboratory of Structural Biology, Chemistry and in free state, and the 1.47 Å crystal structure in complex Molecular Biophysics of Rosario (MPLbioR) with H3K14ac peptide which reveals the structural basis for The misfolding of proteins into a toxic conformation is pro- the recognition of unmodified R2 and acetylated K14 on hi- posed to be at the molecular foundation of a number of stone H3. Moreover, the results of chromatin immunopre- neurodegenerative disorders including Alzheimer and Parkin- cipitation (ChIP) and RT–PCR assays indicate that PHD12 son’s disease. One common and defining feature of pro- facilitates the localization of MOZ onto the promoter locus tein misfolding diseases is the formation and deposition of of the HOXA9 gene, thereby promoting the H3 acetylation amyloid-like fibrils. The aggregation of the protein alpha- around the promoter region and further up-regulating the synuclein (AS) and Aβ peptide are recognized as critical HOXA9 mRNA level. Our findings suggest that the combina- steps in the etiology of Parkinson and Alzheimer diseases, torial readout of the H3R2/K14ac by PHD12 might represent respectively. The study of the structural and toxic mecha- an important epigenetic regulatory mechanism that governs nisms related to amyloid formation is critical to advance in transcription and also provide a clue of cross-talk between the design of a therapeutic strategy. The identification of the MOZ complex and histone H3 modifications. aggregation inhibitors and the investigation of their mecha- References nism of action are fundamental in the quest to mitigate the pathological consequences of amyloid formation. By the com- 1. Qin Su, et al. J Biol Chem., 2011, 286(42):36944-55. bined application of a battery of NMR techniques we have 2. Lei Liu, et al. J Struct Biol. 2012, 180, 165-173. addressed structural and molecular unresolved details related to the mechanistic basis behind the inhibitory effects of anti- 3. Yu Qiu, et al. Genes & Development., 2012, 26(12):1376- amyloud compounds 91. ACKNOLEDGEMENTS: Financial support from ANPCyT, CONICET, Laboratorios Pförtner, Fundación Bunge y Born, Tuesday – May 21st Max Planck Society and Alexander von Humboldt Founda- IDPs, Folding and Misfolding tion is acknowledged. Chair: Gianluigi Veglia Room Gávea B UP053: Caught in Action: Selecting Peptide Ap- tamers Against Intrinsically Disordered Proteins in 13 OP051: New methods based on C direct detection Live Cells to study intrinsically disordered proteins 1Jacqueline Washington, 1Sergey Reverdatto, 1David Burz, ∗ Isabella C. Felli 2Kathleen McDonough, 1∗Alexander Shekhtman CERM and Department of Chemistry “Ugo Schiff”, Univer- 1State University of New York at Albany, Albany, NY, USA, sity of Florence, Italy 2Wadsworth Center, NY Department of Health, Albany, NY Recent progress in NMR instrumentation in parallel to the Intrinsically disordered proteins (IDPs) or unstructured seg- growing interest in understanding the functional role of pro- ments within proteins play an important role in cellular phys- tein intrinsic disorder and flexibility have stimulated the de- iology and pathology. The low cellular concentration, mul- velopment of a variety of new NMR methods to study intrinsi- tiple binding partners, frequent posttranslational modifica- cally disordered proteins (IDPs)[1]. The high flexibility and tions and presence of multiple conformations make it difficult largely solvent exposed backbone typical of IDPs influence to characterize IDP interactions in intact cells. We propose NMR parameters causing reduced chemical shift dispersion to use a combination of peptide aptamers selected by using and efficient amide solvent exchange. These constitute gen- the yeast two hybrid scheme and in-cell NMR to identify eral features of IDPs that need to be taken into account in potent binders to transiently structured IDPs and unstruc- 13 the design of NMR experimental approaches. C detected tured segments at atomic resolution. Since both selection NMR experiments now offer a valuable tool to address these and characterization of peptide aptamers take place inside peculiar features of IDPs. The experimental variants to im- the cell, only physiologically relevant conformations of IDPs 13 prove the performance of C detected NMR experiments are targeted. The method is demonstrated by using peptide to study IDPs include the design of multidimensional experi- aptamers selected against the prokaryotic ubiquitin-like pro- ments[2], the exploitation of longitudinal relaxation enhance- tein, Pup, of the mycobacterium proteasome system. Two ment[3], the design of experiments to alleviate the problem peptide aptamers, which bind to opposite sides of a transient of extensive cross peaks overlap[4]. The new experiments are helix of Pup, have vastly different effects on the survival rate demonstrated on a paradigmatic IDP, α-synuclein. of mycobacteria. This technology can be applied to study the elusive action of IDPs and unstructured segments within [1] Felli I.C., Pierattelli R., IUBMB Life. (2012), 64, 473-81 folded proteins under near physiological conditions. [2] Bermel W., Bertini I., Felli I.C., Gonnelli L., Koźmiński W., Piai A., Pierattelli R., Stanek J., J.Biomol.NMR, . ACKNOLEDGEMENTS: This work is supported by NIH (2012), 53, 293-301 grant R01GM085006 to A.S..

[3] Bermel W., Bertini I., Felli I.C., Pierattelli R., UP054: Phosphorylation Of The Intrinsically Disor- J.Am.Chem.Soc. (2009), 131, 15339-45, Bertini I., Felli I.C., dered Unique Domain Of c-Src Studied By In-Vivo Gonnelli L., Kumar M.V.V., Pierattelli R., ChemBioChem. Real-Time NMR (2011) , 12, 2347-52, Bertini I., Felli I.C., Gonnelli L., Kumar M.V.V., Pierattelli R., Angew Chem Int Ed Engl. (2011), 50, 1,2Irene Amata, 2Mariano Maffei, 1Ana Igea, 1Angel R. 2339-41 Nebreda, 2∗Miquel Pons 1 [4] Bermel W., Bertini I., Chill, J., Felli I.C., Haba N., Kumar Signaling and Cell Cycle Laboratory, Institute for Research 2 M.V.V., Pierattelli R., ChemBioChem. (2012) , 13, 2425- in Biomedicine, Barcelona, Spain, Biomolecular NMR, 2432, Bermel W., Bruix M., Felli I.C., Kumar M.V.V., Pier- Organic Chemistry Department, University of Barcelona, attelli R., Serrano S., J. Biomol. NMR (2013) ,e-pub Barcelona, Spain Complex phosphorylation and dephosphorylation patterns at OP052: NMR-based Structural Biology in Brain Dis- multiple sites are essential for regulation of protein activ- eases ity in the cytoplasm of living cells. Intrinsically disordered 1,2∗ regions are especially rich in post-translational modification Claudio O. Fernández sites. NMR offers the unequaled possibility to study proteins 1Universidad Nacional de Rosario (UNR), Instituto de Bi- at atomic resolution in real-time and in living cells. The ologia Molecular y Celular de Rosario (IBR-CON, 2Max Unique domain of c-Src (USrc) has several Serine and Thre-

35 Abstracts of Talks – Methods for Organic Materials onine phosphorylation sites. We have used Xenopus laevis Tuesday – May 21st intact oocytes and oocytes and eggs extracts as a model sys- Methods for Organic Materials tem to study the phosphorylation of USrc. The Src family Chair: Klaus Schmidt-Rohr members are non-receptor protein kinases composed by three Rooms Turmalina/Topázio folded domains (SH3, SH2 and kinase) and the intrinsically disordered Unique domain, which links the folded core to membrane surfaces. While the folded domains show a high OP056: 14N-1H Solid-State NMR Spectroscopy of degree of homology within the family, the disordered region Organic Solids is exclusive to each member and swapping of the Unique do- ∗ main between some of the family members exchange their Steven P. Brown functional roles. Previous NMR studies from our group iden- Department of Physics, University of Warwick, UK tified structurally singular regions in the USrc; a lipid in- 14 1 N- H heteronuclear multiple-quantum correlation teraction region has also been discovered within USrc using (HMQC) spectra obtained at high magnetic field (850 MHz) NMR and biochemical assays. These evidences suggest a di- and under fast MAS (up to 60 kHz) are presented. Specifi- rect involvement of USrc in c-Src activity. cally, a modified version of the pulse sequence presented by Gan et al. [1] that utilizes rotary resonance recoupling (R3) at the n = 2 condition (ν1 = 2νR) is employed to recouple the NH dipolar coupling: by using short ( 100 µs) or longer ( 500 In this study, by using fast NMR techniques combined with µs) recoupling times, spectra showing one-bond NH con- non uniform sampling, we were able to detect and monitor in nectivities or additionally longer-range NH proximities are real-time the phosphorylation of the intrinsically disordered obtained [2-6]. Applications to supramolecular self-assembly 15N-labeled USrc in the heterogeneous cellular environment, [2], a dipeptide [3] and pharmaceuticals [4-6] are presented, at 4 different sites presenting different kinetics. Moreover, with the methodology being particularly well suited to prob- the in-extract experiments are also performed in experimen- ing nitrogen containing intra- and intermolecular hydrogen tally modified conditions by enriching the system of specific bonds. Moreover, proof for the molecular level mixing in kinases and phosphatases inhibitors. Phosphorylation pat- co-crystals [5,6] and an amorphous dispersion [6] is proven terns identified by NMR have been compared with results via the observation of specific correlation peaks and changes from Mass Spectrometry experiments. This work represents in the 14N shift via the marked sensitivity of the isotropic an example of the creative potential of modern NMR to guide second-order quadrupolar shift. biological research and help to understand the essential role of intrinsically disordered domains in signaling events in Eu- [1] Z. H. Gan, J. P. Amoureux and J. Trebosc, Chem. Phys. karyotes. Lett. 2007, 435, 163. [2] A. L. Webber, S. Masiero, S. Pieraccini, J. C. Burley, A. S. Tatton, D. Iuga, T. N. Pham, G. P. Spada and S. P. Brown, J. Am. Chem. Soc. 2011, 133, 19777.

OP055: NMR of Intrinsically Disordered Proteins: [3] A. S. Tatton, J. P. Bradley, D. Iuga and S. P. Brown, Z. Tools for Increasing Sensitivity and Resolution Phys. Chem. 2012, 226, 1187.

1Zsofia Solyom, 1Melanie Schwarten, 1Sophie Feuerstein, [4] A. S. Tatton, T. N. Pham, F. G. Vogt, D. Iuga, A. J. 2Dieter Willbold, 3Michael Plevin, 1∗Bernhard Brutscher Edwards and S. P. Brown, CrystEngComm 2012, 14, 2654. 1Institut de Biologie Structurale, CEA-CNRS-UJF, Greno- [5] K. Maruyoshi, D. Iuga, O. N. Antzutkin, A. Alhalaweh, ble, France, 2Institute of complex systems, Structural Bio- S. P. Velaga and S. P. Brown, Chem. Commun. 2012, 48, chemistry, FZ Jülich, Germany, 3Department. of Biology, 10844. University of York, UK [6] A. S. Tatton, T. N. Pham, F. G. Vogt, D. Iuga, Intrinsically disordered proteins (IDPs) have been shown to A. J. Edwards and S. P. Brown, Mol. Pharm. 2013, play important roles in regulatory and signaling processes 10.1021/mp300423r where the structural flexibility allows the protein to adapt to and interact with a large number of distinct molecular OP057: Separated Local Field NMR as a tool for partners. NMR spectroscopy provides a unique tool to study probing molecular dynamics in organic solids IDPs in solution at atomic resolution. However, due to their low chemical shift dispersion, such NMR studies remain chal- 1Marcio F. Cobo, 2Anja Achilles, 1Gregório C. Faria, lenging. In addition, fast solvent exchange results in line bo- 2Detlef Reichert, 2Kay Saalwächter, 1∗Eduardo Ribeiro raodening of amide proton resonances. Other concerns are de Azevêdo often limited sample stability, low sample concentrations to 1Universidade de São Paulo, São Carlos-SP, Brazil„ avoid protein aggregation, and substantial peak intensity het- 2Martin-Luther-University Halle Wittenberg, Halle, Ger- erogeneities in the NMR spectra. Therefore, sensitive NMR many pulse schemes are required to detect correlation peaks also for the sites with the lowest signal intensity; high dimen- Developments on 1H-13C separated local field experiments sional (≥ 3D) NMR experiments with long acquisition times and their recoupled variants are discussed as tools for charac- in all dimensions are needed in order to resolve overlapping terizing molecular motions in organic systems. Experimental correlation peaks; and last but not least fast acquisition tech- results, spin dynamics simulations and analytical treatments niques are mandatory to enable multidimensional data ac- based on the average hamiltonian theory and Anderson-Weiss quisition in a reasonable amount of time. Here we show that (AW) approximation were used to demonstrate the possibil- BEST-TROSY experiments provide significant advantages in ity of using these experiments to extract motion parameters terms of experimental sensitivity and spectral resolution as for each chemical site resolved in a MAS spectrum. Effects demonstrated for several IDPs varying in length from about that compromise the precise quantification of the dynamical 100 to 270 residues. BEST-TROSY allows recording of high- parameters, such as, homo and hetero decoupling imperfec- resolution 3D spectra in only a few hours acquisition time. tions, rotational resonance recoupling, rf inhomogeneity, etc., In addition, we present NMR tools that provide site-specific are also discussed together with ways of avoiding them. Fur- amino-acid type information (HADAMAC, iHADAMAC, iso- thermore, based on the CHn dipolar pattern decomposition, HADAMAC, and Pro-HN experiments). These experiments a multi-gaussian approach that improves the motion descrip- are particular helpful for sequential resonance assignment of tion by AW treatment to CH2 and CH3 groups is proposed, large IDPs, as exemplified for the intrinsically disordered re- so this analytical treatment can be used to obtain a more gion of non-structural protein 5A (NS5A) of hepatitis C virus. precise description of chain motions in complex materials.

36 Abstracts of Talks – MRI/In-Vivo

The methods are demonstrated in model samples as well as [1] Jain, S.; Bjerring, M.; Nielsen, N. C.: Efficient and Robust in studies of molecular motions in polymer systems. Heteronuclear Cross-Polarization for High-Speed-Spinning Biological Solid-State NMR Spectroscopy. The Journal of ACKNOWLEDGMENTS: FAPESP(2009/18354-8- Physical Chemistry Letters 2012, 3, 703-708. 2008/1675-0) and CAPES-DAAD PROBRAL (330-9)

OP060: NMR crystallography of small molecules: UP058: Low-field 1H-NMR investigations to reveal from ionic solids to pharmaceuticals local properties of polymer networks ∗Luís Mafra, Sérgio M. Santos 1∗Maria Ott, 2Roberto Pérez-Aparicio, 1Horst Schneider, 2Paul Sotta, 1Kay Saalwächter Department of Chemistry, University of Aveiro, CICECO, Portugal 1Institut für Physik – NMR, Martin-Luther-Universität Halle-Wittenberg, Betty-Heimann-Str. 7, D-0, 2Laboratoire Crystal structure determination from diffraction experiments Polymères et Matériaux Avancés, CNRS/Rhodia,85 avenue is one of the most important and complete sources of struc- des Frères Perret, F-69192 Sai tural information to a broad range of scientific domains, from chemistry and materials science to geology and physics. We use solid-state 1H-NMR techniques to unravel the molec- Diffraction-based experiments are, however, prone to errors, ular level mechanisms of the elastic properties of polymeric limited to certain particle sizes and depend on crystal qual- networks. This unique property makes the material indis- ity. Obtaining the crystal structure solution in powders is pensable in widespread applications, not only in the field of far more complicated and using powder diffraction methods material science but also in biomedical and pharmaceutical alone may lead to geometrical ambiguities and to an appar- engineering. ently well-refined structure with the wrong crystal packing Multiple-Quantum NMR experiments enable to reveal the arrangement. absolute values and the distributions of the average inter- Here, we address these challenges presenting a new hybrid ap- and intra-segmental residual dipolar couplings [1,2], which proach for crystal structure solution of powdered solids based are directly related to the local forces acting at the ends of on the combination of solid-state NMR/solution NMR, pow- the polymer chains [3]. der XRD and an ensemble of computational-assisted struc- We investigate the local deformation of highly homogenous ture solution tools including an genetic algorithm based on networks of Natural Rubber by testing the local response evolution-inspired operators repeatedly applied to popula- in respect to macroscopically applied forces and the impact tions of possible crystal structure solutions that evolve to of embedded nano-scaled particles. Such filler particles are eventually produce the best new "child" candidates. This al- well-known to lead to improved mechanical properties and gorithm also imposes space group symmetry to narrow the we investigate their effect on the local stresses in the net- search space, thus forcing the possible solutions to belong work matrix. We are able to proof the existence of matrix to the same crystallographic space group and crystal system overstrain in filled rubber samples in agreement with simple previously estimated from XRD powder pattern indexation 1 hydrodynamic models and to monitor strain induced network and validated by the algorithm. H Chemical shifts are also inhomogeneities and the occurrence of highly stressed chains. used in the structure-refinement step as pseudo-forces act- ing on the models, leading to the lowest-energy structure. [1] K. Saalwächter, Progr. Nucl. Magn. Reson. 51 (2007), 1 This methodology, which avoids the use of time-consuming ab initio [2] W. Chassé, J. L. Valentín, G. D. Genesky, C. Cohen, K. chemical shift calculations, is successfully applied to Saalwächter, J. Chem. Phys.134 (2011), 044907 challenging cases involving multiple component crystals com- posed by flexible molecules such as a trihydrate β-lactamic [3] J.-U. Sommer, W. Chassé, J. L. Valentín, K. Saalwächter. antibiotic and an alkyl-substituted imidazolium-based ionic Phys. Rev. E 78 (2008), 051803 solid. ACKNOLEDGEMENTS: Supported by Fundação UP059: Efficient Heteronuclear Cross Polarization para a Ciência e a Tecnologia (FCT) - project Techniques for Solid State NMR PTDC/QUIQUI/100998/2008, Portuguese National NMR Sheetal Kumar Jain, Morten Bjerring, ∗Niels Christian Network (RNRMN), CICECO, FEDER, COMPETE, and Nielsen University of Aveiro. SMS acknowledges FCT for his post- doc grant. Aarhus University

A new and highly efficient approach for heteronuclear coher- st ence transfer in solid-state NMR spectroscopy under high Tuesday – May 21 speed spinning conditions is presented along with its adi- MRI/In-Vivo abatic derivative. RESP IRAT ION CP[1] has been demon- Chair: Michael Garwood strated as a very simple experiment with easier experimen- Room Ônix tal set up and significantly more robustness towards im- perfections such as rf inhomogeneity, misadjustments and OP061: Parallel imaging with nonlinear gradient sample induced variations. This rotor synchronized set up fields exploits both ±1 or ±2 Fourier components of the dipo- lar coupling with different orientation dependencies and ∗Gigi Galiana, Leo Tam, Dana C. Peters, R. Todd Consta- leads to significantly higher transfer efficiencies than state- ble of-the art techniques including ramped and adiabatic cross- Yale University polarization experiments with long durations of intense rf irradiation. Furthermore the efficiency of this method can Sequences that encode the spatial information of an object be improved by combining this with an amplitude modu- using nonlinear gradient fields are a new frontier in MRI, lated adiabatic scheme. The results are described analyt- with potential to provide lower peripheral nerve stimulation, ically, numerically and experimentally for biological solids. windowed fields of view, spatially-varying resolution, curved We demonstrate sensitivity gains of factors of 1.3 and 1.8 slices that mirror physiology, and, most importantly, highly using RESP IRAT ION CP for typical 1H→15N and 15N→13C undersampled parallel imaging. O-Space imaging, a method transfers and a combined gain of a factor of 2-4 for a typi- that acquires projections along ring-shaped isocontours cen- cal NCA experiment for biological solid-state NMR. A com- tered at different locations in the field of view, has been shown parison of these transfer efficiencies with adiabatic RESPI- to produce good reconstructions from dramatically under- RATIONCP will be presented along with its application to sampled multichannel datasets. The ring-shaped isocontours quadrupolar nuclei. of the O-Space sequence appear to better complement the az-

37 Abstracts of Talks – MRI/In-Vivo imuthal spatial encoding provided by typical receiver arrays. [3] Ferreira, M.F.; Mousavi, B.; Ferreira, P.M.; Martins, However, the details of this complementarity have been more C.I.O.; Helm, L.; Martins, J.A.; Geraldes, C.F.G.C., Dalton difficult to specify. Trans., 2012, 41, 5472 – 5475.

We present a simple and intuitive framework that describes [4] Pinho, S.L.C.; Laurent, S.; Rocha, J.; Roch, A.; Delville, image encoding with nonlinear gradients in the language of M.-H.; Carlos, L.D.; Elst, L.V.; Muller, R.N.; Geraldes, k-space. Focusing on O-Space imaging, we show how the dat- C.F.G.C., J. Phys. Chem. C, 2012, 116, 2285-2291. apoints relate to k-space, the role of coil encoding, and how [5] Figueiredo, S.; Moreira, J.N.; Geraldes, C.F.G.C.; the gradient trajectory can be used to predict image quality, Rizzitelli, S.; Aime, S.; Terreno, E.; Chem. Comm., 2011, as is often done for Fourier encoded images. Parallels be- 47, 10635-10637. tween the O-Space strategy and that of compressed sensing will also be discussed. Experimental results, in phantoms, in [6] Figueiredo, S.; Cutrin, J.C.; Rizzitelli, S.; De Luca, E.; vivo, and with a human head insert, demonstrate high quality Moreira, J.N.; Geraldes, C.F.G.C.; Aime, S.; Terreno, E. reconstruction of highly undersampled datasets. Molecular Imaging and Biology, in press.

We also show how the framework described above can be used UP063: Magnetic Resonance Microimaging of a to design trajectories that address clinical practicalities, such Swelling Gel as multiecho acquisition. The analysis suggests that multi- echo O-Space should employ a modified acquisition order and 1Wilson Barros Jr., 1Eduardo N. de Azevedo, 2∗Mario timing offsets to smooth T2 weighting across the dataset. Engelsberg Combined with filtering strategies in the reconstruction, we 1Universidade Federal de Pernambuco, Recife, are able to minimize artifacts while still retaining relaxation 2Universidade Federal de Pernambuco, Caruaru contrast in a turbo spin echo type acquisition. The formation of contour patterns in the heterogeneous growth of layered structures is of considerable interest for OP062: Relaxivity Optimized Metal Based MRI understanding the morphogenesis of many structures, in- Molecular Imaging Agents cluding living organisms. We study the time evolution of surface pattern formation in a swelling spherical gel using ∗Carlos F.G.C. Geraldes magnetic resonance microimaging of water molecules migrat- Department of Life Sciences, Center of Neurosciences and ing into the gel[1]. Relaxation-weighted images permit to Cell Biology and Coimbra Chemistry Center, University of map the local evolution of the swelling ratio, which can be Coimbra identified with the determinant of the deformation gradi- The superb spatial resolution of Magnetic Resonance Imag- ent tensor. The breaking of spherical symmetry caused by ing (MRI) has made it a fundamental clinical diagnostic tool. the growth of an instability produces a surface undulation However, its relatively low sensitivity may limit its use in whose wavelength depends upon the width of the region be- Molecular Imaging. High relaxivity and specificity of tar- tween the surface and the unswollen core of the gel[1, 2]. By geted MRI contrast agents (CAs) are currently the most controlling the degree of cross linking within a thin surface important objectives in the development of such diagnostic layer of the gel it is possible to monitor the transition from imaging tools. The design and relaxivity optimization proce- an undulating folding pattern to the formation of singular dures of Gd3+- chelates as CAs will be discussed using a few creases. examples. The effect of various molecular parameters of the 3+ The swelling kinetics is examined with special emphasis on Gd - chelates on the r1 relaxivity optimization, such as the the time evolution of the solvent distribution. We compare inner-sphere water exchange rate and the rotational correla- our microimaging experimental results with the predictions in vivo tion dynamics, will be illustrated [1,2] The evaluation of a nonlinear poroelastic theory[3] by numerically solving the T of the 1- contrast effect of some of these systems by DCE- nonlinear equations of evolution within a spherically symmet- MRI in Wistar rats will be illustrated [2]. ric approximation. The use of nanosized platforms is an effective way to enhance [1] W. Barros Jr., E. N. de Azevedo, and M. Engelsberg, Soft the efficacy of such contrast agents, by increasing the number Matter, 2012, 8, 8511. of imaging agents reaching the target ("cargo effect"). Sev- eral types of nanoparticles (NPs) have been explored for this [2] J. Dervaux , Y. Couder, M. A. Guedau-Boudeville, and objective. Among the inorganic ones, gold NPs decorated M. Ben Amar, Phys. Rev. Lett., 2011, 107, 0181103. 3+ with relaxivity optimized Gd - chelates [1] originate high [3] W. Hong, X. Zhao, J. Zhou, and Z. Suo, J. Mech. Phys. r1 relaxivities in the MRI imaging frequency range due to Solids, 2008, 56, 1779. the slow rotational dynamics of the NPs and the concentra- tion of many chelates at the NP surface [3]. Other inorganic ACKNOWLEDGMENTS: CNPQ, FACEPE NPs, with high r2 relaxivities, originate high negative con- trast in T2w MRI images, especially at high magnetic fields. UP064: Early Detection of Pancreatic Cancers & Examples of this are core-shell γ-Fe2O3@SiO2 NPs [4]. Na- Brain Tumors by Active-Feedback Controlled MR ture inspired platforms, such as yeast-cell wall NPs (YCWP), 1 2 2 1∗ are very efficient carriers of multimodal imaging probes which Zhao Li, Chaohsiung Hsu, Lian-Pin Hwang, Yung-Ya can be exploited as positive MRI contrast agents for in vitro Lin cell labelling and in vivo cell tracking applications [5,6]. 1Department of Chemistry and Biochemistry, UCLA, 2Department of Chemistry, National Taiwan University ACKNOLEDGEMENTS: Financial support from FCT (Por- tugal) and EU COST TD1004 Actions is acknowledged. Introduction: Early detection of high-grade malignancy, such as pancreatic cancers (PC) and glioblastoma multi- REFERENCES forme (GBM), using enhanced MRI techniques significantly increases not only the treatment options available, but also [1] Ferreira, M.F.; Martins, A.F.; Martins, J.A.; Ferreira, the patients’ survival rate. For this purpose, a conceptually P.M.; Tóth, É.; Geraldes, C.F.G.C. Chem. Commun. 2009, new approach, termed "Active-Feedback Controlled MR", 6475-6477. was developed. An active feedback electronic device was homebuilt to implement active-feedback pulse sequences to [2] Ferreira, M.F.; Martins, A.F.; Martins, C.I.O; Tóth, generate avalanching spin amplification, which enhances the É.; Rodrigues, T.B:, Calle, D.; Cerdán, S.; López-Larrubia, weak magnetic-field perturbations from magnetic nanoparti- P.; Martins, J.A.; Geraldes, C.F.G.C. Contrast Media Mol. cles in targeted PC or malignant physiological conditions in Imaging, 2013, 8, 40-49. GBM.

38 Abstracts of Talks – Plenary Session

Theory and Methods: The general principles of the Department of Biochemistry and CRUK Cambridge Insti- "Active-Feedback Controlled MR" can be found in our publi- tute, University of Cambridge, UK cations [1-4] (and references therein). Here, its specific appli- Patients with similar tumor types can have markedly differ- cations to early tumor detection were developed and demon- ent responses to the same therapy. The development of new strated. (i) First, an active-feedback electronic device was treatments would benefit, therefore, from the introduction of home-built to generate feedback fields from the received FID imaging methods that allow an early assessment of treatment current. The device is to filter, phase shift, and amplify the response in individual patients, allowing rapid selection of the signal from the receiver coils and then retransmit the modi- most effective treatment. fied signal into the RF transmission coil, with adjustable and programmable feedback phases and gains. The MR console We have been developing methods for detecting the early computer can execute the active-feedback pulse sequences to responses of tumors to therapy, including magnetic reso- control the trigger signal, feedback phase/gain, and the du- nance (MR) imaging of tumor cell metabolism using hyper- ration of the feedback fields, allowing us to utilize the active polarized 13C-labeled cellular metabolites. We have shown feedback fields in novel ways. (ii) Next, an active-feedback that exchange of hyperpolarized 13C label between lactate pulse sequence was developed for early tumor detection and and pyruvate can be imaged in animal models of lymphoma was statistically tested on in vivo mice tumor models. In and glioma and that this flux is decreased post-treatment. essence, the enhanced tumor contrast arises from "selective We showed that hyperpolarized [1,4-13C]fumarate can be self-excitation" and "fixed-point dynamics" generated by the used to detect tumor cell necrosis post treatment in lym- bulk water 1H under active feedback fields. Use the sensitive phoma and that both the polarized pyruvate and fumarate detection of magnetic nanoparticles as an example. A small experiments can detect early evidence of treatment response flip-angle (q=5-10o) RF pulse tilts the sample equilibrium in a breast tumor model and also early responses to anti- magnetization. Since the averaged transverse magnetization vascular and anti-angiogenic drugs. Fumarate can also be is mainly contributed from the bulk water 1H spins, the re- used to detect necrosis in other tissues, such as the kid- sulting active feedback field possesses a frequency closer to ney. We have shown that tissue pH can be imaged from 1 13 − that of the bulk water H spins which are distant from the the ratio of the signal intensities of hyperpolarized H CO3 13 dipole center. By "selective self-excitation", the feedback and CO2 following intravenous injection of hyperpolarized 1 13 − field tilts the bulk water H spins more effectively towards H CO3 and that tumor redox state can be determined by the stable fixed-point, –z-axis (assume feedback phase 180◦), monitoring the oxidation and reduction of [1-13C]ascorbate while the 1H spins near the dipole center are less affected and [1-13C]dehydroascorbate respectively. Recently we have due to resonance mismatch. This "selective self-excitation" shown that we can monitor tumor glycolysis by measuring the process continues and enlarges the contrast between the lon- conversion of hyperpolarized [U-2H,U-13C]glucose to lactate. gitudinal magnetization of the 1H spins in bulk water and Labeled lactate production was higher in the tumor than in those near the dipole center. Maximum contrast in the lon- surrounding normal tissue and was markedly decreased at 24 gitudinal magnetization can be achieved and locked when all h after treatment with a chemotherapeutic drug. spin magnetizations evolve to the fixed point: all align along –z in this case. Tuesday – May 21st Results (1): Early Detection of Pancreatic Cancers: Anti- Plenary Session CA 19-9 antibodies were conjugated to NH2-PEG-coated Chair: José Figueroa-Villar magnetic nanoparticles. The antigen binding capacity to CA Room Gávea A 19-9 over-expressing cell lines (BxPC3) was confirmed by in vitro MR cellular images. In vivo images of human pancre- atic tumors from nude mouse xenografts show that, while T2- PL066: Recent contributions for Zero-, Low-, and weighted image cannot clearly locate the magnetic nanopar- High-field NMR ticles, the active-feedback images successfully highlight the ∗ Tito J. Bonagamba magnetic nanoparticles distribution with a close correlation with iron-stained histopathology. Instituto de Física de São Carlos - Universidade de São Paulo, São Carlos, Brazil Results (2): Early Detection of Glioblastoma Multiforme: Recently our group decided moving to some specific applica- Stage-1 orthotopic GBM mouse models infected with human tions of zero-, low-, and high-field NMR, not only because U87 cell line were imaged. While both T2 parameter images they could drive us to rich physical problems, but also be- and T2-weighted images by spin echo successfully locate the cause we could transfer to them methods applied for high- GBM tumor, our active-feedback images and decay constant resolution NMR. mapping provide 4-5 times of improvements in GBM tumor contrast through sensitively imaging the susceptibility varia- In the case of zero-field NMR, employed to study mag- tions due to irregular water contents and deoxyhemoglobin. netic materials, using only two arbitrary rf pulses, we ob- tained multiple-quantum (MQ) echoes, which were used to Discussion and Conclusion: In vivo PC and GBM mouse construct the spectra. To understand the data observed, models validated the superior contrast/sensitivity and ro- we proposed a method for selecting each MQ echo based bustness of the "Active-Feedback MR" for early tumor de- on phase-cycling and time-averaging, and a model in which tection. Statistical results (N>10) for PC and GBM mouse there are two regions along the sample with different inho- models at various cancer stages, alternative active feedback mogeneous spectral line broadenings. These methods offered pulse sequences with further improved performance, and ac- complementary physical information about the nuclear spin tive feedback pulse sequences for enhanced R1/R2-weighted interactions and the properties of the magnetic materials. images will also be presented. In the case of low-field NMR, used to study porous me- Reference: dia, we are going to discuss new procedures to perform 1D [1] Science 290, 118 (2001) [2] Magn. Reson. Med. 56, 776 and 2D T2xT2 exchange experiments, which allow observing (2006) [3] Magn. Reson. Med. 61, 925 (2009) [4] J. Phys. only the migrating fluid molecules. In the 2D method, which Chem. B 110, 22071 (2006) employs two CPMG T2 encoding stages separated by a z- store mixing time, the signals from the molecules that do not OP065: Detecting Tumor Treatment Response using migrate are subtracted from the raw NMR data, after pro- Metabolic Imaging with hyperpolarized 13C-labeled cessing the 2D Inverse Laplace Transform. This procedure Cell Substrates eliminates the intense diagonal ridge, providing an accurate study of the migrating molecules. In the 1D method, instead ∗Kevin M. Brindle, Mikko I. Kettunen, Tiago B. Ro- of varying the number of π pulses at the first CPMG stage, drigues, Eva M. Serrao, Brett W.C. Kennedy, De-en Hu it is kept fixed and small enough to act as a short T2-filter.

39 Abstracts of Talks – Amyloids

Thus, varying the mixing time in a wide range provides a ton channel formed by the M2 protein of the influenza virus, set of 1D T2 distributions, which shows simultaneously the and the fusion peptide and transmembrane (TM) domain effects of both exchange and full T1 relaxation. of the paramyxovirus, parainfluenza virus 5 (PIV5). MAS NMR yields a rich panel of structural and dynamic informa- Finally, , we are going to in the case high-field NMR tion that give insight into the proton-conduction mechanism present some NMR tools developed for quantum information of M2 and the basis of membrane curvature generation by studies. the fusion peptide. Distance measurements, exchange spec- 1 AKNOWLEDGEMENTS: IFSC, USP, FAPESP, CNPq, and troscopy and H chemical shifts revealed the proton exchange CAPES. kinetics and hydrogen-bonding of the proton-selective histi- dine in M2. The distances between the histidine and the OP067: Magnetic resonance studies of topological gating tryptophan indicate cation-pi interactions at low pH, materials which shed light on the low proton flux of this channel. Mu- tation of Ser31 to Asn in the M2 TM domain causes drug re- ∗Louis Bouchard sistance and affects proton conductivity. Our measurement UCLA of the conformation and proton-exchange dynamics of the S31N mutant in the apo state and when bound to a novel In recent years the emergence of gapless topologically pro- drug sheds light on the mutant properties. Outside the TM tected edge states in the solid state has led to searches for new domain, the M2 cytoplasmic tail is important for virus assem- phases of condensed matter in new and existing materials. bly and budding. We have investigated the structure of the The protected edge states in topological insulators are due cytoplasmic domain using 2D and 3D NMR, spectral edit- to the combination of spin-orbit coupling and time-reversal ing, and computational modeling. Similar curvature genera- invariance. Examples of exotic phenomena include the quan- tion is the key function of viral fusion peptides such as the tum anomalous Hall effect, fractional quantum anomalous PIV5 fusion peptide, which merge the lipid membranes of Hall effect, fractional time-reversal invariance, topological the virus and the target cell. Our studies of the conforma- Kondo insulator, topological crystalline insulator and the tion and lipid-interaction of the PIV5 fusion peptide indicate topological magneto-electric effect. The observation of topo- that the peptide structure depends sensitively on the mem- logical phases requires tools which can probe nanoscale elec- brane composition. We suggest a correlation between the FP tronic and magnetic phenomena at the surface of the mate- conformations and various stages of membrane fusion. rial, or at some interface located deep inside the material. Radioactive ion beam spectroscopy uses low energy ions im- ACKNOLEDGEMENTS: NIH grants GM088204 and planted at specific depths in the material to report on the GM066976 local properties. I shall discuss ongoing experiments at TRI- UMF whereby spin-polarized muon and lithium ion beams nd have emerged as useful depth-resolved reporters of physical Wednesday – May 22 properties in topological materials and heterostructures in- Amyloids volving topological insulators. New results from conventional Chair: Ana Paula Valente solid state NMR studies will also be discussed. Room Gávea A

OP068: Hyperpolarized Molecular Sensing and De- tection OP070: Amyloid aggregates and large soluble pro- tein complexes 1,2∗Vikram S. Bajaj 1,2,3Sam Asami, 1,2,3Juan-Miguel Lopez del Amo, 1,2,3Andi 1Department of Chemistry and California Institute of Quan- Mainz, 1,2,3Muralidhar Dasari, 3Uwe Fink, 4Marcus Fän- titative Biosciences, University of California, Berkeley, drich, 5Erich E. Wanker, 5Jan Bieschke, 6Tomasz Religa, 2Materials Sciences Division, Lawrence Berkeley National 6Lewis E. Kay, 1,2,3∗Bernd Reif Laboratory 1Technische Universität München (TUM), Department I will survey our efforts to develop and employ magnetic reso- Chemie, 2Helmholtz-Zentrum München (HMGU), 3Leibniz- nance contrast agents for molecular recognition that use hy- Institut für Molekulare Pharmakologie (FMP), 4Max- perpolarized xenon as a reporting medium. These agents Delbrück Centrum für Molekulare Medizin (MDC), 5Max- are based on molecular or supramolecular guest-host com- Planck-Forschungsstelle für Enzymologie der Proteinfaltung, plexes that bind xenon in reversible exchange, the proto- Martin-Luther Universität Halle-Wittenberg, 6University of typical examples being the cryptophanes. Because bound Toronto, Department of Medical Genetics and Microbiology xenon is spectroscopically distinct from xenon in the bulk, we are able to exploit chemical exchange saturation trans- Perdeuteration and back-substitution of exchangeable pro- tons in microcrystalline proteins in combination with recrys- fer (CEST) for additional signal amplification. Further, we 1 1 have increased the sensitivity and biocompatibility of these tallization from D2O containing buffers reduces H, H dipo- cryptophane-based sensors by covalently attaching them in lar interactions such that amide proton line widths on the or- many copies to macromolecular scaffolds, among them the der of 20 Hz are obtained (Chevelkov et al., 2006). Aliphatic capsids of bacteriophage and MS2. Next, we have developed protons are either accessible via specifically protonated pre- new, non-cryptophane supramolecular agents, useful both as cursors or by using low amounts of H2O in the bacterial sensors and reporters of chemical processes in vivo and in growth medium (Asami et al., 2010). This labeling scheme vitro. For chemical sensing in portable devices, we are devel- is applied to amyloid aggregates like fibrils formed by the oping combinatorial methods to produce sensors targeted to Alzheimer’s disease β-amyloid peptide (Aβ) (Linser et al., specific small molecule analytes. These sensors will be used 2011). We present data on solid-state NMR studies of drug in platforms that contain microfabricated analogues of high induced Aβ aggregates focussing in particular on the interac- field NMR instrumentation including xenon gas polarizers tions between Aβ and the polyphenolic green tea compound and optical NMR detectors. epigallocatechin-gallate (EGCG). We show that MAS solid- state NMR techniques are applicable for the structural char- acterization of large soluble protein complexes (Mainz et al., PL069: Structures and mechanisms of viral ion chan- 2009), in case the tumbling correlation time exceeds the rotor nels and fusion peptides from solid-state NMR period. Experimental results are presented for the small heat ∗Mei Hong shock protein αB crystallin (600 kDa) as well as for the 20S proteasome core particle in complex with its 11S activator Iowa State University (1.1 MDa). I will present on-going studies of the structure, dynamics and mechanism of action of two viral membrane proteins, the pro- References

40 Abstracts of Talks – Amyloids

[1] Asami S, Schmieder P & Reif B (2010) High resolution 1H- alterations in metal ion homeostasis may play a pivotal role detected solid-state NMR spectroscopy of protein aliphatic in the progression of AS amyloid assembly and the onset of resonances: Access to tertiary structure information. J Am PD. Elucidation of the structural basis directing AS-metal Chem Soc 132: 15133–15135. interactions and their effect on AS aggregation constitutes a key step towards understanding the role of metal ions in AS Chevelkov V, Rehbein K, Diehl A & Reif B (2006) Ultra- amyloid formation and neurodegeneration. The structural high resolution in proton solid-state NMR at high levels of properties of the AS-metal complexes were determined by the deuteration. Angew Chem Int Ed 45: 3878-3881. combined application of nuclear magnetic resonance (NMR) [2] Linser R, Dasari M, Hiller M, Higman V, Fink U, Lopez and electron paramagnetic resonance (EPR), circular dichro- del Amo J-M, Handel L, Kessler B, Schmieder P, Oesterhelt ism (CD) spectroscopy, and matrix-assistedlaser desorption D, Oschkinat H & Reif B (2011) Proton detected solid-state ionization mass spectrometry (MALDI MS). This work pro- NMR of fibrillar and membrane proteins. Angew Chem Int vides a comprehensive view of recent advances attained in Ed 50: 4508-4512. the metallobiology of AS amyloid diseases. A hierarchy in AS-metal ion interactions has been established: while diva- [3] Mainz A, Jehle S, van Rossum BJ, Oschkinat H, Reif, B. lent metal ions interact at a non-specific, low-affinity binding (2009). Large Protein Complexes with Extreme Rotational interface at the C-terminus of AS, copper binds with high Correlation Times Investigated in Solution by Magic-Angle- affinity at the N-terminal region and it is the most effec- Spinning NMR Spectroscopy. J Am Chem Soc 131, 15968– tive metal ion in accelerating AS filament assembly.[1-4] The 15969. strong link between metal binding specificity and its impact on aggregation is discussed here on a mechanistic basis. A OP071: Amyloid Fibrils: Towards a Molecular Level detailed description of the structural features and coordina- Picture with Solid-State Nuclear tion environments of copper to AS is presented and discussed ∗ in the context of oxidative cellular events that might lead to P. K. Madhu the development of PD. These new findings of the structural Tata Institute of Fundamental Research and metallobiology of PD are discussed via a comparative Aβ peptides are interesting models for investigating differ- analysis with the binding and affinity features of metal ions ent aspects of amyloid aggregation. On the basis of amyloid to the beta amyloid peptide of Alzheimer’s disease.[5] Over- cascade hypothesis, which has dominated amyloid disease re- all, the research findings presented here support the notion search for the past two decades, the main therapeutic strate- that perturbations in the metabolism of metal ions may be a gies have aimed either to prevent the aggregation of Aβ, or to common upstream event in the pathogenesis of neurodegen- remove toxic oligomeric and fibrillar species of Aβ. Since Aβ erative processes.[6] is ordinarily produced in the brain, and there is no proof that References Aβ overproduction underlies sporadic AD, the pursuit of for- mer strategy demands a thorough understanding of all the [1] Rasia et al, Proc. Natl. Acad. Sci. U S A, 2005, 102, neurochemical factors that initiate Aβ deposition in brain. 4294-4299. One such factor is the presence of metal ions, especially Zn2+. 2+ [2] Bertoncini et al, Proc Natl Acad Sci U S A, 2005, 102: Zn plays important roles in normal physiology of brain and 1430-1435. is also considered to be a major neurochemical factor asso- ciated with Aβ aggregation and AD. Observations like high [3] Binolfi et al, J. Am. Chem. Soc., 2008, 130,11801-11812. Zn2+ concentrations in senile plaques found in the brains of Alzheimer’s patients and evidences emphasising the role of [4] Binolfi et al, J. Am. Chem. Soc., 2011, 133, 194-196. 2+ Zn in Aβ-induced toxicity have triggered interest in un- [5] Valiente-Gabioud et al , J. Inorg Biochem. , 2012, 117:334- 2+ derstanding the nature of Zn -Aβ interaction. Of the two 341. strategies mentioned earlier, the later generally involves us- age of external agents/drugs which can make Ab follow such [6] Binolfi et al, Coord. Chem Rev., 2012, 256:2188-2201. aggregation pathways which yield non-pathological species Acknowledgements of Aβ. In this regard, curcumin, a small phenolic compound and a common Asian spice, has been found to ameliorate Financial support from ANPCyT, CONICET, Laboratorios the effects of Aβ induced neuro-degeneration in AD models. Pförtner, Fundación Bunge y Born, Max Planck Society and A structural understanding of how curcumin interacts with Alexander von Humboldt Foundation is acknowledged. Aβ can provide a significant impetus to such efforts, and we are exploring this aspect with SSNMR spectroscopy. In this OP073: Structural studies of an engineered mimic of work, we have studied both the properties and the molecu- neurotoxic amyloid-β protofibrils lar structure of both Aβ40 and Aβ42 aggregates co-incubated 1 2 3 with curcumin. We will present here key results from both Christofer Lendel, Morten Bjerring, Andrei Filippov, 3,4 2 1∗ of these studies. Oleg N. Antzutkin, Niels Christian Nielsen, Torleif Härd UP072: Metallobiology of neurodegenerative dis- 1Dept. of Molecular Biology, Swedish University of Agricul- eases: Structural and mechanistic basis behind the tural Sciences (SLU), Uppsala, Sweden., 2Dept. of Chem- acceleration of amyloid protein assembly istry, Aarhus University, Aarhus, Denmark., 3Chemistry of 1,2 1,2 3 Interfaces, Luleå University of Technology, Luleå, Sweden., M.C. Miotto, A.A. Valiente Gabioud, Andres Bi- 4 4 5 1,2∗ Dept. of Physics, Warwick University, Coventry, United nolfi, L. Quintanar, Christian Griesinger, Claudio O. Kingdom. Fernández Structural and biochemical studies of the aggregation of the 1Instituto de Biología Molecular y Celular de Rosario (IBR- 42-residue amyloid-β peptide (Aβ42) are important to under- CONICET), Universidad Nacional de Rosario, 2Max Planck stand the mechanisms of Alzheimer’s disease, but research Laboratory of Structural Biology, Chemistry and Molecular is complicated by aggregate inhomogeneity and instability. Biophysics of Rosario (MPLPbioR, 3Department of NMR- We previously engineered a hairpin form of Aβ, called Aβcc, assisted Structural Biology, In-cell NMR, Leibniz Institute of which forms stable protofibrils that do not convert into amy- Molecular Pharmacology, 4Centro de Investigación y de Es- loid fibrils [1]. A large number of biochemical and biophysi- tudios Avanzados (Cinvestav), 5Department of NMR-based cal experiments indicate that the morphology and structure Structural Biology, Max Planck Institute for Biophysical of Aβ42cc protofibrils are very similar to those of wild type Chemistry Aβ42 aggregates. Like Aβ42 aggregates, Aβ42cc protofibrils Alpha-synuclein (AS) aggregation is associated to neurode- also induce apoptosis in neuroblastoma cell lines and atten- generation in Parkinson’s disease (PD). At the same time, uate spontaneous synaptic activity. Aβ42cc protofibrils are

41 Abstracts of Talks – Dynamics and Recognition

therefore a good mimic for wild-type Aβ42 protofibrils. The to NMR studies due to the availability of low-lying excited molecular structure of Aβ42cc protofibrils is studied using electronic states. These features are strongly related to the solid-state NMR spectroscopy as part of the effort to under- physiological requirements of these copper centers to perform stand the molecular basis for protofibril neurotoxicity. The efficient electron transfer or oxidation chemistry. 13 C NMR spectrum of Aβ42cc is partially well resolved with The binuclear copper sites CuA and T3 display particularly resonance line widths of less than 1.5 ppm and resonances of fast electron relaxation rates which are due to low-lying ex- residues 16-17 and the C-terminal fragment 31-41 have been cited states that can be populated at room temperature and assigned. NMR chemical shifts and intramolecular 13C-13C contribute to the reactivity of the metal site. Other mag- correlations indicate a β-strand containing hairpin structure netic techniques, such as EPR, ENDOR and MCD, normally of the C-terminal of Aβ42cc protomers in the fibril. Inter- recorded at cryogenic temperatures, are able to monitor ex- molecular 13C-13C and 13C-15N correlations then suggest clusively the ground state. NMR in solution, instead can shed that protomers in the protofibril are packed by interacting light on the availability of these invisible electronic states. We C-termini. These data allow us to construct models of the have carried on detailed studies in the CuA site, involved in packing of Aβ42cc protomers within protofibrils. long range electron transfer in terminal oxidases. [1] A. Sandberg et al. (2010) Stabilization of neurotoxic NMR discloses the fact that the CuA site can exist in two al- Alzheimer amyloid-β oligomers by protein engineering. Proc ternate ground states with different orbital symmetry, which Natl Acad Sci USA 107, 15595-15600. are invisible to other techniques. We show that thermal fluctuations may populate two alternative ground-state elec- Wednesday – May 22nd tronic wave functions optimized for electron entry and exit, Dynamics and Recognition respectively, through two different and nearly perpendicular Chair: Rodolfo Rasia pathways. These findings suggest a unique role for alternative Room Gávea B or "invisible" electronic ground states in directional electron transfer. Moreover, we show that this energy gap and, there- fore, the equilibrium between ground states can be fine-tuned OP074: Probing Protein Conformational Flexibility by minor perturbations, suggesting alternative ways through with Pulsed EPR Spectroscopy which protein–protein interactions and membrane potential Xi Huang, I.M.S. de Vera, Manuel Britto, Mandy E. Black- may optimize and regulate electron–proton energy transduc- ∗ tion. burn, Angelo M. Veloro, Jamie L. Kear, Gail E Fanucci University of Florida References: HIV-1 Protease (HIV-1 PR) is an essential enzyme for gener- [1] Abriata et al., J.Am.Chem.Soc., 131, 1939–1946 (2009). ating infectious HIV virus particles and is a target in the fight [2] Zaballa et al., J.Am.Chem.Soc., 132, 11191–11196 (2010). against HIV infection. Current therapeutic inhibitors, which are designed against HIV-1 PR subtype B found in North [3] K. Lancaster et al., J.Am.Chem.Soc., 134, 8241-53 (2012). America and Europe, are less potent inhibitors against other subtypes. Thus, understanding how the polymorphisms al- [4] M.E.Zaballa et al., Proc.Natl.Acad.Sci USA, 109, 9254-9 ter protein structure, flexibility and interaction with PIs may (2012). provide insights for optimizing drug candidate against highly [5] L.A.Abriata et al., Proc.Natl.Acad.Sci USA, 109, 17348- variable targets. 53 (2012). Results from pulsed EPR studies reveal that drug pressure se- lected mutations and natural polymorphisms alter the HIV-1 UP076: NMR Studies of the Loop Dynamics of the PR conformation ensemble. We are currently assessing corre- Nitrophorins lations among the fractional occupancy of the conformational ∗F. Ann Walker, Dhanasekaran Muthu, Robert E. Berry, ensemble of nominally four different conformational states Hongjun Zhang with enzyme activity and drug resistance. Additionally re- cent NMR relaxation results show that the natural polymor- The University of Arizona phisms also modify the protein backbone dynamics. The nitrophorins (NP) are 8-stranded β-barrel ferriheme pro- We are also investigating how specific single and double natu- teins (lipocalin fold) from the saliva of the blood-sucking bug, ral polymorphisms induce altered flap conformation sampling Rhodnius prolixus, which bind and sequester NO in the insect as seen in other subtypes, which may impact divergent path- salivary gland for up to a month, and release it into the tis- ways of drug resistance. An examination and comparison sues and capillaries of a victim, when found. Lipocalins are of deposited X-ray structures of HIV-1PR variants implies generally known to be fairly rigid proteins, yet the ligands that altered salt-bridge formation of select substitutions may they sequester are able to bind and to be releasedat biolog- account for pulsed EPR results of CRF_01 A/E, which con- ically relevant rates . There are two pairs of NPs with high sistently reveal a novel flap conformer that is more curled sequence identity, NP1 and NP4 (90%) and NP2 and NP3 compare to subtype B. This curled conformation is observed (79%); the overall sequence identity is 39%. All four pro- in many of our pulsed EPR distance profiles, but has not teins release NO slowly at the low pH of the salivary glands been observed in MD simulations of subtype B. We believe (koff = 0.02-0.03 s-1 at pH 5.0, but NP1 and NP4 release the altered salt bridge provides a structural basis for the EPR NO much more rapidly at the higher pH of human tissues observations. (7.3-7.4) (koff = 1.1 and 1.6 s-1, respectively), while NP2 shows a much smaller increase in release rate (koff = 0.093 s-1), and NP3 behaves similarly. The dynamics of the A-B OP075: NMR Spectroscopy reveals alternative and G-H loops of these proteins are believed to be responsible Ground States in Copper Proteins for the differences in the two pairs of proteins, yet the G-H ∗Alejandro J. Vila, Luciano A. Abriata, Andrés Espinoza- loops are identical in length for all four (7 residues), while the Cara, Marcos N. Morgada, María-Eugenia Zaballa A-B loops of NP1,4 are one amino acid longer than those of NP2,3 (11 vs. 10 residues). We are investigating the picosec- Institute for Molecular and Celular Biology (IBR), Univer- ond to nanosecond and microsecond to millisecond dynamics sity of Rosario, Argentina of these proteins by NMR techniques. Analysis of the chemi- NMR of oxidized copper proteins has been hampered by the cal shifts of A-B loop residues of NP2 at pH 5.0 suggests that unfavorable electron relaxation times of the Cu2+ ion which the A-B loop is partially helical (and closed), while the loop induce fast relaxation rates in nearby nuclei, rendering them has an extended structure at pH 7.3. Dynamic motions of in- undetectable. However, this limitation applies only to T2 dividual A-B loop residues at pH 7.3 are much greater than copper sites, while T1, T3 and CuA centers are amenable at pH 6.5 or 5.0, but in no case are the motions correlated.

42 Abstracts of Talks – Workshop: NMR on Porous Media

Little dynamics are seen for the G-H loop at any pH, and net pay as a result of the high complexities in the rock and there appears to be no pH-dependent change in that loop’s fluids properties. Such uncertainty in the nest pay estimates conformation. Thus the behavior of the A-B loop is of most can cause huge errors in calculating the oil and gas reserves interest as we investigate NP2 in the NO-off form (high-spin of a reservoir. Fe(III)) and compare these results to those for NP4. This presentation will discuss the effectiveness of applying the ACKNOWLEDGEMENTS: The support of the U. S. Na- MRI logging technology in measuring and estimating critical tional Institutes of Health grant HL054826 is gratefully ac- petrophysical parameters which helps in better characteriz- knowledged. ing the oil and gas reservoirs and provide improved net pay hydrocarbon volume and better reserves estimates. It also OP077: EPR Method Development for Addressing demonstrates the value of using the MRI technology in deter- Protein Structure and Dynamics mining fluid types, including distinguishing between bound water and free water, as well as differentiating between gas 1∗ 1 1 Gunnar Jeschke, Yevhen Polyhach, Tona von Hagens, and liquid hydrocarbon and even detecting differences be- 2 2 Carsten Dietz, Harald Paulsen tween heavy and light oil. This article also illustrates the 1ETH Zurich, 2University of Mainz latest interpretation techniques and applications of the MRI technology in the oil and gas industry. Distance distribution measurements in the nanometer range by pulsed EPR techniques have recently become popular in structural biology, mainly for their applicability to proteins OP079: Transport and Reaction in Porous Media: of any size in almost any environment. Furthermore, access New Information Obtained Using Compressed Sens- to distance distribution rather than only mean distances pro- ing and Bayesian Methods vides valuable additional information on disordered parts of ∗Lynn F. Gladden, D. J. Holland, Mick D. Mantle, J. a structure. Due to the matching of the accessible distance Mitchell, Andrew J. Sederman, A. B. Tayler range between about 2 and 8 nm with typical protein dimen- sions coarse information on structure and structural changes University of Cambridge, Department of Chemical Engineer- can be obtained from a small number of measured distances. ing and Biotechnology This contribution assesses the potential and limitations of Understanding transport processes in porous media, with and such methodology on the example of the major light harvest- without simultaneous reaction, is central to many areas of ing complex LHCII of green plants. In particular, we dis- the process industries, the pharmaceutical industry and, of cuss site-resolved information folding of the protein and self- course, oil recovery. This presentation will give an overview assembly of the complex with chromophores and structural of the opportunities offered by implementation of compressed information on the disordered part of the N-terminal domain. sensing and Bayesian magnetic resonance (MR) techniques, By combining the enhanced sensitivity of our high-power Q- with illustrations taken from the fields of gas-liquid flows band setup with a double-tag LHCII purification strategy in porous media (i.e., catalytic reactors), gas-liquid bubbly developed by our collaboration partners in Mainz our groups flows, and the structural characterisation of porous media. have been able to measure distance distributions in a single Both compressed sensing and Bayesian methods exploit our protomer of the LHCII trimer. Finally, we address artifacts ability to undersample the data acquisition but still recover in distance distributions that may arise from the presence of the information of interest. This opens up opportunities for more than two spin labels in a nanoobject, a situation that is acquiring data at much higher spatial and temporal resolu- commonly encountered for protein oligomers. We show that tions. It follows that new measurements may now also be simple power scaling of time-domain data can reveal and sup- implemented on low magnetic field hardware. press such artifacts. ACKNOWLEDGEMENTS: We thank Christoph Dockter and Andre Mueller (University of Mainz, A first example shows how compressed sensing MR acquisi- Germany) for providing further samples of LHCII, Alexander tions have been implemented to enable gas and liquid velocity Volkov (MPI for Polymer Research Mainz) for further mea- maps of the gas-liquid flow fields within packings of particles, surements, Adelheid Godt and Muhammad Sajid (Bielefeld such that both flow fields are acquired at the same spatial University) for providing model compounds, and Enrica Bor- resolution. These data provide new information which can dignon and Rene Tschaggelar for contributions to the high- be incorporated into numerical flow simulation codes used to power Q-band setup. predict reactor behavior. A second example uses a Bayesian MR methodology to size gas bubbles in gas-liquid bubbly flows. This approach is sensitive to changes in bubble shape nd Wednesday – May 22 and can measure bubble sizes at high gas voidages where opti- Workshop: NMR on Porous Media cal techniques cannot be used. Compressed sensing combined Chair: Vinicius Machado with spiral imaging has also been used to image 3D vector Rooms Turmalina/Topázio flow fields, with an acquisition time of 16 ms, capturing es- sentially ’real time’ images of the flow field around bubbles as they rise within stagnant water. OP078: Improved Estimation of Oil and Gas Re- serves Using Magnetic Resonance Imaging “MRI” Finally, we demonstrate two low field measurements which Technology - Applications and Case Histories employ the Bayesian approach: the sizing of spherical parti- ∗ cles using an Earth’s Field NMR apparatus, and the deter- Maged Fam mination of grain sizes in rocks at 1T. HALLIBURTON, Bogota, Colombia ACKNOWLEDGEMENTS: EPSRC, ExxonMobil, Johnson With the continuing increase in demand for oil and gas as Matthey, Microsoft Research Connections a vital component of the world’s supply of energy, it is im- portant to be able to account for every small quantity of UP080: Pore Sizes Distribution of Unconsolidated reserve through accurate determination of net pay hydrocar- Geomaterials bon volume for each well drilled. The initial estimates of hydrocarbon quantity in a reservoir are usually established 2,3∗Marcos Montoro, 1,3Lucas C. Cerioni, 3Daniel José through formation evaluation using conventional wireline or Pusiol LWD logs. Those conventional logs, such as resistivity, den- 1Spinlock SRL, 2Universidad Nacional de Córdoba, sity, neutron, and sonic, are traditionally used to estimates 3CONICET important reservoir parameters such as porosity, permeabil- ity, water saturation and net hydrocarbon volume (or net Pore size and pore sizes distribution of soils and rocks control pay). Sometimes, however, the use of such conventional logs many hydraulic and mechanical properties of natural geoma- may provide a high degree of uncertainty in the estimation of terials such as hydraulic conductivity and shear resistance.

43 Abstracts of Talks – Small Molecules and Methods

Pore size and sizes distribution of rocks depend on their structual parameters that allow the conformational, config- mineral composition and the geological processes involved urational, and constitutional analysis of small molecules [1]. in their formation, while for unconsolidated materials such If chiral alignment media are used, even the distinction of as soils, depend on grain sizes, grain sizes distribution and enantiomers is possible with the approach. overburden pressure. The aim of this project is to corre- late transversal relaxation time (T2) with grain size of loose Novel technical developments, like the introduction of use- sandy soils in systems of single and double porosity. In this ful alignment media, imaging of alignment, useful pulse se- research we applied Time Domain Nuclear Magnetic Reso- quences, and methods for data interpretation will be pre- nance (TD-NMR) to measure the magnetization decaying sented. A special focus will be the identification of meso curve of samples composed of selected sizes of sand grains, compounds compared to a racemic mixture of corresponding poor graded sands, silty sands, silt and clay. This was per- enantiomers, that cannot be distinguished by optical means. formed by a CPMG pulse sequence using a low field (12 [1] for a review see e.g.: G. Kummerlöwe, B. Luy, Annu. Rep. MHz) spectrometer (SLK-100). Obtained results show that NMR Spectrosc. 68, 193-232 (2009). transversal relaxation time and its distribution are strongly correlated with grain sizes and their distribution. These results allow improving the knowledge about pore sizes dis- OP083: New NMR strategies for the accurate mea- tribution when needed for modeling porous materials using surement of small heteronuclear coupling constants numerical techniques such as pore networks. ∗Teodor Parella ACKNOLEDGEMENTS: CONICET, SECyT-UNC, Servei de Ressonància Magnètica Nuclear, Universitat FONTAR, FONCYT Autònoma de Barcelona, Catalonia

OP081: Diffusion dynamics in porous media Several pulse schemes will be presented for the measurement of heteronuclear coupling constants in small molecules. Im- ∗Yi-Qiao Song portant aspects such as the simplicity and the accuracy of the Schlumberger measurement, its general applicability, the extraction of mul- tiple information in a single NMR experiment or the simulta- Porous media is known to greatly affect the behavior of neous determination of the J sign will be discussed. It will be NMR properties of the fluids inside. Interactions between shown how three different J-editing methods (IPAP, E.COSY the solid surface and the spins have been the key source of and J-resolved) can be implemented in a single NMR experi- enhanced spin relaxation inside the porous media. Diffusion ment to provide spin-state-edited 2D cross-peaks from which of a molecule is also known to be affected compared either simultaneous measurement of different homonuclear and het- due to interaction with other molecules or the surrounding eronuclear coupling constants can be performed. In addition, solid structures. Both effects have been explored to study the the interference of homonuclear J(HH) evolution and its ef- porous structure in order to understand the fluid flow. These fect on distorted cross-peaks will be evaluated in broadband effects are in general determine the average surface-to-volume versions of the HMBC-IPAP and HSQMBC-IPAP experi- ratio (SVR) of porous materials. ments. Examples on the application of the proposed methods In addition to reduced diffusion constant, the diffusion prop- to other heteronuclei will be also provided. agator can be significantly different from bulk diffusion such ACKNOWLEDGEMENTS: Financial support for this re- as its Gaussian form. This behavior is reflected in diffusion search provided by MICINN (project CTQ2009-08328) is behavior (such as kurtosis) as well as relaxation. For exam- gratefully acknowledged. We also thank to the Servei de ple, slow diffusion could cause multiple relaxation peaks in Ressonància Magnètica Nuclear, Universitat Autònoma de NMR relaxation spectrum for a single pore size. However, Barcelona, for allocating instrument time to this project. in real porous materials, pore size distribution also results in multiple or broad relaxation times. As a result, it is often dif- ficult to differentiate the intrinsic complex diffusion behavior UP084: Experimental measurement and theoretical from the presence of pore size distributions. assessment of fast lanthanide electronic relaxation in solution with four series of isostructural complexes We have studies several two-dimensional NMR methods in or- der to distinguish the presence of complex diffusion and the 1Alexander M. Funk, 1∗David Parker, 1Peter Harvey, pore size distributions. For example, we have found unique 1Alan M. Kenwright, 2Pascal H. Fries features in the T1-T2 correlation experiment that unambigu- 1Durham University, 2CEA, Grenoble ously identify the slow diffusion behavior irrespective of the pore sizes. Also we design a 3-pulse-gradient echo (a version The rates of longitudinal relaxation for ligand nuclei in four of the Double-Pulsed-Field-Gradient) experiment that could isostructural series of lanthanide(III) complexes have been quantify the non-Gaussian diffusion behavior (such as kurto- measured by solution-state NMR at 295 K at five magnetic sis). Using analytical and numerical solutions of the diffusion fields in the range 4.7 to 16.5 T. The electronic relaxation dynamics and numerical simulation we have demonstrated time T1e is a function of both the lanthanide ion and the these effects and found them consistent with experimental local ligand field. It needs to be considered when devising re- results. These new methods allow the investigation of porous laxation probes for magnetic resonance applications since it media to extend beyond the language of average SVR. affects the nuclear relaxation, especially over the field range 0.5 to 4.7 T. Analysis of the data, based on Bloch-Redfield- Wangsness theory describing the paramagnetic enhancement nd Wednesday – May 22 of the nuclear relaxation rate, has allowed reliable estimates Small Molecules and Methods of electronic relaxation times, T1e, to be obtained using iter- Chair: Jochen Junker ative minimization methods. Values were found in the range Room Ônix 0.10 to 0.63 ps, consistent with fluctuations in the transient ligand field induced by solvent collision. A refined theoretical model for lanthanide electronic relaxation beyond the Red- OP082: Structure Determination Using Anisotropic field approximation is introduced, that accounts for the mag- NMR Parameters nitude of the ligand field coefficients of order 2, 4, and 6 and ∗Burkhard Luy their relative contributions to the rate 1 / T1e. Despite the considerable variation of these contributions with the nature Karlsruhe Institute of Technology of the lanthanide ion and its fluctuating ligand field, the the- Anisotropic NMR parameters like residual dipolar couplings ory explains the modest change of measured T1e values and (RDCs), residual quadrupolar couplings (RQCs), and resid- their remarkable statistical ordering across the lanthanide se- ual chemical shift anisotropies (RCSAs) are very powerful ries. Both experiment and theory indicate that complexes

44 Abstracts of Talks – Challenging Systems of terbium and dysprosium should most efficiently promote terconversion between ensemble members have be measured paramagnetic enhancement of the rate of nuclear relaxation. by low temperature relaxation dispersion, dielectric relax- ation [3] and temperature jump SAXS and compared to MD References: results [4]. In addition correlated motion [5] across b-strands [1] Funk, A. M.; Fries, P. H.; Harvey, P.; Kenwright, A. M.; is characterized by novel optimized cross correlated relax- Parker, D., J. Phys. Chem. A, 2013. accepted ation experiments. Further, with high-power relaxation dis- persion measurements [6] it is possible to characterize motion [2] Walton, J. W.; Carr, R.; Evans, N. H.; Funk, A. M.; Ken- kinetically to one digit ms time scales. With these measure- wright, A. M.; Parker, D.; Yufit, D. S.; Botta, M.; De Pinto, ments motion on the time scale of 10 ms at 35◦C is detected S.; Wong, K. L., Inorg. Chem. 2012, 51 (15), 8042. uniformly in the backbone (detected on the amides) and side chains (detected on methyl groups) of ubiquitin. The ampli- [3] Harvey, P.; Kuprov, I.; Parker, D., Eur. J. Inorg. Chem. tude of the relaxation dispersion can be reproduced assuming 2012, 12, 2015-2022. a backbone conformation dependent variance in the popula- tion of the side chain rotatmers. Since the backbone con- OP085: Structural Analysis of Small Organic formational changes occur on the 10 ms time scale, also the Molecules Assisted by Residual Dipolar Couplings side chain rotamer redistribution occurs on this time scale. Finally, progress made towards the fitting of the four state ∗Roberto R. Gil recognition model of Dsk2-UBA with ubiquitin will be dis- Carnegie Mellon University cussed. The 2D structure of most small molecules can be in principle ACKNOLEDGEMENTS: Max Planck Society, ERC grant straightforwardly determined by manual or automatic analy- agreement number 233227 sis of a set of experimental data that includes the molecular formula, a series of 1D and 2D NMR experiments providing [1] Peti, W., Meiler, J., Brüschweiler, R. and Griesinger ,C. trough-bond connectivity (COSY, TOCSY, HSQC, HMBC (2002) Model free Analysis of Protein Backbone Motion from and ADEQUATE/INADEQUATE based experiments), and Residual Dipolar Couplings. J. Am. Chem. Soc. 124, 5822- chemical shift predictions. This is the main concept embed- 5833 ded in automatic structure elucidation programs. Once the 2D structure is available, the determination of the relative [2] Lange, O., Lakomek, N. A., Farès, C., Schröder, spatial arrangement (configuration and preferred conforma- G.,Becker, S., Meiler, J., Grubmüller, H., Griesinger, C., tion) of all atoms in the molecule is a more challenging task de Groot, B.: Recognition dynamics up to microseconds re- that it is commonly addressed in NMR by using NOE and vealed from an RDC-derived ubiquitin ensemble in solution. 3J coupling constants analysis, as well as recent develop- Science, 2008, 320, 1471-1475 13 ments on the application of DFT calculation of C chemical [3] Ban, D., M.F. Funk, R. Gulich, D. Egger, T. M. Sabo, shifts. However, it is difficult to assess how many samples are K.F.A. Walter, R. B. Fenwick, K. Giller, F, Pichierri, B.L. sitting on the laboratory’s refrigerators waiting for an inde- de Groot, O. F. Lange, H. Grubmüller, X. Salvatella, M. pendent methodology that could lift some of the ambiguities Wolf, A. Loidl, R. Kree, S. Becker, N.-A. Lakomek, D. Lee, generated by the use of conventional NMR methods. The de- P. Lunkenheimer, C. Griesinger: Kinetics of Conformational velopment of the application of Residual Dipolar Couplings Sampling in Ubiquitin. Angew. Chem. Int. Ed. 50, 11437- (RDCs) to the configurational and conformational analysis 11440 (2011) of small molecules has matured enough in the recent years to perform this task is an almost straightforward way, with- [4] Shaw, David E.; Maragakis, Paul; Lindorff-Larsen, out even the need of using NOE and 3J coupling analysis, as Kresten; Piana, Stefano; Dror, Ron O.; Eastwood, Michael it will be presented here for the analysis of rigid and semi- P.; Bank, Joseph A.; Jumper, John M.; Salmon, John K.; rigid small molecules. Most of the results presented here Shan, Yibing; Wriggers, Willy. Atomic-Level Characteriza- use Poly(methylmethacrylate) (PMMA) based flexible gels, tion of the Structural Dynamics of Proteins. Science 330, whose degree of alignment can be easily tuned by variable 341-346 (2010) and reversible compression. The application of RDCs to the [5] Fenwick, R.B., Esteban-Martin, S ., Richter, B ., Lee, D ., analysis of flexible molecules is still a field of research plenty Walter, K.F.A., Milovanovic, D. Becker, S., Lakomek, N.A., of room for development and a generally accepted approach Griesinger, C ., Salvatella, X.: Weak Long-Range Correlated is not available yet. Motions in a Surface Patch of Ubiquitin Involved in Molecu- lar Recognition. Journal of the American Chemical Society Wednesday – May 22nd 133, 10336-10339 (2011) Challenging Systems [6] Ban, David, A. D. Gossert, K. Giller, S. Becker, C. Chair: Roberto Salinas Griesinger and D. Lee: Exceeding the limit of dynamics stud- Room Gávea A ies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead. Journal of Magnetic Res- OP086: Protein Dynamics: Its Kinetics and Impli- onance, 221, 1- 4 (2012) cations for Function 1 1,2 1 3 OP087: Distance measurements in biomolecules us- David Ban, Colin Smith, Michael Sabo, R. Bryn Fen- ing Gd3+ spin labels wick, 1Korvin F. A. Walter, 1Claudia Schwiegk, 1Stefan Becker, 3Xavier Salvatella, 1Berend L. de Groot, 1Donghan ∗Daniella Goldfarb 1∗ Lee, Christian Griesinger Weizmann Institute of Science, Rehovot, Israel 1Max Planck Institute for Biophysical Chemistry - NMR Methods for measuring nanometer scale distances between based Structural Biology, 2Max Planck Institute for Bio- specific sites in biomolecules (proteins and nucleic acids) and physical Chemistry - Theoretical and computational bio- their complexes are essential for analysis of their structure physics, 3ICREA and Institute for Research in Biomedicine and function. In the last decade pulse EPR techniques, Barcelona mainly pulse double-electron-electron resonance (DEER), has The possibility to explore dynamics of proteins specifically been shown to be a very effective for measuring distances be- ubiquitin which promiscuously recognizes many binding part- tween two spin labels attached to a biomolecule. DEER is ners will be presented based on the accurate measurement of routine for distances up to 5 nm and with some extra effort anisotropic parameters such as residual dipolar couplings [1]. and favorable conditions distances as high as 8 nm can be ac- This approach allows to characterize at unprecendented de- cessed. So far such measurements have been applied mostly tail the ground state ensemble of the protein [2]. Rates of in- to biomolecules labeled with nitroxide stable radicals. Re-

45 Abstracts of Talks – Metabolomics and Chemometrics cently we have introduced a new family of spin labels that has been involved in the onset of Parkinson’s disease (PD) are based on Gd3+ chelates attached to proteins using site through the conversion of its native monomeric state into directed spin labeling, similar to the common method of la- beta-sheet rich amyloid fibrils in dopaminergic neurons [1]. beling proteins with nitroxide spin labels. Gd3+ spin labels So far, most of the structural studies aimed to understand are particularly attractive for high field DEER measurements this conversion were conducted on isolated protein samples, such as W-band (95 GHz, 3.5 T). The benefits such S=7/2 under conditions that differ substantially from the crowded spin labels in combination with high field offer are the high in vivo environments of intact cells. The question remains sensitivity that reduces the amount of the biomolecule needed whether the features observed for AS in vitro correlate with by more than an order of magnitude and the lack of orien- its cellular behaviour. Until recently, there were no means of tation selection that allows straight forward data analysis. looking into live cells with high enough resolution to address Here we will present Gd3+- Gd3+ distance measurements such important unresolved issues. The development of In-cell in models compounds and their applications to study trans- NMR spectroscopy techniques changed this notion [2]. Here, membrane peptides in model membranes. The chemical and we present high-resolution In-cell NMR data on the structural spin physics requirement for the ultimate Gd3+ label will be and dynamic properties of AS in five different mammalian cell discussed. lines that also include dopaminergic neurons of the Substan- tia nigra. By using a novel approach to efficiently deliver iso- UP088: Detection of light induced intermediates of topically enriched protein samples into the cytosol of cultured photoreceptor membrane proteins by in-situ photo- mammalian cells we were able to record highly reproducible irradiated solid-state NMR In-cell heteronuclear NMR spectra. Moreover, residue spe- cific dynamic information from relaxation experiments was 1∗ 1 1 1 Akira Naito, Yuya Tomonaga, Tetsuro Hidaka, Hiroki obtained. By directly comparing these in-cell NMR results 1 1 2 Yomoda, Teruki Makino, Izuru Kawamura, Yuki Sudo, with AS data from different in vitro environments mimicking 3 3 4 Akimori Wada, Takashi Okitsu, Naoki Kamo intracellular viscosity and macromolecular crowding, we are 1Yokohama National University, 2Nagoya University, 3Kobe in the process of delineating physical and biological contribu- Pharmaceutical University, 4Matsuyama University tions to AS’s different in vivo behaviours. Results emerging from this work contribute to the understanding of the native Photoreceptor retinal proteins usually absorb photon to conformations of AS and lays the ground to further perform generate photo-isomerization, and consequently change the high-resolution in situ investigations under conditions that structure and dynamics of proteins. To generate retinal iso- lead to intracellular aggregation and neurodegeneration as merization, we have developed the photo-irradiation system observed in Parkinson’s disease. equipped to the solid-state NMR spectrometer [1]. Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a [1] Lashuel, H. A. et al (2012) Nat. Rev. Neurosci. 14, 38-48 negative phototaxis receptor of Natronomonus pharaonis and forms a 2:2 complex with the cognate transducer (pHtrII), [2] Ito, Y., and Selenko, P. (2010) Curr. Opin. Struct. Biol. which transmits the photosignal into cytoplasm. Light ab- 20, 640-648. sorption of ppR initiates trans-cis photo-isomerization of the retinal chromophore followed by cyclic chemical reaction con- OP090: NMR studies of the structure and dynamics sisting of several intermediates (K, L, M and O). The M in- of protein-RNA interactions in gene regulation termediate is thought to be an active state for signal trans- duction. 1,2∗Michael Sattler We have successfully trapped and observed the M interme- 1Institute of Structural Biology, Helmholtz Zentrum diate by using newly developed photo-irradiated solid-state München, Germany, 2Munich Center for Integrated Pro- NMR system. 13C NMR signal from [20-13C] retinal-ppR and tein Science and Biomolecular NMR, Department Chemie, ppR/pHtrII revealed that multiple M-intermediates (M1, M2 TUM, Germany and M3) with 13-cis, 15-anti retinal configurations coexisted We are studying the structures, dynamics and molecular in- under the continuously photo-irradiated condition [1]. Fur- teractions of multi-domain proteins and protein complexes ther, since the life time of M1 state was much longer than involved in the regulation of splicing, and the modulation of those of the other M states, this M1 state could be distin- these interactions by posttranslational modifications. Early guished from the other M states and assigned as N interme- steps during the assembly of the spliceosome involve the diate. binding of SF1 and the U2AF heterodimer to the 3’ region of SrSRI (Salinibacter ruber sensory rhodopsin I) is a eubac- the pre-mRNA. We have recently shown that recognition of terium rhodopsin and acting multiple function as attractant the poly-pyrimidine-tract RNA at the 3’ splice site involves and repellent phototaxis. In the photocycle of SrSRI, M in- a conformational shifts between closed and open arrange- termediate functions as attractant and P intermediate by ab- ments of the tandem RRM domains of U2AF65 (Mackereth sorbing second blue light as a double photon process shows et al, Nature 2011) Currently, we are exploring the poten- as repellement. 13C NMR signal of M intermediate in SrSRI tial contributions of conformational selection and induced fit was successfully trapped by illuminating 520 nm light, and mechanisms for these interactions. We also investigate the the configuration of retinal was revealed to be 13-cis, 15-anti. role of tandem serine phosphorylation for the molecular in- P intermediate was trapped by illuminating second 365 nm teractions of SF1 and U2AF and present structural analysis light as a double photon process. of the SF1-U2AF65 complex.

Photo-intermediate in photo-receptor membrane proteins is For these studies we employ an efficient protocol for deter- now possible to trap by using in-situ photo-irradiated solid- mining the quaternary structure of multi-domain proteins state NMR. and complexes in solution by combining experimental data [1] Y. Tomonaga et al. Biophys. J. 2011, 101, L50-L52. derived from solution state NMR as well as Small Angle X- ray and/or Neutron Scattering (SAXS/SANS) experiments. Starting from available structures of individual domains or UP089: High resolution conformational description complex subunits, chemical shift perturbations and NOEs of Alpha-synuclein inside neurons using mammalian are used to define domain interfaces. Overall domain ar- In-cell NMR rangements are defined by long-range distance restraints ob- Andres Binolfi, Beata Bekei, Francois-Xavier Theillet, tained from paramagnetic relaxation enhancements (PRE) Honor M. Rose, ∗Philipp Selenko using spin-labeled proteins and/or RNA or solvent PREs. Small Angle Scattering data provide complementary infor- Leibniz Institut für Molekulare Pharmakologie (FMP-Berlin) mation to validate the NMR derived structures and provide The intrinsically disordered protein alpha-synuclein (AS) complementary information about the overall shapes

46 Abstracts of Talks – Metabolomics and Chemometrics

Wednesday – May 22nd Sugar cane spirit – The most consumed beverage in Brazil Metabolomics and Chemometrics after beer is sugar cane spirit, which is produced from the sugar cane juice fermentation. On the other hands there are Chair: Ana Gil other spirit products also from sugars fermentation (grape, Room Gávea B honey, banana and apple) which have higher values in the marked. Using 1H NMR, SNIF-NMR and chemometrics it OP091: Deciphering Cancer Metabolism for Drug was possible to discriminate the five types of spirits sold in Discovery Brazil.

∗Ulrich L. Günther, Christian Ludwig, Katarzyna ACKNOLEDGEMENTS: FAPESP, CAPES, FINEP and Koczula, Farhat Khanim, Chris Bunce CNPq University of Birmingham, UK UP093: Visualizing metabolic system catalyzed Metabolomics has gained significant ground in Biomedicine, by microbial ecosystem using statistical correlation including the field of cancer research. This is in part because analysis metabolomics observes a phenotypical end point, reflecting small changes in various pathways linked to metabolism. Ap- 1,2,3,4∗Jun Kikuchi plications range from typical diagnostics to drug discovery. 1RIKEN Plant Science Center, 2Biomass Engineering Pro- While the potential diagnostic value is increasingly accepted, gram, RIKEN, 3Nagoya University, 4Yokohama City Uni- the use of metabolomics for understanding the mechanisms versity of drug actions has been realized only more recently. Such developments are very closely linked to the fact that the as- Within the field of metabonomics, measurement of system pect of altered metabolism in cancer has been rediscovered. responses over time is usually important to obtain a picture Although known since Otto Warburg’s research in the 1920s, of metabolic dynamics after or during the application of some metabolism has been neglected for a ling time. input to an environment, such as foods in animal guts, be- cause the variations in each metabolite have intrinsic behav- Now we find altered metabolism in different places of iors. We have previously developed an approach that applies metabolic processing, and we learn how metabolism is af- metabonomics to monitor metabolic dynamics in microbial- fected by drugs. This has been studied in AML and CML animal, as well as microbial-microbial interactions in the gut (acute and chronic myeloid leukemia) cells, where we ob- system [1-3]. In addition to this, the composition of the gut serve unforeseen changes in metabolism following a new drug microbiome is highly variable, and its diversity can be sig- combination. A pronounced metabolic effect arises from the nificantly affected by alterations in diet. Here firstly, we re- generation of high levels of reactive oxygen species, which port an approach to evaluate the intestinal variation and to chemically modify several metabolites. predict metabolic pathways of major microbial symbionts af- fected by the variation of plant polysaccharide intakes. The For this metabolic analysis we combined metabolomics with covariance of structural variation in gut microbiome and host isotopic tracer based metabolic flux analysis, using 13C- metabolism was visualized based on the correlation with the labelled metabolic precursors. In this context NMR has a sig- denaturing gradient gel electrophoresis (DGGE) profiles and nificant advantage as it can observe site-specific label incor- the urine/feces NMR profiles [4]. Thus, time-dependent vari- poration, revealing unforeseen effects, in particular in Krebs’ ations and timed responses should be considered and applied cycle metabolites. in the development of a visualization method when evaluating and characterizing metabolic dynamics in microbial ecosys- OP092: Food NMR applied to typical Brazilian food tems. Similar approach can be applied for not only in the products gut system, but also in industrial process, such as changes ∗Antonio Gilberto Ferreira in metabolic dynamics of methane fermentation sludge upon different polysaccharide addition [5]. Our approach provides Federal University of São Carlos - Chemistry Department a foundation for evaluation of systemic effects of inputting Brazil is undoubtedly one of the major producers and ex- nutrients that are of relevance to both heath and green in- porters of primary products such as iron ore, soybeans, coffee, novation for opening up a new window that will clear up concentrated orange juice and in less extent exotic products metabolic dynamics in the complex microbial communities such as sugar cane spirit, honey and fruit pulp. from the gut to industrial process environments. With the purpose to have better knowledge of the chemi- References cal constitution in different matrix, NMR was used to iden- 1. Fukuda et al., Nature 469, 543-547 (2011). tify/characterize the majority chemical compounds and also to draw a chemical profile considering variety, seasonality and 2. Fukuda et al., Nature (revised). regionality in different products analyzed using chemometrics 3. Nakanish et al., J. Proteome Res. 10, 824-831 (2011). tools. The technique SNIF- NMR (Site Specific Natural Iso- topic Fractionation studied by Nuclear Magnetic Resonance) 4. Date, Y. et al., PLoS ONE (submitted). has also been used to quantify the 1H/2H isotopic relation. 5. Date, Y. et al., J. Proteome Res. 11, 5602-5610 (2012). Orange Juice – Although Brazil is the largest exporter of concentrated orange juice, domestically this form of drink is UP094: The Use of Magnetic Resonance in Pollution not consumed on a large scale being preferred fresh squish Assessment: Theory and Applications orange juice. With the objective to evaluate the products ∗ degradations, five different species/varieties of orange were Oliver A.H. Jones studied considering the effect of temperature and storage RMIT University time. Using 1D and 2D NMR spectra was possible to charac- terize the compounds responsible for the juice modification. Nuclear Magnetic Resonance (NMR) has great potential for use in the both the measurement of toxicants and the as- Honey – Brazil is not a major honey producer when com- sessment of their effects on biological organisms, including pared to other countries but there is a way to fake the product humans. In this presentation we discuss the reasons why sold in local shops which is difficult to detect. It is possible to NMR is useful for this type of research and present a number feeding bees with syrups of different origins. Using honey of of applications which use NMR based metabolomics (a newly Apis mellifera collected from orange, eucalyptus, sugar cane emerging field of research concerned with the comprehensive plantations and wild trees, in São Paulo state, it was pos- characterization of the small molecule metabolites in biologi- sible to distinguish four types of honey using 1H NMR and cal systems) to study the effects of both organic and inorganic SNIF-NMR. pollutants on a range of terrestrial and aquatic species. We

47 Abstracts of Talks – Workshop: NMR on Porous Media

also introduce community metabolomics - the use of the study field and large B1 surface loops to obtain non-invasive mea- of biochemical profiles of whole microbial communities living surements of groundwater. This technology shares many in contaminated soils from various sites with very different commonalities with other NMR techniques in porous media, physicochemical characteristics (levels of metal contamina- including the valuable potential to determine critical flow and tion, underlying geology and soil type) using NMR. While storage parameters. There are also certain unique aspects of some of these site differences may also have been caused by the measurement configuration and shallow Earth environ- additional abiotic factors (such as soil type or pH), pattern ment that pose distinct challenges requiring innovative solu- recognition analysis of the data showed that both site- and tions. Two challenges that have been the focus of research contaminant- specific effects on the metabolic profiles could and progress in recent years are obtaining accurate resolution be discerned and individual sites could be resolved on the of subsurface relaxation times and estimating permeability. basis of their individual metabolic profiles. This is the first Resolving relaxation times in surface NMR is complicated by time whole microbial communities from environmental sam- the fact that B1 is grossly inhomogeneous within the resonant ples have been studied as opposed to lab cultures of single volume and pulses are relatively long. Previous approaches isolates. The work is of importance since the identification of have preferred simple single-pulse FID measurement schemes, biomarkers indicative of a defined response to a pollutant, or with variable pulse amplitudes, to resolve water content and pollutants, before major outward changes become apparent, T2* with depth. Recognizing that the T2* response can be could potentially have applications in a range of a number dominated by magnetic mineralogy, we have developed im- of areas including soil chemistry, contaminated land assess- proved acquisition schemes to resolve relaxation parameters ment and re-mediation, the water industry, agriculture and more sensitive to permeability: T1 (using double-pulse se- ecology. quences), T2 (using CPMG-based sequences), and T1-T2* distributions (using two-dimensional sequences). Meanwhile, OP095: Rapid-dissolution 13C DNP in kinetics of cel- appreciating the advantage of FID measurements to detect lular transmembrane exchange the shortest signal components, we have sought a better un- derstanding of the Earth’s field T2* response of porous media, 1∗ 2 1 Philip W. Kuchel, Guilhem Pagès, Max Puckeridge and have outlined novel approaches to transform FID data 1University of Sydney, 2Singapore Bioimaging Consortium into quasi-T2 distributions. Provided improved determina- tion of relaxation times, challenges remain to estimate per- Rapid-dissolution dynamic nuclear polarization (RD-DNP) meability in groundwater investigations, since shallow Earth with 13C NMR decreases the time in which metabolic events 13 materials differ significantly from reservoir rocks widely stud- can be studied with C-labelled metabolites[1,2,3]. Empha- ied in oilfield NMR. We have demonstrated that coarse, un- sis has been given to enzyme catalyzed reactions in patholog- consolidated aquifer materials can be strongly influenced by ical states of cells, and in a small number of clinical trials for slow-diffusion and bulk-fluid relaxation dynamics, and have the detection of neoplasms. The range of rapid biochemical developed refined models to account for these differences. reactions that are amenable to study by RD-DNP is limited. Further, by complementing surface NMR with newly avail- in vivo New NMR-receptive isotopes are being sought, as are able shallow logging NMR measurements, we have designed probe molecules carrying hyperpolarizable nuclei. However, novel workflows for calibrating permeability models and map- an area unexplored by RD-DNP is membrane transport of ping aquifer properties over wide areas. solutes. Some solutes undergo transmembrane exchange on the 1-second time scale and potential experimental and clin- ical perturbations of the rates are "legion". Some molecules OP097: Magnetic Resonance Imaging of Fluids in yield 13C NMR chemical shifts that differ inside and outside Porous Media cells, thus affording an NMR-based means of measuring their ∗Bruce J. Balcom transmembrane exchange. One such molecule is 13C-urea. We studied the kinetics of its exchange in human erythro- MRI Research Centre, Department of Physics, University of cytes. Mathematica was used to describe the exchange and New Brunswick, Canada NMR-relaxation kinetics. Magnetic resonance is well known in the petroleum research and development world through down hole logging and com- References: panion laboratory MR measurements of bulk fluid proper- [1] J.H. Ardenkjær-Larsen, B. Fridlund, A. Gram, G. Hans- ties. Magnetic Resonance Imaging is less well known, but son, L. Hansson, M.H. Lerche, R. Servin, M. Thaning, K. there is growing recognition that laboratory imaging studies, Golman, (2003) Proc. Nat. Acad. Sci.,100, 10158-10163. with core plug samples, will provide valuable new informa- tion on fluid properties and fluid behavior in the reservoir. [2] M. R. Clatworthy, M. I. Kettunen, D.-E. Hu, R. J. Math- Potential measurements of interest include both core analy- ews, T. H. Witney, B. W. C. Kennedy, S. E. Bohndiek, F. A. sis measurements and studies of model flooding procedures, Gallagher, L. B. Jarvis, K. G. C. Smith, and K. M. Brindle including studies of enhanced oil recovery mechanisms. (2012) Proc. Nat. Acad. Sci., 109 (2012) 13374-13379. This lecture will concentrate on recent MRI technique devel- [3] M. C. D. Tayler, I. Marco-Rius, M. I. Kettunen, K. M. opments, which permit MRI core analysis and model flooding Brindle, M. H. Levitt, and G. Pileio (2012) J. Am. Chem. studies to be undertaken, with an emphasis on techniques ap- Soc. 134, 7668-7671. propriate for samples that have short T2 behaviour. These [4] M. Puckeridge, G. Pagès, P. W. Kuchel (2012) J. Magn. techniques include the SPRITE and hybrid SE SPI methods Reson. 222, 68-73. for simple fluid density imaging and the SE SPI technique for spatially resolved T2 mapping. Translation of these methods nd to a new class of MR compatible metal core holders, for vari- Wednesday – May 22 able temperature and pressure studies, will be a major focus Workshop: NMR on Porous Media of the discussion. Chair: Vinicius Machado Rooms Turmalina/Topázio UP098: Single-scan T1-T2 relaxation correlation ex- periment OP096: Earth’s-field Surface NMR for Groundwater ∗ Characterization Susanna Ahola, Ville-Veikko Telkki University of Oulu 1,2∗Elliot Grunewald, 2Rosemary Knight, 1David Walsh, 2Denys Grombacher, 2Jan O. Walbrecker, 2Katherine Dlubac Power of traditional NMR spectroscopy relies on the versatile chemical information reflected by chemical shifts and cou- 1Vista Clara, Inc., 2Stanford University plings. Multidimensional experiments multiply the chemical Geophysical surface NMR measurements utilize Earth’s B0 resolution and information content, but they also multiply

48 Abstracts of Talks – Materials and Applications the experimental time, restricting the investigations of fast E.S. Ilton, A.R. Felmy, and K. M. Rosso, Langmuir, 28 (18), processes. Frydman et al introduced single-scan multidimen- 7125 – 7128, (2012) sional NMR as a solution for this issue [i]. It has been broadly [4] R.V.F. Turcu, D.W. Hoyt, K.M. Rosso, J.A. Sears, J.H. considered as one of the brightest recent innovations in NMR Loring, A.R. Felmy, J.Z. Hu, J. Magn. Reson, 20120, spectroscopy. http://dx.doi.org/10.1016/j.jmr.2012.08.009, Typically, NMR spectrum of fluids absorbed in materials re- veals little information; it may even consist of a single broad Acknowledgement peak. However, the relaxation time distribution may re- This research was supported by the Carbon Sequestration Ini- veal different environments of the fluid molecules, and con- tiative (CSI) funded by Laboratory Directed Research and sequently provide detailed information about the structure Development (LDRD) at Pacific Northwest National Labo- of the material. As in traditional NMR spectroscopy, resolu- ratory (PNNL) tion and information content of the relaxation time experi- ment can be increased by a multidimensional approach such OP100: Efficient measurement of T1/T2 ratio in as T1-T2 correlation experiment [ii]. A 2D Laplace inver- porous media sion is used to extract 2D relaxation time spectrum from the ∗ experimental data. Original T1-T2correlation experiment is Martin D. Hürlimann a 3D experiment, and, like in traditional multidimensional Schlumberger – Doll Research, USA NMR, the long experiment time restricts its applicability in the study of fast processes. The measurement of the T2 relaxation properties of fluid filled porous media has long been used a fast method for Inspired by Frydman’s single scan multidimensional NMR ap- the characterization of the geometry of the pore space. The proach, we introduce a novel method for single scan measure- distribution of relaxation times reflects the distribution of ment of T1-T2 relaxation correlation spectrum. The typical pore sizes in the sample. More detailed information can be experiment time is a few seconds, depending of the relaxation obtained from the measurement of the T1-T2 distribution time range in the sample. In many practical cases, the ex- function. In many cases, it is found that the T1 and T2 periment is fast enough for the study of dynamic systems. relaxation times are highly correlated, but interestingly, the We demonstrate that the method works by representing the ratio T1/T2 is often larger than unity, even at low fields. An 2D relaxation correlation spectra and showing that the re- increased ratio indicates the presence of slow motion. The sults are in agreement with the relaxation time distributions value of the T1/T2 ratio gives insight into the interaction measured by conventional methods. between fluid molecules and the pore surfaces or allows the [i] A Tal, L Frydman, Prog. Nucl. Mag. Res. Sp. 57 (2010) detection of molecular aggregates in the fluid, e.g. micelles 241-292 and other multi-molecular clusters. Here we discuss a new technique to determine the T1/T2 ratio very efficiently that is [ii] Y-Q Song, L Venkataramanan, MD Hürlimann, M Flaum, applicable even in strongly inhomogeneous field encountered P Frulla, C Straley, J. Magn. Reson. 154 (2002) 261-268. in well logging. The technique is based on a modified CPMG sequence where the refocusing pulses are split into two sep- UP099: High Pressure Magic Angle Spinning NMR arate pulses. In a single acquisition, the sequence generates Capability simultaneously a CPMG-like and a steady-state like signal 1,2∗ 1 1 that are easily separable in the time domain. The relative Flaviu R.V. Turcu, David W. Hoyt, Jesse A. Sears, T1 T2 1 1 1 amplitude of the two signals is directly related to the / John S. Loring, Kevin M. Rosso, Jian Z. Hu ratio. Experimental results confirm the theoretical predic- 1Pacific Northwest National Laboratory, 2"Babes-Bolyai" tions. The analysis of the new sequence shows an interesting University, Romania connection to the Hofstadter’s butterfly. Both theoretically and experimentally, it is shown that the signal power of the Over the last decades a large number of NMR methods have steady-state like signal is sensitive to whether the ratio of the been developed for addressing numerous biological, chemical two pulse spacings is a rational or irrational number. and physical problems across essentially all kinds of scientific disciplines. Magic Angle Spinning (MAS) technique is the most widespread NMR methods and is the only one, which Wednesday – May 22nd allows high resolution NMR spectrum, acquired on solids, Materials and Applications semi-solids, or a mixture thereof [1]. Chair: Hellmut Eckert High pressure and medium temperature magic angle spinning Room Ônix (MAS) NMR capability [2], composed of a novel high pres- sure MAS rotor, a high pressure loading/reaction device for OP101: Following Function in Real Time: New In in situ sealing and re-opening of the rotor’s valve is presented situ NMR Methods for Studying Structure and Dy- This high pressure MAS NMR capability is a powerful tool namics in Batteries and Supercapacitors for in situ structure and dynamic investigations in fields 1 1 1 1 1 Lina Zhou, Michal Leskes, Hao Wang, Alex Forse, John like geological carbon sequestration (GCS), subsurface mi- 2 1 1∗ Griffin, Nicole M. Trease, Elodie Salager, Clare. P. crobiology (1-4). As an example of application, in situ 13C Grey MAS NMR studies of the reaction products and intermedi- 1 ates associated with GCS [3] using different kind of minerals, Chemistry Department, Cambridge University, UK, 2 i.e., forsterite (Mg2SiO4), brucite (Mg(OH)2) and antigorite Chemistry Department, Stony Brook University, USA 2+ (Mg,Fe )3Si2O5(OH)4) reacted with wet scCO2 at various A full understanding of the operation of a chemical energy ◦ temperatures (50–80 C) are reported [4]. storage device such as a battery or supercapacitor requires References that we utilize methods that allow devices or materials to be probed while they are operating (i.e., in-situ). This al- [1] D.W. Hoyt, R.V.F.Turcu, J.A. Sears, K.M. Rosso, S.D. lows, for example, the transformations of the various cell Burton, J.H. Kwak, A.R. Felmy, J.Z. Hu, Prep-Am. Chem. components to be followed under realistic conditions with- Soc., Div. Fuel. Chem. 56 (1), 283, (2011). out having to disassemble and take apart the cell. To this end, the application of new in and ex-situ Nuclear Magnetic [2] D.W. Hoyt, R.V.F.Turcu, J.A. Sears, K.M. Rosso, S.D. Resonance (NMR) approaches to correlate structure and Burton, J.H. Kwak, A.R. Felmy, J.Z. Hu, J. Magn. Reson. dynamics with function in lithium-ion and supercapacitors 212, 378 – 385, (2012) will be described. The in-situ approach allows processes to [3] J.S. Loring, H.T. Schaef, R.V.F. Turcu, C.J. Thompson, be captured, which are very difficult to detect directly by Q.R.S. Miller, P.F. Martin, J.Z. Hu, D.W. Hoyt, O. Qafoku, ex-situ methods. For example, we can detect side reactions

49 Abstracts of Talks – Materials and Applications involving the electrolyte and the electrode materials, sorption convenient sampling for ex situ NMR investigation, for the processes at the electrolyte-electrode interface, and lithium study of methanol crossover and methanol reaction interme- ion dynamics. Ex-situ NMR investigations allow more de- diates in a DAFC with high resolution magic angle spinning tailed structural studies to be performed to correlate local (MAS) NMR spectroscopy. and long-range structure with performance in these systems. If time permits, our work on the identification of electronic, chemical and structural changes of FC components during OP102: Unique Insights into Carbon Capture and each FC assembly and operation process will be presented. Geosequestration of CO2 from In Situ High-Pressure, The work includes the influence of hot pressing on the pore High-Temperature NMR structures of PEM investigated by residual quadrupolar split- 2 J. Andrew Surface, Jeremy Moore, Mark Conradi, ∗Sophia ting in H NMR spectra of D2O in PEM and by freezing-point E. Hayes depression of water in nano-sized pores, and on the effects of Nafion ionomers on the local density of electronic states of Washington University in St. Louis surface Pt of Pt particle electrode catalysts explored by 195Pt We have constructed hardware to conduct in situ high- NMR techniques. pressure, elevated-temperature study of heterogeneous mix- in situ tures of solids, liquids, gases, and supercritical fluids. The Present work demonstrates that 1) NMR studies on objective is to simulate processes at geologically relevant pres- real DAFCs can be done, 2) alcohol oxidation reaction mech- in situ ex situ sures and temperatures, monitoring the kinetics of CO2 sorp- anism can be studied by the and NMR tech- tion and the stability of the sequestration within geologic niques, and 3) various NMR methods can be successfully ap- materials. Specifically, our aim is to monitor CO2 uptake plied for the identification of electronic, chemical and struc- in both ultramafic rocks and in more porous geological ma- tural changes of FC components during each FC assembly terials to understand the mechanisms of physisorption and and operation process. chemisorption as a function of temperature and pressure. A further aim is to spectroscopically characterize and quantify UP104: Local Order and Cation Distribution in CO2 in supercritical, "dissolved" (in salt water), and "min- Mixed-Alkali Phosphate Glasses eral" CO2 (mineral carbonation) phases. Thus, the results 1∗José Fabian Schneider, 1Jefferson Tsuchida, 2Hellmut will allow NMR to be used for quantitative characterization 2 of the phases and their chemistry. We have already developed Eckert, Rashmi Deshpande 1 CO2 spectroscopy to analyze the phase of CO2 (liquid, super- Instituto de Física de São Carlos, Universidade de São critical, or gas) and its conversion into other forms, such as Paulo, 2Institut für Physikalische Chemie, Westfälische carbonic acid, bicarbonate, and carbonate species When CO2 Wilhelms-Universität Münster reacts with the calcium or magnesium in a rock sample, such 13 The mixed-ion effect (MIE) is the dramatic non-linear varia- as peridotite, the C chemical shift, linewidth and lineshape, tion in the values of transport properties in glasses due to re- and relaxation times will change dramatically. This change striction in ionic mobility resulting from the cation mixing. A can be monitored in situ and provide instantaneous and con- satisfactory explanation of this phenomenon has been elusive tinuous characterization that maps the chemistry that is tak- during decades. Metaphosphate glasses AxB1−xPO3 with ing place. On the pathway to MgCO3 formation, there are a two mobile monovalent cations A+ and B+ are archetypical number of phases of Mg(OH)x(H2O)y(CO3)z that are appar- examples of MIE in d.c. conductivity. The deepest known re- ent. The in situ NMR experiments consist of heterogeneous duction in conductivity is observed in mixed Rb-Li metaphos- mixtures of rock, salty brine solution, and moderate pressure phates, attaining 8 orders of magnitude with respect to the CO2 gas at elevated temperatures. Results are compared to single-cation glasses. In contrast, reductions of less than 1 or- those from Raman spectroscopy of aquo- hydro- carbonato- der of magnitude are observed in Ag-Na metaphosphates. A magnesium species. comprehensive analysis of local order and alkali distribution Reference: in mixed metaphosphates is presented here, covering simi- lar/dissimilar cation pairs as Na-Li, Na-Ag, Na-K, Na-Rb, "In Situ Measurement of Magnesium Carbonate Formation Na-Cs, and Li-Rb, Li-Cs, Ag-Cs. The aim of this study is 13 from CO2 Using Static High-Pressure and -Temperature C the identification of cation segregation or random mixture, NMR", J. Andrew Surface, Philip Skemer, Sophia E. Hayes, which are configurations critical to determine conductivity, and Mark S. Conradi, Environ. Sci. Technol. 2013, 47, 119– and the possible dependence with size mismatch or differ- 125. DOI: 10.1021/es301287n ences in ionic potential. A broad set of NMR techniques was applied: 31P, 23Na, 7Li and 133Cs high-resolution NMR, UP103: Direct Alcohol Fuel Cells Investigated In 109Ag-NMR, 23Na Triple-Quantum-MAS, REDOR/SEDOR Situ and Ex Situ by NMR Spectroscopy between 31P-23Na, 7Li-6Li, and 7Li-133Cs, 87Rb quadrupole 23 1,2∗ Carr-Purcel-Meiboom-Gill, and Na-NMR spin-echo decay. Oc Hee Han The structural picture emerging from these results reveals 1Korea Basic Science Institute, 2Chungnam National Uni- that each cation site retains its structural specificities, as in versity single-cation glasses. However, a common structural adjust- Fuel cells are an ideal primary energy conversion device for ment on the cation environment was discovered: the compres- remote site locations, automobiles, and mobile electrical de- sion of the O environment around the smaller cation of the vices such as mobile phones and laptops. But direct alcohol pair, as its concentration diminishes. The mixture of cations fuel cells (DAFCs) are challenged to reduce alcohol crossover species at atomic scale is generally random, even in glasses and to develop less expensive catalysts and electrolyte mem- having the biggest cation mismatch as Li-Cs, but some devi- branes. Understanding fundamental aspects of DAFCs such ations indicating short-range like-cation preferences were ob- as the mechanisms of alcohol oxidation and crossover would served for Na-K, Na-Cs and Na-Rb. These findings support provide the direction to overcome the challenges. recent modeling procedures pointing to a fundamental rela- tion between the dynamic MIE and structure in phosphates, We developed the toroidal cavity detectors for in situ observa- which might be universal in all type of glasses. tion of DAFC by NMR spectroscopy, and demonstrated that the functional difference of anode catalysts such as Pt and ACKNOLEDGEMENTS: FAPESP, CNPq, CAPES PtRu could be detected. The anode exhaust of DAFC was analyzed by liquid state 13C NMR spectroscopy for quantita- OP105: Nanoscale Imaging of Electron and Nuclear tive correlation of the amount of electrical current generated Spins to the quantities of each chemical product. The membrane ∗ electrode assembly (MEA) composed of triple-polymer elec- Jörg Wrachtrup trolyte membrane layers was designed, thereby facilitating a 3rd Institute of Physics, University of Stuttgart, Stuttgart,

50 Abstracts of Talks – DNP

Germany [1] Nicholson J.K. and Lindon, J.C. (2008) Nature 455 1054- Increasing the sensitivity of magnetic resonance towards sens- 1056. ing single electron and nuclear spins is a prominent goal in [2] Swann, J.R. et al (2011) PNAS 108 4523-4530. NMR and EPR methodology. In addition in opens the way towards using quantum physics itself to increase sensitivity [3] Nicholson, J.K. et al (2012) Science 336 1262- 1267. beyond the standard quantum limit. In the recent past dia- [4] Kinross, J. et al (2011) Lancet 377 (9780) 1817-1819. mond defect centers have proven to be exquisitely sensitive sensors for sample spins and detection of single electron spins [5] Nicholson, J.K. et al (2012) Nature doi:10.1038/nature11708. or as few as 102 nuclear spin magnetic moments have become [6] Clayton, T.A. et al (2006) Nature 440 1073-1077. possible [1]. The talk will describe approaches to image such small spin ensembles for example to achieve subcellular reso- [7] Clayton, T.A. et al (2009) PNAS 106 14728-14733. lution in MRI imaging of cells [2] or eventually the detection [8] Mirnezami, R. Nicholson, J.K. and Darzi, A. (2012) New of single proteins. Eng. J. Med. 366 (6) 489-491. [1] T. Staudacher et al. "Nuclear magnetic resonance spec- [9] Holmes, E. et al (2008) Nature 453 396-400. troscopy on a (5nm)3 volume of liquid and solid samples", Science (2013) in press PL107: New contraptions/gadgets/techniques for [2] S. Steinert et al. "Magnetic spin imaging under solid-state NMR ambient conditions with sub-cellular resolution" (2012) ∗ arXiv1211.3242 K. Takegoshi Department of Chemistry, Graduate School of Science, Ky- Thursday – May 23rd oto University, Kyoto, Japan Plenary Session When I was Post.Doc., I could only use 4 phases for rf pulses Chair: Beat Meier with constant amplitude/phase/frequency. The size of data Room Gávea A storage was only a few MB and it took 45 min to process 2D data. . . At present, rf amplitude/phase/frequency can be var- ied continuously. The storage size is almost infinite and the PL106: Spectroscopy in the Clinic – from dreams to time for data analysis is too quick to finish a cup of coffee. reality We should fully enjoy these developments and I shall dis- ∗Jeremy K. Nicholson cuss recent advance of hardware, data treatment, and pulse sequences developed in my group. The topics may be covari- Department of Surgery and Cancer, Faculty of Medicine, Im- ance data processing for HETCOR,[1] elemental analysis by perial College London NMR,[2] a cryo-coil MAS probe for 1H-13C double resonance Systems biology and advanced spectroscopic tools can be ap- experiment,[3] a tear-less way of adjustment of magic angle, plied at both individual and population levels to understand air-less MAS, etc. If time allows, I shall also present analyti- integrated biochemical function in relation to disease patho- cal formalism on the derivatives of the density matrix and the genesis. Metabolic phenotyping offers an important window observed signal with respect to the experimental parameters on systemic activity and both advanced spectroscopic ap- varied.[4] proaches can be used to characterize disease processes and responses to therapy [1]. There is now wide recognition that [1] Takeda, Kusakabe, Noda, Fukuchi, and Takegoshi, Phys. the extensive cross-talk and signalling between the host and Chem. Chem. Phys. 14 (2012) 9715. the symbiotic gut microbiome links to both to responses to [2] Takeda, Ichijyo, and Takegoshi, J. Magn. Reson. 224 therapy and disease risk factors and indeed these also mod- (2012) 48. ulate drug toxicity [2,3]. Such symbiotic supra-organismal interactions greatly increase the degrees of freedom of the [3] Mizuno, et al. to be published. metabolic system that poses a significant challenges to fun- [4] Momot and Takegoshi, J. Magn. Reson., 221 (2012) 57. damental notions on the nature of the human diseased state, the aetiopathogenesis of common diseases and current sys- rd tems modelling requirements for personalized medicine. We Thursday – May 23 have developed scalable and translatable strategies for "phe- DNP notyping the hospital patient journey" [4,5] using top-down Chair: Christian Griesinger systems biology tools that capitalizes on the use of both Room Gávea A NMR and MS based metabolic modelling and pharmaco- metabonomics [6,7] for diagnostic and prognostic biomarker OP108: Magnetic resonance is no longer the world’s generation to aid clinical decision-making at point-of-care. least sensitive form of spectroscopy Such diagnostics (including those for near real-time applica- tions as in surgery and critical-care) can be extremely sensi- 1,2∗Geoffrey Bodenhausen tive for the detection of diagnostic and prognostic biomarkers 1Ecole Normale Supérieure, Paris, 2Ecole Polytechnique in a variety conditions and are a powerful adjunct to conven- Fédérale, Lausanne tional procedures for disease assessment that are required for future developments in "precision medicine" including un- There seem to be only three obstacles to the relentless ex- derstanding of the symbiotic influences on patient state [8]. pansion of the scope of contemporary magnetic resonance: Many biomarkers also have deeper mechanistic significance sensitivity, sensitivity, and sensitivity. Dynamic nuclear po- and may also generate new therapeutic leads or metrics of larization, which can enhance NMR signals by two orders of efficacy for clinical trial deployment. Furthermore the com- magnitude in solids, and by five orders of magnitude in liq- plex ands subtle gene-environment interactions that gener- uids, appears ideal to lift these hurdles. Our laboratory is ate disease risks in the general human population also ex- the only one in the world that has been working successfully press themselves in the metabolic phenotype, and as such, on the development of DNP in both solids and liquids side the NMR-based "Metabolome Wide Association Study" ap- by side. In solids, we have pioneered the characterization of proach [9] gives us a powerful new tool to generate disease organic molecules grafted on silica surfaces in view of study- risk biomarkers from epidemiological sample collections and ing heterogeneous catalysts, using 13C, 29Si and 27Al NMR. for assessing the health of whole populations. Such popula- The effects of spin diffusion have been investigated in porous tion risk models and biomarkers an also feedback to individ- silicates. By selective enrichment of a protein that is part ual patient healthcare models thus closing the personal and of the Ribosome with 13C and 15N, we have been able to public healthcare modelling triangle. observe signals of this 80 kDa protein complex. In liquids,

51 Abstracts of Talks – DNP many contributions have been made to improve the so-called as nanoparticulate silica, metal-organic-frameworks (MOF), "dissolution DNP" process by using cross-polarization at low catalyst precursors or organometallic systems,... Here we will temperatures and high fields, by scavenging radicals during present the most recent developments in DNP SENS, and transport, and by reducing losses due to relaxation in low in particular show how this allows the acquisition of mul- fields. tidimensional 1H-29Si and or 1H-13C correlation spectra of active surface species. The multi-dimensional spectra yield detailed three-dimensional structural information of tethered OP109: Dissolution Dynamic Nuclear Polarization of surface species.[3] The structural information obtained by Large Molecules DNP SENS opens the way to a more rational design of sup- ∗Christian Hilty, Mukundan Ragavan, Hsueh-Ying Chen, ported catalysis. Youngbok Lee, Haifeng Zeng, Hlaing Min [1] A. Lesage, M. Lelli, D. Gajan, M. A. Caporini, V. Texas A&M University, Chemistry Department, USA Vitzthum, P. Mieville, J. Alauzun, A. Roussey, C. Thieuleux, Solid-to-liquid state dynamic nuclear polarization (DNP) has A. Mehdi, G. Bodenhausen, C. Copéret, L. Emsley, J. Am. become a popular way of enhancing the signals in liquid Chem. Soc. 2010, 132, 15459-15461; state NMR and MRI by several orders of magnitude. Up [2] M. Lelli, D. Gajan, A. Lesage, M. A. Caporini, V. to now, the most prominent applications of solid-to-liquid Vitzthum, P. Mieville, F. Heroguel, F. Rascon, A. Roussey, state DNP have been for the observation of small molecules. C. Thieuleux, M. Boualleg, L. Veyre, G. Bodenhausen, C. Using highly polarized spin systems, chemical reactions and Copéret, L. Emsley, J. Am. Chem. Soc. 2011, 133, 2104- other dynamic processes can be studied in one single real- 2107; time experiment, even if far from equilibrium. In addition to small molecules, NMR has long been applied to determine [3] M. Samantaray, J. Alauzun, D. Gajan, S. Kavitake, A. the structure and function of macromolecules, including pro- Mehdi, L. Veyre, M. Lelli, A. Lesage, L. Emsley, C. Copéret, teins. Challenges in making use of the large signal gain of C. Thieuleux J. Am. Chem. Soc. 2013 in press solid-to-liquid state DNP in these applications, however, in- clude the need for dissolution and injection of such samples, UP111: Sensitivity enhancement in solution NMR as well as spin relaxation that occurs between polarization through dynamic nuclear polarization of encapsu- and NMR measurement. We present strategies that enable lated proteins the use of dissolution DNP for direct observation of macro- molecules by high-resolution NMR spectroscopy. In a first 1Kathleen G. Valentine, 2Guinevere Mathies, 1Nathaniel V. experiment, we transfer polarization from a hyperpolarized Nucci, 1Igor Dodevski, 1Sabrina Bédard, 1Matthew Stetz, ligand to a protein through the Nuclear Overhauser effect. 2Thach V. Can, 2Robert G. Griffin, 1∗A. Joshua Wand The resulting spectrum contains signals that are specifically 1University of Pennsylvania, 2Massachusetts Institute of enhanced in the vicinity of the binding site. The efficiency Technology of intermolecular polarization transfer depends on the kinet- ics of binding, cross-relaxation rates and spin diffusion rates Solution NMR has contributed significantly to studies of the within the protein. In a second experiment, we directly hy- structural and dynamic aspects of proteins and the informa- perpolarize a model protein, the ribosomal protein L23 from tion inherent in NMR phenomena offers much more. Yet, T. Thermophilus. Hyperpolarization occurs either in the na- despite tremendous advances in technology, experimental de- tive state or the chemically denatured form. Biosynthetically sign and analytical strategies, solution NMR remains funda- produced proteins are readily labeled with stable isotopes mentally restricted due to its inherent insensitivity. Thus, including 2H, which reduces spin relaxation. Final signal en- it seems important to improve the sensitivity of the solu- hancements of 102-103 are obtained in the NMR spectrome- tion NMR experiment in order to reduce experiment time, ter, which permit the observation of the re-folding process of lower the absolute quantities of sample required and open a the protein in real-time. lower concentration regime where proteins of limited solubil- ity can be accessed. With this in mind there has been a re- vival of dynamic nuclear polarization (DNP). In solution, it is UP110: DNP Surface Enhanced NMR (SENS): a thought that such DNP transfer will occur primarily through Tool for Structural Investigation of Supported Catal- the Overhauser effect. The basic strategy is to saturate the ysis electronic transition of a stable free radical and transfer this 1∗Moreno Lelli, 1Alexandre Zagdoun, 1David Gajan, non-equilibrium polarization to the hydrogen spins of water, 1Aaron J. Rossini, 4Olivier Ouari, 4Paul Tordo, 3Chloé which will in turn transfer polarization to the hydrogens of Thieuleux, 2Christophe Copéret, 1Anne Lesage, 1Lyndon the dissolved macromolecule. Unfortunately, technical as- Emsley pects of this approach seem to prove fatal to the idea in its current form. The primary reason is that the frequency of 1Centre de RMN à Très Hauts Champs, Université de Lyon the electron transition of suitable radicals lies in the sub- (CNRS/ENS Lyon/UCB Lyon 1), 2Department of Chem- THz spectrum where water absorbs strongly and results in istry, ETH Zürich, Switzerland, 3Institut de Chimie de catastrophic heating of the sample. In addition, the residence Lyon, Université de Lyon (CNRS-Université Lyon 1-ESCPE time of water on the surface of the protein is too short for Lyon), 4Aix-Marseille Université, CNRS, Institut de Chimie efficient transfer of polarization. Here we take advantage of Radicalaire (ICR), Marseille the properties of solutions of encapsulated proteins dissolved In recent years, our group has demonstrated that high-field in low viscosity solvents. Such samples are largely transpar- DNP in MAS solid-state NMR at low temperature ( 100 ent to the subTHz frequencies required and thereby avoid K) can be applied to investigate the structure of surfaces significant heating during saturation of the electronic transi- of materials. In this approach, which we dubbed DNP sur- tion. Nitroxide radical is introduced into the reverse micelle face enhanced NMR spectroscopy (SENS), the polarizing system in three ways: covalently attached to the protein; co- agent (for example an organic di-nitroxide radicals such as valently attached to a surfactant embedded in the reverse bCTbK, etc.) is introduced into the sample by incipient micelle shell; and free in the aqueous core. DNP experiments wetting impregnation with a radical solution. Upon irra- at the X-band EPR frequency have yielded initial enhance- diation with microwaves the DNP effect enhances polariza- ments from the nitroxide embedded in the surfactant to the tion of the 1H nuclei of the solvent and the surface, and water on the order of -35. In addition, we find that the hy- this enhanced 1H polarization can then be transferred to dration properties of encapsulated proteins allow for efficient the surface hetero-nuclei (13C, 15N, 29Si or 27Al) by cross- polarization transfer from water to the amide hydrogens in a polarization (CP).[1,2] The SENS method can be extended protein. Thus the merging of the reverse micelle technology to a large set of solvents, thus extending the nature of the with DNP demonstrates the potential to provide a significant accessible systems to include many modern materials, such increase in the sensitivity of solution NMR spectroscopy of

52 Abstracts of Talks – Dynamics and Recognition proteins and other bio-macromolecules.Supported by the NIH ∗Gerhard Wagner and the NSF. Department of Biological Chemistry and Molecular Pharma- cology, Harvard Medical School, Boston OP112: Dissolution DNP: Theoretical Considera- Solution NMR spectroscopy is ideally suited for studies of tions and Experimental Implementation dynamic and weak interactions of proteins. We have fo- Grzegorz Kwiatkowski, Alexander Karabanov, ∗Walter cused on several macromolecular complexes involved in pro- Köckenberger tein synthesis. A central interest in my laboratory is to under- stand the mechanisms by which eukaryotes initiate transla- University of Nottingham tion of mRNA. This process is highly regulated by numerous Recent progress made in the experimental implementation of weak and transient protein interactions. We have used NMR sensitivity enhancement strategies based on dynamic nuclear and other techniques to characterize these interactions and polarization has attracted much attention in the NMR com- the effect of inhibitors that prevent such interactions. We munity. Theoretical models of the DNP mechanism are use- found that inhibitors of the interaction between the transla- ful to provide insight into optimal conditions for such experi- tion initiation factors eIF4E and eIF4G discovered with high ments. However the theoretical quantum mechanical descrip- throughput screening bind eIF4E at a site distinct from the tion of the complex spin dynamics is limited to calculations eIF4G-binding site and act allosterically causing small but using only small representative systems consisting of up to 20 decisive rearrangements of the target protein. In addition, spins [1]. Here we present a new strategy that makes it pos- we have characterized the 68 kDa complex between the RNA sible to calculate the spin dynamics of DNP experiments for helicase eIF4A and the scaffold protein eIF4G as well as the large spin ensembles consisting of a network of several thou- interactions between initiation factors eIF1, eIF1A and eIF5. sand coupled spins. Building on previous work [2] we demon- We have shown that these interactions control start-codon strate that it is possible under certain conditions to overcome recognition. In a related project we are working on assembly- the exponential scaling of the required equations and to re- line mega-enzymes that synthesize natural products using a duce the dimensions for a system of N nuclear spins from 4N set of four domains. Reprogramming of these systems may to N coupled rate equations that describe the polarization enable production of new antibiotics. dynamics for each individual spin. The huge reduction of the required equations arises from the use of a quantum mechan- To enable structural and functional studies of these large ical projection technique related to a formalism proposed by systems we have optimized procedures for spectroscopy Nakajima and Zwanzig [3]. with large proteins. This includes more efficient pulse se- quences, and optimization of methods for non-uniform sam- We demonstrate numerically the validity of this approach by pling (NUS). We have developed new reconstruction methods comparing results derived from solving the master equation for NUS data that make reconstruction of sparsely sampled in the full Liouville space to simulations obtained from our high-resolution 3D and 4D data sets feasible. novel formalism. We show then how realistic build-up times for the experimentally observable nuclear spin polarization can be obtained from simulations involving 103 13C spins ar- OP114: Allosteric Motions in periplasmic binding ranged in a cubic lattice around a single electron. The use of Proteins characterized by NMR Spectroscopy this model enables us to study the transport of polarization ∗ Gabriel Ortega, Tammo Diercks, Oscar Millet between the electron and the nuclear spins and to distinguish between direct transfer of polarization from the electron to CIC bioGUNE, Parque Tecnológico de Vizcaya, Spain the nuclear spins and an indirect transfer via nuclear spin Protein function, structure and dynamics are intricately cor- diffusion. related, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of By varying the parameter of this model we gain further in- dynamics related to function (functional dynamics). We sight into the optimal conditions that need to be fulfilled for have applied NMR to investigate the functional dynamics in the generation of maximal nuclear spin polarization. In this two homologous periplasmic sugar binding proteins with bi- theoretical study we explicitly refer to the problems encoun- domain composition: E.coli glucose/galactose (GGBP) and tered by experimentalists working with hyperpolarized 13C ribose (RBP) binding proteins. In contrast to their structural and discuss how to design the optimal DNP experiment.We and functional similarity, we observe a remarkable difference report also on recent progress in the development of a DNP in functional dynamics: for RBP, the absence of segmental NMR spectrometer based on a unique dual centre magnet motions allows only for isolated structural adaptations upon that makes it possible to transfer the sample in solid state carbohydrate binding in line with an induced fit mechanism; between a cryogenic environment and and a probehead kept on the other hand, GGBP shows extensive segmental mo- at ambient temperature. bility in both apo and holo states, enabling selection of the [1] Y. Hovav, A. Feintuch and S. Vega, "Theoretical aspects most favorable conformation upon carbohydrate binding in of dynamic nuclear polarization in the solid state – The solid line with a population shift mechanism. Collective segmental effect", J. Magn. Reson.,207(2), 176–189, 2010 motions are controlled by the hinge composition: by swap- ping two identified key residues between RBP and GGBP [2] A. Karabanov , A. van der Drift , L. J. Edwards , I. we also interchange their segmental hinge mobility, and the Kuprov and W. Köckenberger, "Quantum mechanical sim- doubly mutated GGBP* no longer experiences changes in ulation of solid effect dynamic nuclear polarization using conformational entropy upon ligand binding while the com- Krylov-Bogolyubov time averaging and a restricted state- plementary RBP* shows the segmental dynamics observed space", Phys. Chem. Chem. Phys. 14(8), 2658-2668, 2012 in wtGGBP. Most importantly, the segmental inter-domain [3] H.-P. Breuer and F. Petruccione, The Theory of Open dynamics always increase the apparent substrate affinity and Quantum Systems, Oxford University Press, 2007. thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding.

Thursday – May 23rd ACKNOLEDGEMENTS: Support was provided from The Dynamics and Recognition Department of Industry, Tourism and Trade of the Govern- Chair: Martin Billeter ment of the Autonomous Community of the Basque Country, Room Gávea B from the Innovation Technology Department of the Bizkaia County, from the Ministerio de Economía y Competititvi- dad (CTQ2009-10353/BQU; CTQ2012-32183 and CSD2008- OP113: Use of Solution NMR for Studies of Proteins 00005). G.O. acknowledges a fellowship from the Ministerio and Protein Complexes Involved in Protein and Pep- de Ciencia y Tecnología. tide Synthesis

53 Abstracts of Talks – New Solution NMR Methods

UP115: Atomistic Descriptions of Protein Dynamics with biochemical and crystallographic approaches, we are de- on Multiple Timescales From NMR Chemical Shifts termining how HIV RT recognizes this complex, and how this ∗ ternary complex can be targeted by novel therapeutics. Sec- Paul Robustelli ond, viruses express RNAs to circumvent host responses to in- Columbia University fection. Using NMR we are determining the structure of a 30 kDa RNA from adenovirus that inhibits the innate immune NMR chemical shifts are highly sensitive probes of molecular response protein PKR, and how the RNA binds and inhibits structure. This work illustrates two approaches for utilizing this kinase. Our results show how NMR merged with other the structural information contained in chemical shifts to ob- techniques can illuminate the complex functions of RNAs in tain atomistic descriptions of the structural fluctuations of viral infection. proteins on the ns-µs and millisecond timescales.

In the first approach, semi-empirical NMR chemical shift pre- OP117: High-Resolution Protein Structures by diction methods are used to evaluate the dynamically aver- Magic-Angle Spinning NMR aged values of backbone chemical shifts obtained from unbi- ased molecular dynamics (MD) simulations of proteins on the ∗Chad M. Rienstra, Marcus D. Tuttle, Gemma Comel- ns-µs timescale [1]. MD-averaged chemical shift predictions las, Andrew J. Nieuwkoop, Ming Tang, Lindsay J. Sperling, generally improve agreement with experimental values when Robert B. Gennis, Julia M. George compared to predictions made from static X-ray structures University of Illinois at Urbana Champaign and a detailed analysis of the structural dynamics and confor- mational changes associated with the improvements provide We present multidimensional solid-state NMR methods for support for specific motional processes in proteins. Chemical protein structure determination, examining alpha-synuclein shifts are sensitive reporters of fluctuations in backbone and fibrils and the membrane protein DsbB. side chain torsional angles, aromatic ring positions, and the For alpha-synuclein, resonance assignments of several iso- geometries of hydrogen bonds. Improved chemical shift pre- forms demonstrate that the major secondary structure motifs dictions result from population-weighted sampling of multiple observed in the wild type protein are retained in the A30P conformational states and from sampling smaller fluctuations and A53T (Parkinson’s disease early onset) mutants, and fur- within conformational basins. thermore conserved upon fibril incubation on phospholipid In the second approach, chemical shifts and anisotropic re- vesicles. Spectra of intermediate states give insights into the straints obtained from relaxation dispersion experiments are sequence of fibril assembly. We propose structural models used to determine the structure of a sparsely-populated, on- consistent with the available long-range distance restraints. pathway folding intermediate that folds on the millisecond For DsbB, we have developed a high-resolution structural timescale [2]. CPMG measurements are used to measure model by combining experimental X-ray and solid-state NMR backbone chemical shifts, RDCs, and RCSAs of the 2% pop- with molecular dynamics (MD) simulations. We embedded ulated folding intermediate of the A39V/N53P/V55L Fyn the high-resolution DsbB structure, derived from the joint SH3 domain and the structure of the intermediate is cal- calculation with X-ray reflections and solid-state NMR re- culated using a recently developed chemical shift restrained straints, into the lipid bilayer and performed MD simula- molecular dynamics structure calculation protocol [3]. The tions to provide a mechanistic view of DsbB function in the structure provides a detailed characterization of the non- membrane. Further, we validated the membrane topology of native interactions stabilizing an aggregation-prone interme- DsbB by selective proton spin diffusion experiments, which diate under native conditions and insight into how such an directly probe the correlations of DsbB with water and lipid intermediate can derail the folding pathway and initiate fib- acyl chains. NMR data also support the model of a flexible rillation. periplasmic loop and an interhelical hydrogen bond between These results illustrate that NMR chemical shifts can be used Glu26 and Tyr153. to provide atomistic descriptions of protein motions on mul- This work was supported by NIH grants R01-GM073770 and tiple timescales. R01-GM075937. [1] P Robustelli, KA Stafford, AG Palmer III, JACS, 134, 6365-6374 (2012) Thursday – May 23rd [2] P Nuedecker, P Robustelli, A Cavalli, P Walsh, P Lund- New Solution NMR Methods strom, A Zarrine-Afsar, S Sharpe, M Vendruscolo, LE Kay, Chair: Julien Wist Science, 336, 362-366 (2012) Rooms Turmalina/Topázio [3] P Robustelli, KJ Kohlhoff, A Cavalli, M Vendrusculo, Structure, 18, 1-11 (2010) OP118: Broadband Inversion of 1JCC Correlations in 1,n-ADEQUATE Spectra UP116: Viral RNAs and Infection 1∗Gary E Martin, 1Mikhail Reibarkh, 1R. Thomas Joseph D. Puglisi, Aaron Coey, ∗Elisabetta Viani Williamson, 2Wolfgang Bermel Department of Structural Biology & Stanford Magnetic Res- 1Merck Research Laboratories, 2Bruker BioSpin onance Laboratory, Stanford University ADEQUATE experiments are a family of "out-and-back" Viruses use RNAs in multiple ways during their infection and 2D NMR experiments that begin with an initial magneti- 1 replication. Using NMR spectroscopy combined with other zation transfer via 1JCH followed by either a JCC (1,1- n biophysical approaches, we have explored two general strate- ADEQUATE) or JCC (1,n-ADEQUATE) magnetization gies that RNAs and RNA-protein interactions are harnessed transfer. The magnetization is transferred back along the to enhance viral infection. First, we have determined the same pathway and detected via the initial proton-carbon het- structural basis for initiation of reverse transcription in HIV, eronuclide pair. The 1,1-ADEQUATE experiment exclusively by which the viral genomic RNA is converted to a DNA copy gives correlations between adjacent protonated or protonated 1 by the viral enzyme reverse transcriptase (RT); this is the and non-protonated carbons (via JCC ) that are equivalent 2 first step in viral replication after infection. The process is to JCH correlations in HMBC spectra. Hence, for proto- initiated from a specific RNA-RNA complex formed by the nated adjacent carbons, the experiment is analogous to the Lys 2 3 genomic RNA and a host tRNA 3. We have monitored J, J-HMBC and H2BC experiments. However, for adja- the conformation of this 40-70 kDa RNA-RNA complex us- cent non-protonated carbons, 2J,3J-HMBC and H2BC can- ing heteronuclear NMR spectroscopy. We determined the not yield any correlations. In comparison, magnetization n global fold of this RNA complex, and showed the presence of transfer in the 1,n-ADEQUATE via JCC is typically with 4 key RNA contacts that stabilize the conformation. Combined n=3, very usefully affording the equivalent of JCH HMBC

54 Abstracts of Talks – New Solution NMR Methods

correlations. The down side of the 1,n-ADEQUATE experi- nance frequency and determined according to Eq. (1): σX = 1 ment is that JCC correlations unavoidably "leak" into the 1 – (νX γHe/νHeγX )(1 – σHe) spectrum and must be identified by some means. where ν, µ and γ are the resonance frequencies, the nuclear A modified 1,n-ADEQUATE pulse sequence employing asym- magnetic moments and the gyromagnetic ratios of helium- metrical delays was first investigated as a means of selectively 3 and another observed nucleus (X), respectively. Eq. (1) 1 inverting JCC correlations in 1,n-ADEQUATE spectra. The can be used for the shielding measurement of any nucleus initial method should probably best be described as "semi- whenever the gyromagnetic ratio of the given nucleus is ac- 1 selective" in that while it did invert most JCC correlations, curately known. The isolated helium-3 atom serves as the some correlations would still have positive intensity due to the primary reference standard of shielding and can be applied oscillatory nature of the amplitude transfer function. Worse, for all the magnetic nuclei. Moreover the vantage point of the 1 a mismatch of actual JCC coupling constants and the ampli- absolute shielding scale can be transferred from helium-3 to tude transfer function could potentially result in a spectrum another reference standard, such as deuterated lock solvent, 1 where most JCC correlations are not inverted. and one can conveniently use in practice this procedure as σ ν ν σ 1 described by Eq. (2): X = 1– CX ( X / D)(1– *D) A new, dual-optimization, inverted JCC 1,n-ADEQUATE experiment will be described that provides uniform inversion where σ*Drepresents the shielding of deuterons in selected 1 of JCC correlations across the range of 29-82 Hz. Even liquid solvent [1] and CX parameters are known for the ma- 1 2 11 13 15 17 more usefully, relative to the original version of the experi- jority of light magnetic nuclei: H, H, B, C, N, O, 19 29 31 ment, the dual-optimization version affords significantly bet- F, Si and P. ter signal-to-noise for the same acquisition time. Using 1.7 As shown the chemical shifts can be entirely replaced by mm cryoprobe technology, the experiment can be successfully the measurement of shielding parameters and this alterna- applied to samples of 1 mg and smaller. tive method of standardization of NMR data has numerous distinct advantages. OP119: Highlighting Change (1) It unifies multinuclear methods into one NMR spec- 1∗ 2 Eriks¯ Kupče, Ray Freeman troscopy because the values of magnetic shielding have the 1Bruker Ltd, Coventry, UK, 2Jesus College, Cambridge, UK same molecular meaning independently of observed nuclei. Two-dimensional NMR has revolutionized modern NMR (2) The experimental shielding values determined this way methodology, not least because it offers a simple, clear display are ready for direct comparison with the results of quantum- of correlation information in a format well suited to the spa- chemical calculations for the same molecular objects. tial imagination of chemists. We demonstrate an entirely new (3) The precision of such measurements is in every case ex- version of this technique, where the "evolution" dimension is actly the same as for the appropriate chemical shifts because derived by the Radon transform, instead of the more famil- it is based on the same reading of resonance frequencies. iar Fourier transform. The result is a two-dimensional chart that maps changes in the spectrum induced by molecular in- (4) The new method allows the measurement of first order teractions or external physical influences. For instance, if a isotope effects in shielding as it was shown for hydrogen iso- given chemical shift depends on temperature, the correspond- topomers [3]. ing correlation peak is displaced in the vertical dimension by an amount determined by the temperature coefficient. (5) There is no need to use any additional reference stan- dard if NMR experiment is carried out with deuterated lock It is important to note that no sensitivity is sacrificed be- solvent. cause the experiment benefits from the multiplex advantage of two-dimensional NMR. The line-shapes have weak skirts (6) Shielding measurements performed relatively to any ex- with characteristic "butterfly" contours, but these are easily ternal or internal deuterated solvent give practically the same converted into the more familiar four-pointed star by a suit- results. able windowing function. We show a simple example where (7) The measurement of shielding can be easily extended on the temperature dependence of NH and NH2 resonances are MAS NMR experiments as it was already shown for 13C spec- highlighted in the 600 MHz proton spectrum of a neuropep- tra [4]. tide, substance P. The reduced temperature coefficient of the G9 HN proton is indicative of intramolecular hydrogen-bond (8) Experimental shielding parameters can be always con- formation. We call this particular application "Temperature- verted into chemical shifts if the shielding of reference com- Induced Projection Spectroscopy (TIPSY)". pound is known. The general technique for mapping change is not limited to All the above conclusions are efficiently illustrated with the the study of temperature effects. Pressure, pH, solvent, asso- results of our recent experimental studies. ciation, hydrogen-bonding, molecular binding, partial align- Support from the National Science Centre (Poland) grant ment, conformational changes, protein folding, or the effect of DEC-2011/01/B/ST4/06588 is acknowledged. added paramagnetic agents can all be addressed under the ap- propriate experimental conditions. Indeed the scheme is not [1] Jackowski K., Jaszunski M., Wilczek M., J. Phys. Chem. restricted to NMR; initial experiments suggest that it can be A 114 (2010) 2471 and references there in. applied to infrared spectra. Because the Radon transform is the essential ingredient of the method, we call it "projection [2] Rudziński A., Pachulski M., Pachucki A., J. Chem. Phys. spectroscopy (PROSPECT)" a term that implies "a survey 130 (2009) 244102-1. of the landscape", "something considered promising" or even [3] Garbacz P., Jackowski K., Makulski W., Wasylishen R., "to explore for gold". J. Phys. Chem. A 116 (2012) 11896. [4] Jackowski K., Makulski W., Magn. Reson. Chem. 49 UP120: Measurements of Nuclear Magnetic Shield- (2011) 600. ing ∗ Karol Jackowski UP121: Optimizing Ionic Liquids for CO2 Capture: Faculty of Chemistry, University of Warsaw, Poland an NMR Approach Recently we have demonstrated the method of 1H and 13C 1Marta Corvo, 1João Sardinha, 2Sonia Maria Cabral shielding measurements available on a standard NMR spec- de Menezes, 3Jairton Dupont, 3Graciane Marin, 4Sandra trometer [1]. It is based on the magnetic shielding of an Einloft, 4Marcus Seferin, 1Teresa Casimiro, 1∗Eurico J. isolated helium-3 atom (σHe = 59.96743 ppm) [2], its reso- Cabrita

55 Abstracts of Talks – New BioSolid Methods

1REQUIMTE,CQFB, Dept. Química, Fac. Ciên- References: cias e Tecnologia, Univ. Nova Lisboa, Portugal, 2PETROBRAS/CENPES, RJ Brazil, 3Dept. Química [1] Carr HY & Purcell EM Phys Rev 94(3):630 (1954); Mei- Orgânica, Inst. Química, Univ. Federal do Rio Grande do boom S & Gill D Rev Sci Instrum 29(8):688 (1965). 4 Sul, Brazil, Fac. Química Pontificia Univ. Católica Rio [2] Smith PES, Bensky G, Álvarez GA, Kurizki G, Frydman, Grande do Sul, Brazil L. Proc. Natl. Acad. Sci. USA, 109, 5958 – 5961 (2012). Global warming has prompted the scientific community to [3] Alvarez, G. A. ; Shemesh, N.; Frydman L; in preparation look for new strategies for CO2 capture and storage. Re- cent studies suggest that ionic liquids (ILs) can be alterna- Acknowledgments: BioNMR EU Grant #261863, EU ERC tive materials for CO2 capture due to their high selectivity Advanced Grant # 246754, Fulbright Foundation (US) for CO2 absorption. ILs physical and chemical properties can be enhanced and modified by both their cationic and anionic rd moieties and this is the reason for their broad range of appli- Thursday – May 23 cations. Considering that ILs properties can be designed to New BioSolid Methods satisfy specific application requirements, the optimization of Chair: Stanley Opella ILs for CO2 capture is a matter of great interest.[1-3] Room Ônix Herein we present a detailed NMR study focused on the eval- uation of the interactions between ILs and CO2. Through OP123: Designing efficient re- and decoupling exper- careful structural modifications we were able to identify CO2- iments for biological solid-state NMR using optimal philic features by the analysis of CO2 solvation in imida- control and effective Hamiltonian methods zolium ILs. Our High Pressure NMR (HPNMR) methodol- ∗Niels Christian Nielsen ogy allows a direct measurement of CO2 solubility in ILs and an in-situ assessment of the relationship of all the species Aarhus University, Aarhus, Denmark involved in the solvation process - cation/anion/CO2. HP- Biological solid-state NMR spectroscopy to an increasing ex- NMR experiments based on NOE and multinuclear diffusion tend relies on a multitude of samples with different isotope- NMR experiments using the PGSE technique combined with labeling patterns and pulse sequences capable of transferring molecular dynamics simulations enabled a molecular based magnetization between different spin species for assignment interpretation for the solvation mechanism. This systematic and establishment of constraints on structure and dynam- study allowed the identification of promising candidates for ics from high-resolution spectra. This calls for advanced re- CO2 capture. and decoupling experiments providing optimal performance [1] X Zhang, X Zhang, H Dong, Z Zhao, S Zhang and Y for the specific mode of operation. Huang Energy Environ. Sci., 2012, 5, 6668- 6681. In this presentation, we will describe systematic design of [2] M Ramdin, TW Loos and TJH Vlugt Energy Environ. efficient re- and decoupling experiments for biological solid- Sci., 2012, 51, 8149-8177. state NMR designed using combinations of optimal control and effective Hamiltonian methods. Focus will be devoted [3] JL Anderson, JK Dixon and JF Brennecke Acc. Chem. to heteronuclear dipolar recoupling methods for recoupling Res. 2007, 40, 1208-1216. of dipolar interactions between spins like 1H, 2H, 13C, 14N, and 15N using optimal control pulse sequences[1] as well The authors would like to thank Petróleo Brasileiro as optimal controlled inspired analytical sequences such as SA – PETROBRAS, Fundação para a Ciência e Tec- RESP IRAT ION CP[2] (and refined variants) and spin-state nologia (FCT) and Ministério da Educação (CQFB selective recoupling.[3] We will also present novel approaches Strategic Project PEst-C/EQB/LA0006/2011 and Project for efficient heteronculear decoupling using refocused CW PTDC/QUI-QUI/098892/2008) for financial support. The (rCW) irradiation.[4,5] NMR spectrometers are part of the National NMR Network (RNRMN) and are funded by FCT. [1] Wei, D.; Akbey, U.; Paaske, B.; Oschkinat, H.; Reif, B.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. 2011, 2, OP122: Old Dog, New Tricks: Methodological NMR 1289. Developments to Characterize Solution-State Struc- [2] Jain, S.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. ture and Dynamics 2012, 3, 2020. Pieter ES Smith, Gonzalo A Álvarez, Noam Shemesh, Ger- ∗ [3] Kehlet, C.; Nielsen, J. T.; Tosner, Z.; Nielsen, N. C. J. shon Kurizki, Lucio Frydman Phys. Chem. Lett. 2011, 2, 543. Department of Chemical Physics, Weizmann Institute of Sci- ence, Israel [4] Vinther, J. M.; Nielsen, A. B.; Bjerring, M.; van Eck, E. R. H.; Kentgens, A. P. M.; Khaneja, N.; Nielsen, N. C. J. NMR utilizes a broad range of manipulations to probe re- Chem. Phys. 2012, 137. laxation effects over a broad range of conditions and scenar- ios – nanomaterials, rocks under the oceans, proteins, and [5] Vinther, J. M.; Khaneja, N.; Nielsen, N. C. J. Magn. Re- disease in humans. One of the earliest and most widely son. 2013, 226, 88. used forms among these manipulations is the Carr-Purcell Meiboom-Gill sequence, proposed over a half century ago for OP124: Spectrally Edited 2D NMR of Carbon Ma- removing inhomogeneous broadenings[1]. This sequence has terials and Proteins found widespread use in NMR and MRI, thanks to its ability 1∗Klaus Schmidt-Rohr, 1Robert L. Johnson, 1Keith J. to free the evolution of a spin-1/2 from linear Iz terms, like 2 2 1 chemical shifts and inhomogeneity effects. We have recently Fritzsching, Jason Anderson, Brent Shanks, Mei Hong introduced a new family of spin-echo sequences based on the 1Department of Chemistry, Iowa State University, selective dynamical recoupling (SDR) experiment, which al- 2Department of Chemical and Biological Engineering, Iowa though fulfilling =0 can still detect site-specific inter- State University actions like chemical shift, thanks to the presence of envi- Techniques for spectral editing of two-dimensional 13C-13C ronmental fluctuations of common occurrence in high-field correlation spectra of 13C-enriched carbon materials and NMR –such as homonuclear spin-spin couplings, chemical ex- proteins will be presented. They remove peak overlap and change[2], or random diffusion.[3] It is conjectured that these greatly simplify spectral assignment. new experiments can be used to characterized functionally relevant "invisible" states in exchanging proteins, and serve Carbon materials produced from renewable feedstocks at as new contrast agent for water in confined media. moderate temperatures are attracting interest for various

56 Abstracts of Talks – New BioSolid Methods applications, including catalyst supports and soil amend- 1Catherine Bougault, 1Lauriane Lecoq, 2Sabine Hediger, ments/carbon sequestration. The aromatic-carbon region 2Hiroki Takahashi, 2Gaël De Paëpe, 3Michel Arthur, of their 13C NMR spectra often exhibits strongly overlap- 1∗Jean-Pierre Simorre ping bands from furans, arenes, and phenols. These can 1Institut de Biologie Structurale CEA-CNRS-UJF Greno- be separated by two complementary combinations of spec- 2 13 13 ble, Laboratoire de Chimie Inorganique et Biologique, CEA tral editing with 2D C- C NMR, applicable to materi- 3 13 13 Grenoble, Centre de Recherche des Cordeliers, INSERM, als made from C-enriched feedstocks such as u- C glu- Paris cose. Exchange with protonated and nonprotonated spectral editing (EXPANSE) identifies protonated aromatics, selected The cell wall is essential for the survival of bacteria. It by short cross polarization, and their nonprotonated bond- gives the bacterial cell its shape and protects it against os- ing partners, selected by dipolar dephasing, while double- motic pressure, while allowing cell growth and division. It quantum/single-quantum NMR with dipolar dephasing of is made up of peptidoglycan (PG), a biopolymer forming the double-quantum coherence selects bonded nonprotonated a multi-gigadalton bag-like structure, and additionally in carbons. Both spectra are free of a diagonal ridge, which has Gram-positive bacteria, of covalently linked anionic polymers many advantages; in particular, cross peaks on the diagonal called wall teichoic acids (WTA). TAs are thought to play can be detected. Combined with the chemical-shift informa- important roles in ion trafficking, host-cell adhesion, inflam- tion, this double spectral editing defines the local bonding mation and immune activation. environment. The machinery involved in the synthesis of this envelop is cru- Spectral editing techniques can also be used to simplify spec- cial and is one of the main antibiotic target. Different protein tral assignment in 13C-13C spectra of uniformly 13C, 15N- as transpeptidase, transpeptidase activator or hydrolase are labeled proteins. CH selection by dipolar DEPT, and CN recruited to maintain the morphogenesis of the peptidogly- suppression by asymmetric 15N-13C REDOR dephasing have can during the bacterial cell cycle. Based on few examples in- been implemented at sufficiently high MAS frequencies ( 10 volved in the machinery of synthesis of the peptidoglycan, we will demonstrates that a combination of liquid and solid-state kHz). For complete (<2%) suppression of rigid-CH2 and CO-N signals, standard 1H-13C (in dipolar DEPT) and 15N- NMR can be a powerful tool to screen for cell-wall interacting in vitro on cell 13C REDOR dephasing proved insufficient, but a lower and proteins and . broader minimum in the dephasing curve was achieved by In particular, structure of the L,D-transpeptidases that re- ◦ asymmetric REDOR with optimized 180 pulse position. sults in b-lactam resistance in M. tuberculosis, has been stud- The C=O region of the CN-free 2D spectrum contains only ied in presence of the bacterial cell wall and in presence of peaks of Asp and Glu, while the CH-selective 2D spectrum antibiotic. The NMR study reveals new insights into the in- retains the signals of Ile, Leu, and Val. The experiments are hibition mechanism. demonstrated on a 56-residue model protein (GB1). In parallel, we have investigated the potential of Dynamic ACKNOWLEDGMENTS: NSF Engineering Center for Nuclear Polarization (DNP) to investigate cell surface di- Biorenewable Chemicals (CBiRC) rectly in intact cells. Our results show that increase in sen- sitivity can be obtained together with the possibility of en- UP125: Recording Quantitative One- and Two- hancing specifically cell-wall signals. It opens new avenues Dimensional 13C NMR Spectra of Microcrystalline for the use of DNP-enhanced solid-state NMR as an on-cell Proteins with Improved Sensitivity investigation tool.

1,2,3∗Piotr Tekely, 1,3Rudra Narayan Purusottam, 1,2,3 OP127: The HIPER project – Very High Sensitivity Geoffrey Bodenhausen Pulse EPR 1 2 Ecole Normale Superieure (ENS), Centre National de la 1∗ 1 1 3 Graham Smith, Hassane El Mkami, Paul Cruickshank, Recherche Scientifique (CNRS), Université Pierre et Marie 1 1 1 1 Curie (UPMC) Robert Hunter, David Bolton, Duncan Robertson, Bela Bode, 2Olav Schiemann, 3Richard Wylde, 4David Keeble, Recording sensitive and quantitative spectra of low-γ nu- 4David Norman clei such as 13C constitutes a long-standing challenge in 1University of St Andrews, Scotland, 2University of Bonn, solid-state NMR spectroscopy. The standard 13C spectra Germany, 3Thomas Keating Ltd, Billingshurst England, are inherently non-quantitative, i.e. showing the integrated 4University of Dundee, Scotland signal intensities which are not proportional to the number of nuclei, when cross-polarization from protons to carbons The HIPER project was originally funded in 2003 under is employed, or, if the relaxation rates are not equal, when the UK’s Basic Technology program with the main technical the delay between successive acquisitions is too short to aim of constructing a very high sensitivity, low dead-time, allow a uniform recovery of the longitudinal 13C magneti- EPR spectrometer that would significantly improve on cur- zation in single-pulse experiments. The most pronounced rent state-of-the-art. This new spectrometer operates at 94 distortions of intensities in the cross-polarization spectra GHz at peak power levels of more than 1 kW with short are observed between proton-carrying carbons on the one pulses, high isolation, fast averaging capabilities, low dead- hand, and carbonyl, carboxyl, or quaternary carbons on the time and a wide instantaneous bandwidth of 1 GHz. Un- other. Additional distortions can also arise because of local like traditional 94 GHz spectrometers that use small resonant variations in internal mobility leading to variations in CP cavities, this system uses non-resonant sample-holders with efficiency and/or rotating-frame relaxation rates. relatively large sample volumes that are comparable to those 13 used in pulsed X-band measurements. This combination of We will show first quantitative C spectra of microcrys- high frequency, high power and relatively high sample volume talline proteins with improved sensitivity as compared with can lead to a spectacular increase in concentration sensitivity single-pulse, fully relaxed spectra. The approach exploits 13 for low dielectric loss samples. Gains approaching two orders the equilibration of C magnetization by low-power PARIS of magnitude have been observed, for good glasses, relative irradiation leading simultaneously to the efficient heteronu- to conventional X-band systems. Such gains in sensitivity clear Overhauser enhancement. We will also show how to combined with large instantaneous bandwidths are particu- record sensitive, fully symmetric 2D homonuclear correlation larly beneficial for popular pulse EPR measurements such as spectra of microcrystalline proteins even at highest available PELDOR, commonly used to characterize large-scale struc- static fields up to 23.5 T (1 GHz for protons). ture in biomolecules, where it is not uncommon to average for 12-24 hours using standard X-band instrumentation. The UP126: Molecular interactions with the bacterial cell new system reduces averaging times and allows samples to be wall by liquid state, standard and DNP solid state measured with excellent signal to noise: at low sample con- NMR. centrations, or with extended time windows to access longer

57 Abstracts of Talks – New BioNMR Methods distances, or with selective orientation dependent excitations glucose concentrations in the blood stream, while activating to quantitatively characterize the relative orientation of spin variants as well as the small molecule therapeutics abolish labels rigidly attached to proteins. In the talk, I will give this kinetic cooperativity and lead to a hyperbolic kinetic examples, on a variety of biological systems, which clearly profile. Despite nearly a half-century of research, the mech- demonstrate these advantages and I will examine how pulse anistic basis for GCK’s homotropic allostery remains unre- EPR (and DNP) instrumentation might evolve even further solved. 17 selectively 13Cδ1-labeled isoleucine methyl groups in the future. and 3 15N1-labeled tryptophan side chains were assigned and used as sensitive NMR probes to monitor the spatial rd and temporal behavior of GCK and its mutants in the ab- Thursday – May 23 sence and presence of glucose. It is found that the small New BioNMR Methods GCK domain is intrinsically disordered, while both small- Chair: Gisele Amorim molecule diabetes therapeutic agents and hyperinsulinemia- Room Gávea A associated GCK mutations display a population shift to- ward a narrow, well-ordered ensemble resembling the glucose- OP128: High Sensitivity Pulse Dipolar ESR and Pro- bound conformation. The NMR data suggest that GCK gen- tein Structure erates its cooperative kinetic response at low glucose con- centrations by using a millisecond disorder-order cycle of ∗Jack H. Freed the small domain as a "time-delay loop", which is bypassed Department of Chemistry and Chemical Biology, Cornell at high glucose concentrations, providing a unique mecha- University, Ithaca, New York, USA nism to allosterically regulate the activity of human glucoki- nase under physiological conditions. We will also discuss re- The importance of and requirements for high-sensitivity cent progress in the characterization of protein dynamics on Pulse Dipolar ESR Spectroscopy (PDS) in measurements of fast and slow time scales by the combined interpretation of distances in the 1 to 10 nm. range will be discussed. As molecular dynamics computer simulations and NMR chem- projects in the determination of complex proteins structures ical shifts with the recent protein chemical shift predictor become more challenging, increasing the sensitivity and ac- PPM (http://spinportal.magnet.fsu.edu/ppm/ppm.html). curacy is required. The relative virtues of double electron- electron resonance (DEER) and Double Quantum Coherence ACKNOWLEDGEMENTS: The National Science Founda- (DQC)-ESR in meeting these objectives is compared. It is tion, the National Institutes of Health, and the American shown how micromolar sensitivity is valuable both for dilute Heart Association. solutions and for accurate data analysis. The need for longer dipolar evolution times to measure longer distances in protein complexes is effectively addressed by the new technique of UP130: A unified conformational selection and in- 5-pulse DEER, which can almost double such times. The ap- duced fit approach to the modelling of protein- plication of such developments to protein structure is demon- peptide interactions. strated in the cases of an integral membrane protein complex ∗ and of locating how a substrate penetrates into the active Mikael Trellet, Adrien Melquiond, Alexandre M. J. J. site of an enzyme. In the former, the sodium and aspartate Bonvin symporter from Pyrococcus hokoshii, GLtPh which forms a Utrecht University, Faculty of Science, Bijvoet Center trimer, has each protomer transporter function by alternating Protein-protein interactions are vital for all cellular mech- between outward-facing can inward-facing states. We showed anisms. Among those, peptide-mediated interactions con- that the conformational ensemble of protomers samples both tribute up to 40% of all protein-protein interactions. Due to states with nearly equal probabilities, indicative of compa- their important role in regulatory and signalling processes, rable energies, and independently of each other. In the lat- peptides are in many cases implicated in diseases. They σ ter, we located (to 2 < 2Å accuracy) how a lysolecithin are also known to block protein-protein interactions, which spin-labeled on the polar-end, interacts with soybean seed makes them interesting leads for protein drug design. lipoxygenase-1. The general approach we developed could be used to locate other flexible molecules in macromolecular Despite the large amount of structural data available, it re- complexes. mains very challenging to predict protein-peptide interactions ACKNOWLEDGEMENTS: Co-workers: Peter Borbat, Elka by homology modelling. This leaves biomolecular docking as Georgieva, Betty Gaffney, and Olga Boudker. Support from one of the few amenable computational technique to predict NIH/NIGMS Grant P41GM103521 and NIH/NIBIB Grant these interactions. Yet, the intrinsic flexibility of peptides R01EB003150. is a major obstacle for any modelling technique. Docking methods are sensitive to conformational changes and gener- ally require high-resolution structures and/or knowledge of OP129: Harnessing the Synergies between NMR and the bound form to perform well. However, it is possible to Computation for the Understanding of Protein Func- narrow down the uncertainty by using information to drive tion the search, allowing concentrating on further refining the con- 1,2Mioara Larion, 1,2,3Roberto Salinas, 4Vitali Tugarinov, formation of the peptide at the interface. 1,2Dawei Li, 1,2Dong Long, 1,2Yina Gu, 2Lei Bruschweiler- Li, 1Brian Miller, 1,2∗Rafael Brüschweiler We have developed HADDOCK (http://haddock.science.uu.nl), a flexible information-driven docking program that distin- 1Department of Chemistry and Biochemistry, Florida State guishes itself from others by the use of experimental and/or University, 2National High Magnetic Field Laboratory, Tal- bioinformatics data to drive the modelling process [1]. HAD- lahassee, FL, 3Institute of Chemistry, University of São DOCK has been widely applied to model 3D structures of Paulo, 4Department of Chemistry and Biochemistry, Uni- protein-protein and protein-nucleic acids complexes and has versity of Maryland also demonstrated strong performance in CAPRI, a blind ex- Glucokinase (GCK) is a 52 kDa monomeric enzyme that periment for the prediction of biomolecular interactions. We catalyzes the rate-limiting step of glucose catabolism in the will present recent results on the application of HADDOCK pancreas. GCK is allosterically regulated by glucose, a fea- to protein-peptide modelling using a benchmark of 103 com- ture that allows the enzyme to functions as the body’s glu- plexes [2]. In the absence of information on the bound form cose sensor. Over 600 mutations were identified in human of the peptide, we achieve near-native predictions for 80% of glk gene, some of which lead to protein activation causing the benchmark by combining the concepts of conformational persistent hypoglycemic hyperinsulinemia of infancy (PHHI). selection and induced fit into our flexible docking approach. Wild-type GCK displays a characteristic sigmoidal response in its enzymatic glucose phophorylation rate to increasing References:

58 Abstracts of Talks – Bio-Solids

[1] de Vries S et al. (2007). Proteins: Struc. Funct. & Thursday – May 23rd Bioinformatic, 69, 726-733. Bio-Solids

[2] London N et al. (2009). Structure, 18, 188-199. Chair: Mônica Freitas Room Gávea B

UP131: Replica Averaging-Replica Exchange OP133: Type-III secretion needles studied by solid- (RARE) Molecular dynamics simulations with NMR state NMR chemical shifts 1 1 1 ∗ Jean-Philippe Demers, Antoine Loquet, Veniamin Carlo Camilloni, Andrea Cavalli, Michele Vendruscolo Shevelkov, 2Nikolaos Sgourakis, 3Rashmi Gupta, 3Michael University of Cambridge Kolbe, 1Karin Giller, 1Dietmar Riedel, 1Christian Griesinger, 2 1 1∗ Solving the behaviour of proteins from the point of view of David Baker, Stefan Becker, Adam Lange statistical mechanics, involves the calculation of the Boltz- 1Max Planck Institute for Biophysical Chemistry, Göttin- mann ensemble. A major goal in the study of complex gen, Germany, 2Department of Biochemistry, University of molecular systems is the generation of ensembles of confor- Washington, USA, 3Max Planck Institute for Infection Biol- mations that represent their Boltzmann distributions. The ogy, Berlin, Germany observable properties of these systems can be then predicted Type-III secretion systems (T3SS) are molecular machines by calculating averages over such ensembles. In principle, found in Gram-negative bacteria that transfer virulence fac- given accurate energy functions and efficient sampling meth- tors to the cytosol of host cells. Based on 2D solid-state ods, the ensembles can be generated. In practice, however, NMR experiments recorded on uniformly, [1-13C]-glucose and often the energy functions are known only approximately and [2-13C]-glucose labeled samples, we have obtained the 95%- the sampling can be carried out only in a limited manner. complete assignment of 13C and 15N chemical shifts for the We describe here a method that simultaneously enables to T3SS needle of Shigella flexneri, responsible for bacillary increase both the accuracy of the energy and the extension dysentery (Demers et al, PLOS Pathogens, 2013, accepted). of the sampling. The method incorporates experimental Very sharp 13C line-widths are observed, ranging from 0.3 data as structural restraints in molecular dynamics simu- to 0.6 ppm in the uniformly labeled sample and from 0.1 lations and exploits the replica exchange approach in the to 0.4 ppm in sparsely labeled samples. Only the first 3 well-tempered ensemble metadynamics framework. We illus- N-terminal residues are flexible; in contrast to the crystal trate the method in the case of a peptide and show that it structure of monomeric needle protein where the first 14 N- enables its free energy landscape to be generated accurately terminal residues are disordered (Deane et al, PNAS, 2006). using only backbone NMR chemical shifts. The secondary structure consists of a kinked α-helix (L12 to A38), a loop (E39 to P44) and a second α-helix which extends OP132: Extending the Empirical Limits of Chemical up to the C-terminus (Q45 to R83). Although rigid, the N- Shifts terminal segment from S2 to T11 does not adopt any conven- tional secondary structure. The solid-state NMR data have Yang Shen, Alex Maltsev, Alexey Mantsyzov, Jinfa Ying, been compared in detail with a recent state-of-the-art high- ∗Ad Bax resolution cryoEM map of the Shigella needle (Fujii et al, Laboratory of Chemical Physics, NIDDK, NIH PNAS, 2012). The secondary structure elements are highly similar to those identified in the homologous needle protein NMR chemical shift assignments are often easily obtained of Salmonella typhimurium for which we have recently deter- and contain important local information regarding biomolec- mined an atomic model (Loquet et al, Nature, 2012), allow- ular structure. Our original approach to take advantage of ing us to propose a common structural architecture for T3SS ad hoc this approach was based on a simple database search needles. Furthermore, we discuss in vivo data that show the to find small, 3-residue fragments that best match the sec- biological relevance of the solid-state NMR structural data. ondary chemical shifts and residue types of the query seg- Finally, we will present proton-detected solid-state NMR ex- ment, culminating in the TALOS program.[1] This program periments on perdeuterated T3SS needles. A set of 2D and ϕ Ψ provided and torsion angle information for about 70% 3D experiments on such samples was used for the unambigu- of the residues, with an error rate <3%. A subsequent fil- ous assignment of the backbone resonances. tering of the TALOS output, using an artificial neural net- work (ANN) derived relation between chemical shifts, residue OP134: Solid-State NMR Structural Studies of Pro- type, and approximate region of Ramachandran space, al- teins Using Paramagnetic Probes lowed extension of predictions to ca 85%, culminating in the TALOS+ program.[2] The remaining 15% for which angles 1Ishita Sengupta, 1Philippe S. Nadaud, 1Min Gao, 1Rajith could not be identified are all in regions outside of regular J. Arachchige, 2Charles D. Schwieters, 1∗Christopher P. secondary structure, and therefore the hardest to identify Jaroniec from regular NMR parameters. By further extending both 1The Ohio State University, Columbus, USA, 2National In- the size of the fragment and the database, and by convert- stitutes of Health, Bethesda, USA ing the search to an almost entirely ANN-based approach, we have reduced the fraction of residues whose backbone an- Structural studies of proteins by magic-angle spinning solid- gles cannot be determined to 10%, without increasing the state NMR are hampered by the relative shortage of dipolar error rate. This TALOS-N program also provides χ1 angle coupling based restraints corresponding to distances signif- information for approximately half of the residues, with an icantly exceeding 5 Å that can be reliably determined us- error rate smaller than 10%. Application to the study of the ing conventional techniques. This paucity of long-distance structure of an intrinsically disordered protein, α-synuclein, structural data can be circumvented by extending the stud- shows significant residue-by-residue variation, despite very ies to include structural analogs of the protein of interest, small secondary chemical shifts. intentionally modified to contain covalently-attached param- agnetic tags at selected sites. In such paramagnetic proteins, [1] Cornilescu, G.; Delaglio, F.; Bax, A. Protein backbone the dipolar couplings between the nuclear spins and the un- angle restraints from searching a database for chemical shift paired electrons of the tag can manifest themselves in NMR and sequence homology J. Biomol. NMR 1999, 13, 289-302. spectra in the form of nuclear dipolar shifts or relaxation en- hancements that depend on the electron-nucleus distance and [2] Shen, Y.; Delaglio, F.; Cornilescu, G.; Bax, A. TALOS+ can be considerable for nuclei located up to 20 Å away from : a hybrid method for predicting protein backbone torsion the paramagnetic center. I will discuss our recent progress in angles from NMR chemical shifts J. Biomol. NMR 2009, 44, the studies of proteins using paramagnetic solid-state NMR 213-223. techniques. The topics will include the rapid backbone fold

59 Abstracts of Talks – Contrast Agents/ Instrumentation refinement for the model protein GB1 using only 4-5 longitu- hitherto poorly understood. What is more, its transient char- dinal 15N PRE restraints per residue recorded using a set of acter renders the study of membrane protein supramolecular cysteine EDTA-Cu(II) variants and backbone dihedral angle organization experimentally very challenging. restraints derived from 13C and 15N chemical shifts, and the development of improved Cu(II)-binding tags for this type of Using the potassium channels KcsA and KcsA-Kv1.3 as ex- structural analysis. amples, we demonstrate how the combination of solid-state NMR and computational approaches, assisted by electrophys- ACKNOWLEDGMENTS: This research was supported by iological measurements, allows dissecting various aspects of US National Science Foundation grants MCB-0745754 (CA- membrane protein supramolecular structure, including, inter REER Award) and MCB-1243461 to C.P.J. alia, annular and non-annular protein – lipid binding, the in- fluence of structural water or protein – protein interactions UP135: 100 kHz Magic-Angle Spinning for Proteins in membranes. 1∗ 2 3 Beat H. Meier, Ago Samoson, Anja Böckmann, OP137: Membranes, crystals, sediments: solid-state 1 1 2 Matthias Ernst, Vipin Agarval, Marie-Laure Fogeron, NMR of large proteins 1Matthias Huber, 1Susanne Penzel, 1Francesco Ravotti 1∗ 2 1 1 2 Anja Böckmann, Beat H. Meier, Britta Kunert, ETH Zurich, Physical Chemistry, Switzerland, Tallinn 2 2 2 3 Andreas Hunkeler, Susanne Penzel, Anne Schütz, University of Technology, Estonia, IBCP, CNRS, Lyon, 1 1 3 France Laurent Terradot Alexandre Bazin, Pierre Falson, Jean- Michel Jault, 1Carole Gardiennet We report protein spectra obtained at 100 kHz MAS and 1Institut de Biologie et Chimie des Protéines, CNRS- describe the hardware requirements as well as spectroscopic Université de Lyon, France, 2ETH Zürich, Physical Chem- techniques adapted to these MAS frequencies that signifi- istry, CH-8093 Zürich, Switzerland, 3IBS, CNRS-CEA- cantly exceed the magnitude of the individual dipolar cou- Université de Grenoble, France plings. Solid-state NMR is an increasingly powerful tool to charac- Despite the small sample amounts - less than 500 micrograms terize challenging proteins. Notably, it can analyze an aston- of protein- well resolved HSQC spectra with good signal-to- ishing variety of states, as proteins inserted in membranes, noise ratio can be acquired in 10 minutes from deuterated crystals and simple sediments. Combined with other biophys- and fully reprotonated (at the exchangeable positions) or ical approaches, NMR thus gives unique insight into protein from fully protonated proteins, using proton detection and structure, and ultimately function. We will illustrate this exploiting the long proton T2’s at 100 kHz MAS. A detailed with two examples. investigation of proton T2 and T2’ values as a function of the MAS frequency is presented. DnaB is a helicase from Helicobacter pilori. It is believed to function as a dodecamer , each subunit having a molecular Even at 100 kHz MAS, homogenous contributions to the mass of 55.7 kDa (488 residues). While EM has revealed the linewidth (in the sense of Maricq and Waugh) are present. envelope of the multimer, high-resolution structures could We characterize them and discuss schemes to average them, only be obtained for the isolated N-terminal and C-terminal e.g. in the context of heteronuclear decoupling. Particular- parts. Indeed, the entire protein today only yields poorly ities arising for MAS at 100 kHz and above are discussed. diffracting crystals, which however give rise to highly resolved Under these conditions, decoupling is most efficiently imple- NMR spectra. This allowed us to compare the fingerprints mented as low-power sequences, which allow to further im- from the isolated domains to the functional protein. Also, prove the signal-to-noise due the reduced interscan delays, in DnaB has different interaction partners, which however do particular if integrated in experiments where all the differ- not co-crystallize. We recently showed that sedimentation ent periods of the multidimensional experiments are imple- can be used as sample preparation, and illustrate how this mented as low-power versions. We discuss the performance of approach can be a promising possibility to study such pro- different schemes and introduce a new decoupling sequence teins and their partners. to further reduce the linewidth in the carbon and nitrogen dimensions. BmrA is a membrane protein of the class of ABC trans- porters. It functions as a dimer of 2x 64.5 kDa (589 residues). These techniques open the perspective for investigations of It is a close bacterial homologue of the P-glycoprotein, which structure and dynamics of proteins using submilligramm is involved in multidrug resistance in humans. In order to amounts of protein, facilitating sample preparation, e.g. by obtain stable preparations showing a high protein/lipid ra- cell-free expression. tio of BmrA for solid-state NMR studies, we reconstituted it in its native lipids extracted from B. subtilis. The obtained UP136: Determining supramolecular organisation of samples yield very good signal/noise ratio and linewidths, ion channels by solid-state NMR and computational which do not correlate with sample homogeneity as analyzed methods by EM. The possibility to prepare the protein under different conditions in a lipid environment will allow to gain insight 1∗Markus Weingarth, 1Elwin van der Cruijsen, in the conformational changes between the open, closed and 1,2Alexander Prokofyev, 1Eline Koers, 1Alexandre M. J. drug-bound states. J. Bonvin, 2Olaf Pongs, 1Marc Baldus ACKNOWLEDGEMENTS: CNRS, ANR, SNF, CEA 1Utrecht University, Bijvoet Center for Biomolecular Re- search, Utrecht, The Netherlands, 2Saarland University, Faculty of Medicine, Department of Physiology, Homburg, Thursday – May 23rd Germany Contrast Agents/ Instrumentation Protein supramolecular structure invokes the conformation Chair: Carlos Geraldes beyond a single protein. While the protein is held together Rooms Turmalina/Topázio by intramolecular forces comprising strong interactions like covalent bonds, supramolecular structures are often com- OP138: New Directions in Dipolar Imaging to En- posed of weaker intermolecular contacts. The latter ones hance Endogenous Contrast confer great diversity and sensitivity to their environment upon these multi-molecular arrangements. ∗Warren S. Warren, Thomas Theis, Yi Han, Zijian Zhou Duke University Membrane protein are exposed to a partiuclarly complex habitat which crucially modulates protein structure and func- For decades, the classical or mean-field picture describing the tions, however, the molecular basis of such modulations is distant dipolar field (and the coupled spin picture described

60 Abstracts of Talks – Contrast Agents/ Instrumentation by intermolecular multiple quantum coherences (iMQCs)) 1Tel Aviv University, 2Chaim Sheba Medical center has always simplified to an effective internuclear coupling The chemical exchange saturation transfer (CEST) NMR I I proportional to zi zj , differing only by a scale factor of 1.5 method enables to detect low concentrations of metabolites for homomolecular (identical resonance frequency) and het- that contain residues with exchangeable protons such amine, eromolecular interactions. We show both theoretically and amide or hydroxyl. The enhanced sensitivity allows obtaining experimentally that the mathematical framework needs to images of relatively low concentrations of endogenous cellu- be modified for modern applications such as imaging. We lar components or exogenous agents by MRI. 2-deoxyglucose demonstrate new pulse sequences which produce unexpected (2DG) and 2-fluoro deoxyglucose (FDG) are two glucose ana- effects; for example, modulating an arbitrarily small fraction logues that are taken up by cells through the glucose trans- of the magnetization can substantially alter the frequency porter, they undergo phosphorylation catalyzed by hexoki- evolution, so matched gradient pulse pairs (a seemingly in- nase but unlike glucose they and their metabolic products nocuous module in thousands of existing pulse sequences) can accumulate in the cells. The increased glycolysis characteris- alter the time evolution in highly unexpected ways. We also tic of cancer has led to the use of the positron emitter 18-FDG demonstrate improved multiple echo sequences, optimized for for the PET imaging of tumors and their metastases. fatty tissue imaging. In the present work we demonstrate that xerograph breast This approach provides a novel method for imaging local tumors in mice can be imaged with high level of contrast anisotropy in tissue: we model a slab of brain tissue (for using CEST of either 2DG or FDG. During the first 30 min example) as if it were a container of water with an irregular following 2DG or FDG i.v. injection the CEST at the tumors boundary, and then compare observed and predicted iDQC increased gradually, reaching values of over 25% and then re- images to measure the "dipolar surprisal". A similar method mained constant for more than 2 hours. The 2DG-CEST is valuable for enhance field perturbations due to SPIONS, tumor images were similar to the optical and two photon im- long-lived hyperpolarized agents, or other contrast mecha- ages of the mCherry/2-NBDG labeled tumor cells. nisms. The accumulation of 2DG6P and FDG6P and their phos- ACKNOWLEDGEMENTS: NSF, NIH NIBIB phorylated metabolic products in the tumors was quanti- tatively measured using 31P NMR spectroscopy of the ex- OP139: Capturing Signals from Ultrafast Relaxing tracted tumors. The concentration of these metabolites was Spins with SWIFT MRI found to exceed 13mM across the total tumor volume. Thus 2DG/FDG CEST MRI has the potential to replace FDG- ∗Michael Garwood PET for the detection of tumors and metastases, distinguish- Center for Magnetic Resonance Research, University of Min- ing between malignant and benign tumors and monitoring tu- nesota, USA mor response to therapy, without the need for radio-labeled This presentation focuses on a radically different approach isotopes. to MR imaging called SWIFT (sweep imaging with Fourier transformation). SWIFT exploits simultaneous or time- UP141: Physiological Model for the Determination 17 shared excitation and acquisition to preserve signals from of rCMR(O2) and rCBF by O MRI in the Human spins with extremely short transverse relaxation times, T2 ∗ ∗ Daniel Fiat and T2 . In SWIFT, spins are excited using a frequency sweep to minimize the peak RF power needed to excite a Universityof Illinois at Chicago broad and uniform response in the presence of the encod- A novel physiological model was developed that allows to ing gradient. With SWIFT, time domain data (k-space) is replace the previous method which required 17O NMR mea- 17 created from FIDs acquired in a radial manner (i.e., with surements of arterial blood. The model provides H2 O con- T spokes radiating out from k = 0). 1-weighting can be pro- centrations in the body organs, including the brain, the veins duced by appropriately setting the flip angle and repetition and the arteries as a function of rCMR(O2) and rCBF. A time (TR), like in conventional gradient echo (GRE) imag- ∗ least square fit of the model as a function of rCMR(O2) T T 17 ing. However, SWIFT avoids bias from 2 or 2 -weighting and rCBF to O MRI data allows the determination of TE because there is no echo time ( ). The latter feature is be- rCMR(O2) and rCBF. ing exploited in dynamic contrast-enhanced (DCE) MRI to ∗ minimize T2 -bias in the shape of the time-intensity curve Previously determination of Regional cerebral Metabolic and thus to improve estimates of perfusion parameters us- Rate of Oxygen (rCMR(O2)) and Regional Cerebral Blood 17 ing pharmacokinetic modeling. Furthermore, SWIFT pre- Flow (rCBF) was carried out by measurement of O signal 17 17 serves frequency-shifted signals in the vicinity of magnetic ob- intensity by O MRI and the measurement of H2 O concen- 17 jects. For example, magnetically labeled nanoparticles (e.g., trations in the arterial blood as a function of O inhalation 17 SPIOs), which cause signal voids in GRE images, give rise to time during the inhalation and washout periods. H2 O con- positive contrast (bright spots) in SWIFT images. The latter centration measurements in arterial blood were carried out 17 capability is being exploited to track cells, identify therapeu- by O NMR of blood samples withdrawn from the arteries. tic targets (eg, cancer-specific receptors), and to predict ther- For the following reasons it was desired to find alternative apeutic efficacy using magnetic fluid hyperthermia for cancer methods: treatment. Finally, rather unexpectedly, with SWIFT small magnetic susceptibility variations in tissues give rise to fre- 1) Withdrawal of blood from the arteries, although consid- quency shifts that manifest as phase contrast, like that seen ered safe, involves discomfort to the patient and requires in GRE images. Phase contrast is obtained with SWIFT de- skilled physician. TE spite the lack of echo ( =0) and can be visualized without 2) Ideally the NMR measurements should be carried out dur- image post-processing (ie, no need for phase-unwrapping al- ing the inhalation study to correspond to the time of mea- gorithms or high pass filtering). In summary, SWIFT is a 17 17 surement of H2 O cerebral concentrations by O MRI as new tool with many promising prospects for expanding the the metabolism of oxygen in the red blood cells continues to capabilities of MRI. some extent in the test tube. This requires the use of MRI ACKNOWLEDGEMENTS: NIH P41 EB15894, Keck Foun- and NMR equipment during the inhalation study. dation, Komen Foundation The basic equations utilized for the determinations of rCMR(O2) and rCBF using arterial experimental values as UP140: Molecular imaging of tumors using CEST well as the model equations and the model algorithm will be MRI of 2DG and FDG described. 1Michal Rivlin, 2Galia Tzarfaty, 1Judith Horev, 1Ilan The results of the data analysis by means of the model algo- Tzarfaty, 1∗Gil Navon rithm will be presented.

61 Abstracts of Talks – Physics

The results conform with accepted literature values for the OP144: Loschmidt Echoes as Quantifiers of Deco- whole brain. herence, Quantum Phase Transitions and Thermal- ization in Interacting Spin Systems OP142: Magnetic Susceptibility Contrast in Human 1,2∗ 1,2 Brain Horacio M. Pastawski, Patricia R. Levstein 1LaNaIS de RMS, Instituto de Física Enrique Gaviol ∗ Jeff Duyn, Peter van Gelderen, Pascal Sati, Afonso Silva, (CONICET-UNC), 2Facultad de Matemática Astronomía y Daniel Reich, Hellmut Merkle, Jacco de Zwart Física, Universidad Nacional de Córdoba Laboratory of Functional and Molecular Imaging, National The Loschmidt Echo [LE] is the amount of signal recovered Institutes of Health, Bethesda from a spreading or decaying excitation after an imperfect At high magnetic field strength (7T and above), in vivo MRI time reversal procedure [1]. As such, it recognizes a first can visualize human brain structure in exquisite detail. An NMR antecedent in the Hahn Echo and followed by the Magic important aspect of high field MRI is the increased magnetic Eco, the Polarization Echo and the Multiple Quantum Coher- susceptibility contrast, which is originates from the subtle dif- ence experiments. In crystals with dipolar interacting spins it ferences in the extent to which brain tissues becomes magne- has allowed us to quantify the decoherence induced by uncon- tized by application of an external magnetic field. This type trolled environment or experimental errors [2]. Notably com- of contrast allows visualization of neuroanatomical structure plex many-body dynamics makes the system particularly sen- not otherwise visible by imaging and has catalyzed the devel- sitive to environmental disturbances beyond a small thresh- opment of methods to map magnetic susceptibility distribu- old [3]. In this talk, I will summarize, at a tutorial level, the tions in vivo. In this presentation, we will review the major experimental and theoretical results of our group on the LE contributors to magnetic susceptibility contrast, with a focus that fueled the fields of quantum chaos, quantum optics and on observations in white matter. Specifically, contributions classical-quantum correspondence [4]. This also will lead us of iron, myelin and deoxyhemoglobin will be discussed. In to discuss situations where swiping the environment strength addition it will be discussed how axonal fiber bundles may induces non-analytic changes in the dynamical behavior, i.e. introduce orientation-dependent contrast that, under certain quantum dynamical phase transition [QDPT] [5]. In a spec- conditions, allows the distinction of intra-and extra cellular tral analysis a QDPT manifest as the collapse of two spectral water. lines into a short-lived and long-lived state with the same frequency. We briefly discuss how these long-lived states are rd now helping to design quantum memories [6]. More recently, Thursday – May 23 we have shown that, by filtering trivial parts in the dynamics Physics [6], the numerical evaluation of the LE can be a useful tool Chair: Jose Schneider to asses thermalization in interacting many-body systems. Room Ônix [1] Environment-independent decoherence rate in classically OP143: Dynamics and Quantum Information Trans- chaotic systems. R. Jalabert and HMP, Phys. Rev. Lett. 86, port in Nuclear Spin Chains 2490 (2001)

∗Paola Cappellaro [2] Attenuation of polarization echoes in NMR: A test for the emergence of Dynamical Irreversibility in Many-Body Quan- Massachusetts Institute of Technology tum Systems. P.R. Levstein, G. Usaj, HMP, J. Chem. Phys. A promising approach toward a scalable quantum informa- 108„ 2718 (1998) tion processor is a distributed architecture, where quantum spin wires connect small computational nodes. Coherent [3] A Nuclear Magnetic Resonance answer to the Boltzmann- transmission of quantum information over short distances is Loschmidt controversy? HMP, G. Usaj , P. R. Levstein, J. enabled by internal couplings among spins, ideally aligned in Raya and J. Hirschinger. Physica A 283 166 (2000) a one-dimensional (1D) chain. [4] Loschmidt Echo A. Gousev, R.A. Jalabert, HMP Similarities between the transport properties of pure and and D.A. Wisniacki. Scholarpedia 7, 11687 (2012) mixed-state chains enable protocols for the perfect trans- http://www.scholarpedia.org/article/Loschmidt_echo fer of quantum information and entanglement in mixed-state chains [1]. [5] Environmentally induced quantum dynamical phase tran- sition in the spin swap operation, G.A.Álvarez, E.P.Danieli, Owing to their unique geometry, nuclear spins in apatite crys- P.R. Levstein, and HMP, J. Chem.Phys. 124, 1 (2006) tals provide an ideal test-bed for the experimental study of quantum information transport, as they closely emulate an [6] Unforgeable noise-tolerant quantum tokens, F. Pastawski, ensemble of 1D spin chains. In this talk I will describe how N. Y. Yao, L. Jiang, M.D. Lukin, and J. I. Cirac, PNAS 109 NMR techniques can be used to drive the spin chain dynam- (40) 16079 (2012) ics and probe the accompanying transport mechanisms. Recently, we demonstrated initialization and read-out capa- [6] Loschmidt echo as a robust decoherence quantifier for bilities in these spin chains, even in the absence of single- many-body systems P.R. Zangara, A.D. Dente, P.R. Levstein, spin addressability [2]. It thus becomes possible to explore and HMP, Phys. Rev. A 86, 012322 (2012) experimentally the effects of discrepancy from the ideal 1D nearest-neighbor coupling model and the perturbation due to UP145: Origin of Long-Lived Signals in Dipolar Cou- the interaction of the chains with the environment [3,4]. pled Spin Systems

References 1∗Alexej Jerschow, 1Jae-Seung Lee, 2Anatoly Khitrin [1] P. Cappellaro, L. Viola and C. Ramanathan, Phys. Rev. 1New York University, 2Kent State University A 83, 032304 (2011). One typically assumes that one cannot excite narrow lines in [2] G. Kaur and P. Cappellaro, New J. Phys. 14, 083005 a system with a high degree of dipolar broadening. Never- (2012). theless, several examples of this effect were shown in liquid crystals, polymers, and ordinary solids by applying a long and [3] W. Zhang, P. Cappellaro, N. Antler, B. Pepper, D. G. weak rf irradiation (we call this signal long-lived response – Cory, V. V. Dobrovitski, C. Ramanathan and L. Viola, Phys. LLR) [1-4]. Most recently, such signals were also excited in Rev. A 80, 052323 (2009). samples of cortical and trabecular bone [5]. Such long-lived [4] C. Ramanathan, P. Cappellaro, L. Viola and D. G. Cory, signals would naturally be of great utility in imaging rigid New J. Phys. 13, 103015 (2011). samples, or rigid tissue components.

62 Abstracts of Talks – Membrane Proteins

In this paper, we describe how these long-lived signals may [4] Vold, R. L. and Vold, R. R., J. Magn. Res. 13, 38-44 arise as a consequence of the direct excitation of highly cor- (1974) related states. In the presence of small chemical shift differ- ences, cancellations between certain spin operators no longer OP147: Using single spins for quantum computing occur (e.g. between terms IiyIjz and IizIjy), and a narrow and sensing signal could be seen. After the end of the LLR irradiation, ∗ highly correlated terms of the type IiyIjzIkzImz. . . can be Dieter Suter converted into observable magnetization by successive action TU Dortmund of the dipolar couplings between spin i and other spins in- volved in this correlated term. This approach may also ex- Information processing on the basis of quantum mechani- plain why highly-correlated terms lead to long-lived signals, cal systems promises breakthroughs in the processing speed. since, e.g. dipolar couplings between any j, k, m,. . . spins The realization of this potential depends on the availability leave the term IiyIjzIkzImz intact. of suitable quantum systems that must be controllable with high precision and conserve the stored information for the du- These simulations point to the fact that the experimentally ration of the computation. Spins are in many respects ideal observed long-lived signals are most likely due to the gen- quantum bits, and most of the demonstration experiments for eration of these highly correlated spin terms. Experiments quantum information processing have been performed with highlighting the unusual character of the excited signals will spins as qubits. Unfortunately, the most easily accessible be shown as well. spins, nuclear spins in liquids, cannot be initialized into pure quantum states, which is a requirement for many quantum al- References: gorithms. The nitrogen-vacancy center in diamond, however, [1] A.K. Khitrin, J. Magn. Reson. 213 (2011) 22-25. is a spin that allows optical initialization into a nearly pure state as well as the precise readout of single spins by optical [2] A.K. Khitrin, J. Chem. Phys. 134 (2011) 154502-154509. techniques. We use this system for demonstrating basic steps [3] A.K. Khitrin, V.L. Ermakov, and B.M. Fung, J. Chem. of quantum information processing, such as gate operations Phys. 117 (2002) 6903-6906. or protection of quantum information against environmental noise. In addition, these systems can be used as point-like [4] A.K. Khitrin, V.L. Ermakov, and B.M. Fung, Chem. sensors for different environmental variables, such as electric Phys. Lett. 360 (2002) 161-166. and magnetic field, temperature or pressure. [5] B. Zhang, J.-S. Lee, A. Khitrin, A. Jer- th schow, 2012, arXiv:1209.4030 [physics.med-ph], Friday – May 24 http://arxiv.org/abs/1209.4030. Membrane Proteins Chair: Laurent Catoire UP146: High Resolution Para-Hydrogen Induced Room Gávea A Polarization (PHIP) in Inhomogeneous Magnetic Fields OP148: NMR studies on the type IV secretion sys- 1,2∗Lisandro Buljubasich, 1,2Ignacio Prina, 3María Belén tem of Xanthomonas citri Franzoni, 3Kerstin Münnemann, 3Hans Wolfgang Spiess, 1,2 Diorge Souza, Luciana Coutinho de Oliveira, Denize C. Rodolfo Héctor Acosta ∗ Favaro, Cristina Alvarez-Martinez, Chuck Farah, Roberto 1FAMAF – Universidad Nacional de Córdoba, 2IFEG Salinas 3 –CONICET, Max Plank Institut für Polymerforschung Department of Biochemistry, Institute of Chemistry, USP The application of parahydrogen for the generation of hyper- Gram-negative bacteria use specialized supramolecular com- polarization has increased continuously during the last years. plexes to secrete macromolecules across the bacterial cell en- When the chemical reaction that deposits the parahydrogen velope. One such complex is the Type IV Secretion System atoms into the target molecule is carried out at the same field (T4SS) [1]. T4SSs are generally composed of 12 proteins, as the NMR experiment (PASADENA protocol [1]) an anti- VirB1 to VirB11 and VirD4 [2]. The upper layer of the chan- phase signal is obtained, with a separation of the resonance nel of the T4SS consists of fourteen repetitions of an het- lines of only a few Hz. This imposes a stringent limit to the erotrimer formed by VirB7 and the C-terminal domains of required homogeneity of the magnetic field in order to avoid VirB9 (VirB9CT ) and VirB10 [1,2]. We showed previously signal cancellation. In this work we show that the spectrum that the VirB7 of the phytopathogen Xanthomonas citri of the PHIP signal acquired with a Carr-Purcell-Meiboom- (Xac) has an extra C-terminal globular domain (residues 52- J Gil (CPMG) sequence, referred to as -Spectroscopy [2-4], 133) that is absent in the VirB7 of other organisms [3]. This not only presents an enhanced spectral resolution compared finding suggests a structural variation in the T4SS of Xan- to standard the NMR-spectrum, but also avoids partial peak thomonadaceae. In order to obtain further high-resolution cancellation. structural information on Xac’s T4SS, we initiated a study Experimental and numerical simulations concerning the hy- of the three-dimensional structure of the complex formed by CT NT drogenation of Hexene with parahydrogen in PASADENA Xac-VirB9 and Xac-VirB7 . conditions are presented. Acquisition with a digital filter References: is used to select a desired multiplet, namely a partial J- Spectrum acquisition [3]. The performance of the method [1] Chandran, V., Fronzes, R., Duquerroy, S., Cronin, N., is tested on a thermally polarized sample, showing that the Navaza, J., Waksman, G. (2009). Nature 462, 1011–1015. corresponding partial J-Spectra are unaffected by large inho- [2] Zechner, E.L., Lang, S., and Schildbach, J.F. (2012). Phi- mogeneities in the polarizing magnetic field. Finally, limita- los. Trans. R. Soc. Lond. B Biol. Sci. 367, 1073–1087. tions and applicability of the method to obtain either spectral information of the sample or to monitor chemical reactions [3] Souza, Diorge P., Andrade, Maxuel O., Alvarez-Martinez, of very diluted samples will be discussed. Cristina E., Arantes, Guilherme M., Farah, Chuck S., Sali- nas, Roberto K. (2011). PLoS Pathogens 7, e1002031. References

[1] Russell, C. and Weitekamp, D. P., J. Am. Chem. Soc. OP149: Magic Angle Spinning Solid-State NMR 109, 5541-5542 (1987) Studies of Membrane Proteins

[2] Allerhand, A, J. of Chem. Phys. 44 (1966) ∗Vladimir Ladizhansky [3] Freeman, R. and Hill H. D. W, J. Chem. Phys. 54 (1971) University of Guelph

63 Abstracts of Talks – Instrumentation

Magic angle spinning (MAS) solid-state NMR (SSNMR) Friday – May 24th shows great potential for studying structure and dynamics Instrumentation of membrane proteins, which are not readily amenable to X- ray crystallography and solution NMR. In this presentation Chair: Mario Engelsberg we will describe the latest developments in our group, includ- Room Gávea B ing advances in sample preparation procedures for obtaining MAS spectra with optimal spectral sensitivity, experimen- OP152: Robust Multi-Phase Flow Measurements tal strategies for obtaining spectroscopic assignments, meth- Using Magnetic Resonance ods for measuring long-range structural restraints for the de- 1,2∗ 1 termination of high resolution structure and supramolecu- Daniel José Pusiol, Lucas C. Cerioni lar organization of membrane proteins in the lipid environ- 1Instituto de Física Enrique Gaviola IFEG, CONICET, Ciu- ment. We will present high-resolution structure of a seven- dad Universitaria, Córdoba, Argentina, 2SPINLOCK SRL & helical photoreceptor Anabaena Sensory Rhodopsin, and dis- CONICET, Córdoba, Argentina cuss progress towards structural characterization of a eukary- The flow metering of multiphase fluids is an actual technical otic membrane water channel human Aquaporin 1. challenge, especially when flow velocity is in the range of 0.1 – 10 m/sec. The primary complication is the existence of ’phase slip’ between fluid components. Without physically UP150: Comparison of Structure of Cyanobacateria separating individual fluid components of multi-phase flow, and Spinach PSII Studied by PELDOR it is impossible to accurately determine the flow rate of each 1∗ 2 3 component from the measurement of physical properties that Asako Kawamori, Jiang-Ren Shen, Hiroyuki Mino are related to the average fluid displacement in the pipe. A 1AGAPE-Kabutoyama Institute of Medicine, 2Department non-linear system of fluid phase velocity and cross-sectional of Biology, Okayama University, 3Department of Physics, area occupied by each fluid phase is established during multi- Nagoya University phase flow, which cannot be adequately approximated by cor- The Structure of Cyanobacterum has been determined re- relations derived from macroscopic properties such as fluid cently with resolution of 1.9 Å (Umena et al. NATURE density or pressure drop. (2011) 473 55) We have determined the distances between Magnetic resonance is intrinsically sensitive to flow; how- electron transfer components of PSII in Spinach with PEL- ever, current applications to flow measurement are limited DOR( Pulsed Electron Double Resonance) with accuracy of to very low flow rates that are inapplicable to oil-field and 1 Å, 27 Å for YD-YZ ,YD-Mn4 cluster by measurement of other industrial applications. In this paper we discuss real- dipolar interactions between radicals. Usually Electron Spin time quantification NMR-based methods of high-flow rates is not localized and hyperfine constants reflecting spin distri- in multiphase fluids. In particular, we focalize in a recent bution are observed. Point dipolar approximation is not cor- development which employs a new principle that enables ro- rect to determine radical distances. In this report we present bust 3-phase flow metering with a broader operating envelope the distance estimation by taking account of spin distribu- than existing flow meters, has no sensors in the flow stream, tion. Typically distances of chlorophylls and carotenoid from no radioactive source, yet could potentially be produced at YD are reported based on spin distributions, which were de- sufficiently low cost to promote increasing numbers of well rived hyperfine constants of each radical, and crystal struc- head installations. ture data. ACKNOWLEDGMENTS: CONICET, FONTAR, SPIN- LOCK In a cynobacterium the distances obtained by PELDOR are not coincident with those in spinachs but different values are OP153: Using Time Domain NMR to Study Magne- obtained. Several radical pairs produced by light illumination toelectrolysis phenomenon in Situ will be compared for cyanobacteria and spinaches. 1∗Luiz Alberto Colnago, 1Luis Fernando Cabeça, 2Luiza Maria da Silva Nunes, 2Paulo Falco Cobra, 2Bruna Ferreira 1 OP151: Structure Determination of Membrane Pro- Gomes, André de Souza Carvalho teins in their Native Environment of Phospholipid 1Embrapa Instrumentação, São Carlos/SP - Brazil, Bilayers 2Instituto de Química de São Carlos - USP

∗Stanley Opella Recently we demonstrate that time domain NMR (TD-NMR) relaxometry can be a very fast, simple, and efficient tech- University of California, San Diego nique to monitor the variation in the Cu2+ concentration NMR spectroscopy is eminently capable of solving the during an electrodeposition reaction (Anal. Chem. 84, 6351, structures of macromolecules, including proteins, as solids, 2012). In this talk, the effect of NMR magnetic field in whether polycrystalline or amorphous, or in aqueous solu- the electrodeposition reaction (magnetoelectrolysis) will be tion. However, biological processes rarely occur in such clear- presented. The interest in the process is related to the possi- cut physical states. Membrane proteins function in liquid bility of controlling the deposit morphology, mass transport crystalline phospholipid bilayers. They are "immobilized" on and the reaction rate. The effect of the magnetic field on NMR timescales, except for rapid rotational diffusion about electrodeposition reaction has been related to the magneto- the bilayer normal, and some local internal motions, primar- hydrodynamic force (FB ) and the magnetic gradient field ily at the N- and C- termini. A recently developed approach, force (F∇B ). The TD-NMR analyses were performed in Rotationally Aligned (RA) Solid-State NMR is ideally suited SLK-100 0.23 T, TD-NMR spectrometer (Spinlock) and 0.35 for structure determination of membrane proteins; it merges T homemade unilateral NMR sensor. The electrochemical many aspects of Magic Angle Spinning (MAS) Solid-State cell was describe elsewhere. The reactions were performed NMR and Oriented Sample (OS) Solid-State NMR into a without the magnetic field (W) and with magnetic field with method that measures angles between bonds and the bilayer the cell in the center (C) and top border (T) of the bench top normal. Supplemented with other angles and distances, this magnet and at the top of unilateral sensor (U). The W, U, T approach enables the structures of membrane proteins to be and C reactions removed 37, 47, 53 and 60% of the Cu2+ in determined with atomic resolution in proteoliposomes under solution during 1-hour reaction, respectively. The W reaction physiological conditions. Complementary experiments de- was the slowest one because reaction is dependent only on scribe the global and local motions of these proteins in the the convection of the Cu2+ caused by concentration gradient. bilayer environment. Examples of proteins that range from The reactions in the magnetic field were faster due to the single domains with one trans-membrane helix to GPCRs Cu2+ convection caused by the magnetoconvection forces. with seven trans-membrane helices will be described. The C reaction was the fasted one due FB that causes a

64 Abstracts of Talks – Innovative NMR/MRI Methods

flux perpendicular to B0 increasing the mass convection and than modern superconducting magnets. Yet in recent times, consequently the electrodeposition. The U and T reactions compact permanent magnets have been developed for NMR, rate decrease when compared to C, because the magnetic which are light enough to be carried to the object or the pro- field gradient causes a strong F∇B toward the bottom of the duction line for analysis [1]. As NMR relaxation can be mea- cell, reducing the copper concentration at the top of the cell, sured in the inhomogeneous stray field, a portable stray-field around the working electrode. We conclude that TD-NMR NMR device known as the NMR-MOUSE has been developed can be a useful tool to study magnetoelectrolysis in situ. for non-destructive materials testing [2,3]. It is particularly well suited for studying rubber materials and the morphology ACKNOWLEDGMENTS: FAPESP, CNPq and semi-crystalline polymer products as a function of pro- cessing parameters and service time. Current research aims UP154: In situ MR: Pore condensation at elevated at predicting the residual service time of polymer pipes from temperature and pressure such measurement [4]. In another project, the use of a desk- 1∗ 1 2 top MRI magnet is investigated to acquire images of rubber Matthew P. Renshaw, S. Tegan Roberts, Belinda S. Akpa, 1Mick D. Mantle, 1Andrew J. Sederman, 1Lynn F. gaskets within a few seconds, from which the position of the Gladden inner surfaces can be extracted for control the production process [5]. Last but not least, small magnets are employed 1 2 University of Cambridge, University of Illinois at Chicago to measure 1H NMR spectra in real time under the fume hood Magnetic Resonance (MR) methods, being non-invasive and to follow chemical reactions. These and other applications of non-destructive, are ideal for probing heterogeneous catalytic compact, state-of-the art NMR magnets will be reported [6]. systems under realistic operating conditions [1,2]. However, References until recently, the processes studied using MR have been performed under mild conditions (typically < 5 atm and < [1] B. Blümich, F. Casanova, E. Danieli, J. Perlo, St. Appelt, 200 ◦C). We have recently commissioned a fixed-bed reactor, Sub-Compact NMR, in: C.L. Khetrapal, A. Kumar, K.V. Ra- compatible with operation inside a superconducting magnet, manathan (eds.), Future Directions of NMR, Springer (India) which can be operated up to a temperature of 350 ◦C and 2011, pp. 1-10 a pressure of 31 atm while simultaneously performing MR [2] B. Blümich, F. Casanova, J. Perlo, Mobile single-sided experiments. NMR, Progess Nucl. Magn. Reson. Spectrosc. 52 (2008) To demonstrate its capability we have studied the effect of 197–269 confinement in mesopores on the vapour-liquid phase change [3] F. Casanova, J. Perlo, B. Blümich, eds., Single-Sided of cyclohexane to elucidate pore filling and emptying mecha- NMR, Springer, Berlin, 2011 nisms. The understanding of confinement effects, particularly at realistic reaction conditions, is an important consideration [4] R. Kwamen, B. Blümich, A. Adams, Estimation of Self- in heterogeneous catalysis. Diffusion Coefficients of Small Penetrants in Semicrystalline Poymers Using Single-Sided NMR, Macromol. 33 (2012) 943- Isothermal (150 ◦C and 188 ◦C) vapour-liquid phase change 947 cycles of cyclohexane in a bed of titania pellets were con- ducted. 1H spin density images reveal changes in pore satu- [5] E. Danieli, K. Berdel, J. Perlo, W. Michaeli, U. Mas- ration of the pellets as the phase boundary is crossed. Pore berg, B. Blümich, F. Casanova, Determining object bound- confinement effects are exhibited, as liquid remains within the aries from MR images with sub-pixel resolution: Towards in- pores after bulk liquid has vaporised. Pore saturation dur- line inspection with a mobile tomography, J. Magn. Reson. ing the evaporation-condensation cycles exhibits hysteresis, 207 (2010) 53–58 demonstrating the differences between the filling and emp- [6] S. Küster, E. Danieli, B. Blümich, F. Casanova, High reso- tying mechanisms in these systems. The data are analogous lution NMR spectroscopy under the fume hood, Phys. Chem. to N2 adsorption isotherms that characterise pore diameter Chem. Phys. 13 (2011) 13172-13176 and surface area, but are conducted for a relevant species at realistic conditions. th 1 Friday – May 24 Data from these MR images, in conjunction with H T1 and Innovative NMR/MRI Methods T2 relaxation time measurements and pulsed field gradient diffusion measurements, are used to assess the mechansim of Chair: Betty Gaffney pore filling and emptying in addition to revealing information Rooms Turmalina/Topázio about the formation of liquid films. The use of the in situ MR reactor is currently being extended OP156: Relaxometry and dynamics to study heterogenous catalytic reactions. ∗ Claudio Luchinat [1] Gladden et al., Catalysis Today, 155 (2010) 157-163. Magnetic Resonance Center (CERM), University of Flo- [2] Lysova and Koptyug, Chemical Society Reviews, 39 (2010) rence 4585-4601. Field-cycling relaxometry experiments provide longitudinal relaxation rates of sample nuclei (usually abundant, high-γ OP155: Compact NMR of Materials and Processes nuclei) over a wide field range, so that the whole spectral density is experimentally and directly available. Recent ap- ∗ Bernhard Blümich plications of relaxometry comprise the analysis of the spectral RWTH Aachen University density of water protons for the characterization of contrast agents [1], for a better understanding of dynamic nuclear po- Typical NMR spectrometers use superconducting magnets, larization (DNP) experiments [2], and for acquiring infor- which are heavy, expensive and require high maintenance. mation on protein aggregation and dynamics in folded and This is one reason, why the reputation of NMR is perceived intrinsically disordered proteins. The analysis of the collec- to be more demanding than that of many other analytical tive spectral density of protein protons in D2O solutions is methods including IR spectroscopy and DSC. Yet NMR is also feasible [3] to obtain information on protein dynamics in also versatile. It provides spectra for chemical analysis, im- terms of reorientation time and generalized order parameter. ages that give insight into sample heterogeneity and func- tion of devices, and relaxation parameters, which scale with Relaxivity profiles can provide reorientation times of solute mechanical material properties [1]. While superconducting molecules up to the microsecond range, so that the tumbling magnets are common today in NMR, permanent magnets time of proteins as large as hundreds kDa can be accessed. have been used decades before. They were temperature sen- In sedimented proteins [4], they can provide information on sitive, hard to shim, heavy, and produce fields much lower the intervening motions.

65 Abstracts of Talks – Small Molecules and Bioactive Peptides

In the context of the characterization of polarizing agents generated was greater in mitochondria isolated from diabetic for solution DNP experiments, relaxation profiles of solvent muscle. Moreover, ROS production began to increase at a water protons in the presence of nitroxide radicals or para- lower threshold for inner membrane potential in diabetic mi- magnetic metal complexes have been analyzed to obtain the tochondria. In summary, we describe a novel methodology structural and dynamic parameters needed for determining for measuring ATP production and provide new mechanistic the coupling factor, on which the solvent DNP enhancement insight into the dysregulation of ATP production and ROS depends. 1H relaxometry thus represents an easy way to es- in mitochondria of insulin deficient rodents. timate the DNP enhancement at full electron saturation as a function of the applied magnetic field. OP159: Using SABRE as a route to hyperpolariza- [1] Mastarone, Harrison, Eckermann, Parigi, Luchinat, tion in NMR and MRI Meade, J. Am. Chem. Soc. (2011) 133, 5329. 1∗Simon B Duckett, 1Kevin D. Atkinson, 1Alex J. J. 1 2 1 [2] Bennati, Luchinat et al. J. Am. Chem. Soc. (2008) Hooper, Lyrelle S. Lloyd, Gary G. R. Green, Marianna 1 1 1 130, 3254; J. Am. Chem. Soc. (2009) 131, 15086; Fekete, Richard A. Green, Ryan E. Mewis, Louise A. R. Phys.Chem.Chem.Phys. (2010) 12, 5902; Phys. Chem. Highton Chem. Phys. (2012) 14, 502 1York Centre for Hyperpolarisation In Magentic Resonance, 2York Neuroimaging Centre [3] Luchinat, Parigi, J. Am. Chem. Soc. (2007), 129, 1055. In medicine MRI plays a role in non-invasive diagnosis and it [4] Bertini, Luchinat, Parigi, Ravera, Reif, Turano, Proc. also plays a significant role in clinical research. However, MRI Natl. Acad. Sci. USA (2011) 108, 10396. suffers inherently from the underlying physical basis of the method which limits it to the detection of effectively only 1 OP157: The Nitrogen-Vacancy Center In Diamond in every 200,000 hydrogen atoms present in the sample when As A Nanoscale Spin Sensor working at physiological temperatures and a magnetic field strength of 1.5 T as routinely used in clinical MRI. 1Abdelghani Laraoui, 2Florian Dolde, 2Tobias Staudacher, 2Jörg Wrachtrup, 2Friedemann Reinhard, 1∗Carlos A. Hyperpolarization deals with the generation of non- Meriles equilibrium populations of these nuclear spins, and provides 1Department of Physics, CUNY - City College of New York, enhanced sensitivity to both NMR and MRI experiments. New York, 23rd Physics Institute, University of Stuttgart, One variant of this is dynamic nuclear polarization (DNP) 13 Germany which dramatically increases the sensitivity of C detection. The Nitrogen-Vacancy (NV) center in diamond – a spin-1 An alternative approach to achieve substrate hyperpolariza- point defect formed by a substitutional nitrogen adjacent to tion, as pioneered by Weitekamp, involves the use of parahy- a vacancy site – is presently the focus of intense research in drogen, a molecule that exists in a pure magnetic state. How- areas spanning physics, chemistry, and biology. Thanks to ever, it is not parahydrogen itself that is detected in these ex- their long spin coherence times at room temperature, NV periments, but rather reaction products that are formed by a centers within diamond nanostructures are being explored as metal catalyzed hydrogenation. The newly formed molecules a platform for various technologies, most notably nanoscale now contain non-equilibrium spin state populations for nuclei resolution MRI. Here, I will present recent results on the use that are spin coupled to protons that were originally located of near-surface NVs for nuclear spin spectroscopy, including in parahydrogen. Consequently they yield MR signals that the detection of proton spins from an organic film deposited are substantially larger than normal. Indeed 100% polariza- on the diamond crystal surface (the effective sample volume tion has been generated for a pair of metal hydride protons being approximately (5 nm)3). I will also discuss recent ex- using this method. periments with diamond nanocrystals, where we use the NV A newer route to substrate hyperpolarization with parahy- as a probe to detect other, optically-inactive paramagnetic drogen has been termed signal amplification by reversible centers within the particle and on its surface. exchange (SABRE). This approach yields substantial polar- ACKNOLEDGEMENTS: National Science Foundation, Re- ization without the need for chemical modification. It is search Corporation, Alexander von Humboldt Foundation. achieved instead through the temporary binding of a sub- strate and parahydrogen in a suitable transition metal com- UP158: Mitochondrial function in diabetes: Novel plex. This process of bringing together two materials, for ex- methodology and new insight ample nicotine and parahydrogen, into temporary contact en- ables the sharing of their magnetization and hence the spon- ∗Liping Yu, Brian Fink, Judith Herlein, William Sivitz taneous enhancement of the MR signals of what correspond University of Iowa to ultimately the free substrate. This talk will illustrate how this approach is developing in York. Diabetes is a group of metabolic diseases associated with mal- function of mitochondria. Major metrics used to describe th mitochondrial function include oxygen consumption (respi- Friday – May 24 ration), membrane potential (∆Ψ), ATP production, and Small Molecules and Bioactive Peptides generation of reactive oxygen species (ROS) in the form of Chair: Marta Bruix superoxide. Interpreting mitochondrial function as affected Room Ônix by comparative physiologic conditions is confounding since these individual functional parameters are inter-dependent. OP160: NMR Studies of the Self-Association and In this study, we investigated the relationships between the Membrane Binding of Cyclotides parameters of muscle mitochondrial function as affected by insulin deficiency. In particular, we examined ATP produc- ∗David J Craik, Conan Wang, K Johan Rosengren, Anne tion in a unique way that provides new information about the Conibear, Sonia T Henriques function of mitochondria. To achieve our objectives, we de- Institute for Molecular Bioscience, The University of veloped a novel, highly sensitive and specific ATP production Queensland, Australia assay using NMR spectroscopy of mitochondria at clamped levels of ∆Ψ. ROS production can also be assayed simulta- NMR is a powerful technique for determining the structures, neously. The NMR techniques reported in this study proved dynamics and interactions of peptides and proteins. Here far more sensitive than conventional 31P NMR and allowed we describe the application of NMR to the study of cyclic high throughput study of small mitochondrial isolates. Over disulfide-rich peptides. Because of their exceptional stability conditions ranging from state 4 to state 3 respiration, ATP these peptides make valuable leads in drug design applica- production was found to be lower and ROS per unit ATP tions and have been used as templates to stabilize bioactive

66 Abstracts of Talks – Small Molecules and Bioactive Peptides peptide epitopes. Of particular focus are the cyclotides,[1] [7] C. Dalvit, J. M. Böhlen, NMR in Biomedicine 1997, 10, which are 30 amino acid peptides from plants that have 285. a broad range of biological activities, including antimicro- bial, anticancer, anti-HIV and insecticidal activities. The cy- ACKNOWLEDGEMENT clotides are particularly amenable to NMR studies and give This work was supported by ANR (ANR-08-BLAN-273) and well dispersed spectra. We have used a range of NMR tech- Region PACA (APO-G 2009). niques to determine the three-dimensional structures, iden- tify key hydrogen bonding networks, and determine the na- UP162: Structural Basis for the Interaction of Hu- ture of intermolecular interactions of cyclotides, with a par- man β-Defensins 1 and 6 and Its Putative Chemokine ticular focus on membrane binding interactions. NMR stud- Receptor CCR2 and Breast Cancer Microvesicles ies combined with alanine mutagenesis scanning have pro- vided novel insights into the modes of interactions of cy- ∗Viviane Silva de Paula, Robson Q. Monteiro, Fabio C. clotides with membranes.[2] L. Almeida, Ana Paula Valente [1] Craik D J, Swedberg J, Mylne J S, Cemazar M: Cyclotides Universidade Federal do Rio de Janeiro as a basis for drug design. Expert Opinion on Drug Discovery Human β-defensins (hBD) are believed to function as alarm (2012) 7, 179-194. molecules that stimulate the adaptive immune system when [2] Wang C K, Wacklin H P, Craik D J: Cyclotides insert into a threat is present. In addition to its antimicrobial activity, lipid bilayers to form membrane pores and destabilize the defensins present other activities such as chemoattraction of membrane through hydrophobic and phosphoethanolamine- a range of different cell types to the sites of inflammation. We specific interactions. Journal of Biological Chemistry (2012) have solved the structure of the human β-defensins 6 (hBD6) 287, 43884-43898. by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We also investi- Work in our laboratory is supported by grants from the gated the interaction of β-defensin 1 and 6 with microvesicles Australian Research Council (DP0984390) and the National shed by breast cancer cell lines using NMR. Chemical shift Health & Medical Research Council (APP1026501). mapping of the interaction showed that both defensins inter- act with microvesicles but in slightly different way, suggesting OP161: Separative NMR: adapted resolution for an inverse correlation with the aggressiveness potential of the complex mixtures of small molecules cell. Furthermore, molecular docking using restraints derived from the NMR chemical shift data produced a model of the 1,2∗Stefano Caldarelli complex between hBD6 and a peptide derived from the extra- 1 2 Aix Marseille Université,Marseille, France, CNRS UPR cellular domain of CC chemokine receptor 2 (Nt-CCR2) that 2301 Ecole Palaiseau France reveals a contiguous binding surface on hBD6, which com- In this talk, we shall outline the results of two strategies that prises amino acid residues of the α-helix, loop between β2-β3 have been implemented recently in our laboratory as tools and C-terminal. The microvesicles binding surface partially to simplify and ameliorate the analysis of mixtures of small overlaps with the chemokine receptor interface. These data molecules. offer new insights into the structure–function relation of the hBD6–CCR2 interaction and may be helpful for the design The basis of the methods are DOSY and the use of multiple- of novel anti-cancer agents. quantum coherences. ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq - Chromatographic NMR[1, 2] is a matrix-assisted version of DOSY[3], in which the addition of a chromatographic solid phase can induce larger differences of mobility of the mix- OP163: Breaking the barrier: NMR of membrane- ture components, thus simplifying the analysis. We shall active peptides show that the method is capable of discriminating labile ∗Frances Separovic supramolecular species (interchanging chiral ion pairs), which have been elusive to conventional LC.[4] School of Chemistry, Bio21 Institute, University of Mel- bourne, Australia - Multiple-quantum coherences have interesting properties for The interaction of antimicrobial peptides (AMP) and amyloid the study of mixtures. Firstly, they produce spectra with a peptides with cell membranes is contingent on the nature of number of signals inversely proportional to the coherence or- the constituent lipids. Since eukaryotic and bacterial mem- der. This property in itself can produce spectra with very branes are comprised of different proportions of a range of high discriminating power (up to tens of molecules overlap- lipid species with diverse physical properties, we have studied ping in a couple of ppm).[5] In this talk we shall revisit diffu- these membrane-active peptides with membranes of different sion using homonuclear multiple-quantum coherences based lipid types. on J couplings.[6, 7] MQ-DOSY allows to increase the appar- ent diffusion coefficient, by the square of the MQ order. An Maculatin 1.1 is a short AMP secreted from the skin of Aus- interesting side-effect of this is that differences in mobility tralian tree frogs. This peptide has a high activity against also appear to be amplified. Combined with the reduced Gram-positive bacteria but reduced action against Gram- number of resonances produced by MQ-filter, MQ-DOSY negative bacteria and red blood cells. We have studied outperforms regular DOSY, being capable of distinguishing the interactions of maculatin 1.1 with various model mem- isomers with very similar diffusion coefficients. branes mimicking prokaryotic or eukaryotic cells to under- [1] S. Viel, F. Ziarelli, S. Caldarelli, PANS 2003, 100, 9696. stand the selectivity and mechanism of action and the role of lipids. The impact of unsaturated versus saturated fatty [2] G. Pages, C. Delaurent, S. Caldarelli, Angewandte acids, surface charge, presence of cholesterol and heteroge- Chemie-International Edition 2006, 45, 5950. neous compositions were investigated. The peptide adopted α [3] K. F. Morris, P. Stilbs, C. S. Johnson, Analytical Chem- an -helical structure in contact with eukaryotic and prokary- istry 1994, 66, 211. otic membrane-mimics and inserted into neutral membranes, even in the presence of cholesterol, but remained on the sur- [4] M. Reddy G. N, R. Ballesteros-Garrido, J. Lacour, S. Cal- face of negatively charged membranes. The dynamic and darelli, Angewandte Chemie International Edition 2013 structural changes of lipid vesicles mimicking E. coli, S. au- reus or eukaryotic plasma membranes were monitored using [5] M. G. N. Reddy, S. Caldarelli, Analytical Chemistry 2010, solid-state NMR. Maculatin 1.1 promoted a strong response 82, 3266. in saturated lipid model membranes at lower concentration [6] L. E. Kay, J. H. Prestegard, Journal of Magnetic Reso- than with E. coli lipid extracts and disrupted membranes nance 1986, 67, 103. according to their lipid composition.

67 Abstracts of Talks – Plenary Session

The 42-residue amyloid-beta (Aβ42) peptide is a fibrous con- on neural circuits and networks. The success of these studies stituent of amyloid plaques found in the brains of Alzheimer’s are driven by initiatives like the Human Connectome Project disease patients. Although the precise involvement in neu- (HCP), an ambitious effort initiated by the 16 National Insti- rodegeneration is unknown, interactions with neuronal cell tutes of Health (NIH) Institutes and Centers that support the membranes and copper ions may be involved. Our previ- NIH Blueprint for Neuroscience Research, to map the neural ous work has shown that aggregation kinetics and fibril mor- pathways that underlie human brain function and generate phology of Aβ42 is influenced by the lipid compositions of a comprehensive description of the connections among gray membranes, and we now probe the role of copper ions. Cop- matter locations in the human brain at the millimeter scale, per, at and above equimolar ratios, stabilised Aβ42 into non- using MR methodology. It is a remarkable journey from the oligomeric β-sheet structures alone and in the presence of first demonstrations by Isidor Rabi of resonant spin flips in lipids. Solid-state NMR revealed that the peptide, with and an atomic beam experiment in 1933, to the observation of without copper, significantly disrupted lipid headgroups in the Nuclear Magnetic Resonance in condensed matter by Ed brain lipid extract and phospholipid POPC/cholesterol mem- Purcell and Felix Bloch in 1945. brane systems containing 10% (molar) POPS or GM1, as in- 31 dicated by reductions in P chemical shift anisotropy, T1 PL165: Synergy between NMR, cryo-EM and large- and T2 relaxation values. Copper alone had a strong param- 31 scale MD simulations - Novel Findings for HIV Cap- agnetic effect on P relaxation values, which were enhanced sid Function in the presence of Aβ42, suggesting that the peptide does 2 not scavenge copper from the membrane. H NMR spec- ∗Angela M. Gronenborn tra did not indicate copper penetration into the hydrophobic Department of Structural Biology, University of Pittsburgh core region. By contrast, Aβ42 induced small vesicle for- mation and large changes in the headgroup and core regions Mature HIV-1 particles contain a conical-shaped capsid that of POPC/cholesterol systems containing cardiolipin, a mito- encloses the viral RNA genome and performs essential func- chondrial lipid. Our results support a superficial interaction tions in the virus life cycle. Previous structural analysis of Aβ42 with the model brain lipid membranes and demon- of two- and three-dimensional arrays provided a molecular strate the importance of lipid composition on peptide activ- model of the capsid protein (CA) hexamer and revealed three ity. interfaces in the lattice. Using the high-resolution NMR structure of the CA C-terminal domain (CTD) dimer and th in particular the unique interface identified, it was possible Friday – May 24 to reconstruct a model for a tubular assembly of CA pro- Plenary Session tein that fit extremely well into the cryoEM density map. Chair: Sonia Menezes A novel CTD-CTD interface at the local three-fold axis in Room Gávea A the cryoEM map was confirmed by mutagenesis to be es- sential for function. More recently, the cryo-EM structure PL164: Nuclear Spins, High Magnetic Fields, and of the tube was solved at 8Å resolution and this cryo-EM Traveling Waves in Pursuit of Human Brain Func- structure allowed unambiguous modeling and refinement by tion and Connectivity large-scale molecular dynamics (MD) simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer- ∗Kamil Ugurbil of-hexamer elements of spheroidal capsids. Furthermore, the CMRR, University of Minnesota, USA 3D structure of a native HIV-1 core was determined by cryo- electron tomography (Cryo-ET), which in combination with Ever since Plato and Aristotle, the unique abilities of the hu- MD simulations permitted the construction of a realistic all- man brain has been the subject of many philosophers and atom model for the entire capsid, based on the 3D authentic scientists throughout history, including Descartes who pos- core structure. tulated a dualism between mind and matter, and Helmholtz who pioneered contemporary physiology. Today, tremen- In addition, interaction with the innate immune defense dous strides are being made towards deciphering human restriction factor TRIM5α was studied. TRIM5α recog- brain function using experimental observations performed nizes the lattice of the retrovirus capsid through its B30.2 non-invasively and directly on the human brain as it executes (PRY/SPRY) domain in a species-specific manner. Upon its functions. This rapid progress has been possible because binding, TRIM5α induces premature disassembly of the vi- of neuroimaging, and in particular magnetic resonance (MR) ral capsid and activates the downstream innate immune re- imaging of the brain that has rapidly evolved over the last sponse. We have determined the crystal structure of the two decades due to incessant developments in instrumenta- rhesus TRIM5α PRY/SPRY domain that reveals essential tion, ranging from very high field magnets large enough to features for capsid binding. Combined cryo-electron mi- accommodate humans to parallel RF transmission and signal croscopy (cryo-EM) and biochemical data show that the reception, approaches to manipulating spins, and novel sig- monomeric rhesus TRIM5α PRY/SPRY, but not human nal processing schemes. These technological accomplishem- TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein as- nts have permitted mapping of neuronal activity in the hu- semblies, without causing disruption of the capsid. Our data man brain at the scale of elementary computational units and suggests a model for how this factor disrupts the virion core functionally distinct ensembles of such computational clus- and suggests that structural damage of the viral capsid by ters, complemented with "functional" and "anatomical" con- TRIM5α is likely one of the important components of the nectivity data obtained also by MR techniques to inform us mechanism of HIV-1 restriction.

68 Poster Abstracts – Biomolecular Solution NMR

Poster Abstracts

1 gp120 formed by the C4 and the base of the V3, part of Biomolecular Solution NMR which was implicated in Nt-CCR5 binding. An opposing sur- face consisting mostly of RANTES β2-β3 hairpin residues and that is involved in ECL2 binding was found to be sim- MO001: Molecular recognition of complex-type ilar to a surface in the crown of the V3. The chemical and biantennary N-glycans by lectins: epitope selection biosynthetic approaches presented in this study for linking through NMR GPCR surface regions may be widely applicable to investiga- 1 1 2 2 tion of interactions of the extracellular segments of chemokine Ana Ardá, Pilar Blasco, Daniel Varón Silva, Volker 3 4 1 receptors with their respective ligands. Schubert, Sabine André, Marta Bruix, Francisco Javier 3 2 1∗ Cañada, Hans-Joachim Gabius, Carlo Unverzagt, Jesús 13 13 Jiménez-Barbero TH003: Improved C- C isotropic mixing experi- ments for side chain assignment of labeled proteins 1Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 2 3 Universität Bayreuth, Germany, Ludwig-Maximilians- 1Helena Kovacs, 2∗Alvar Gossert University Munich, Germany, 4Instituto de Química-Física 1Bruker BioSpin Switzerland, 2Novartis Pharma Switzer- Rocasolano, CSIC, Madrid, Spain land The molecular recognition of carbohydrates is at the heart Progress in cryogenic probe technology makes it now possi- of essential biological events. N-glycans are common modi- ble to apply 13C-13C-spinlocks at RF field strengths that are fications of membrane/secreted proteins that confer specific sufficient to cover the whole 13C- frequency bandwidth at properties to the associated protein. Their structures are medium magnetic field strengths. Wide bandwidth spinlocks widely diverse. However, they share a common structural mo- on carbon enable the following three experiments that con- tif, the pentasaccharide N-glycan core, whose branching can siderably facilitate the assignment of side chain resonances in give rise to highly complex large glycans. NMR and X-ray labeled proteins and, in particular, allow connecting aromatic crystallography, which provide key information at atomic res- side chains to the protein backbone. olution on biomolecules, have mainly focused, in the protein- carbohydrate interaction field, on acquiring information of 1. HCCH-TOCSY to connect aromatic ring carbons to complexes formed by lectins with small/medium-sized frag- aliphatic carbons ments of natural glycan chains. 2. HCC-TOCSY to detect all carbon frequencies in a single Herein, we would like to show our results in the study of the experiment recognition of two complex-type bianntenary Asn conjugated N-glycans, undeca- and nonadecasacchacarides, by different 3. HCCCONH for large proteins plant lectins by NMR. Our results highlight the effect of epi- To connect the aromatic ring to the aliphatic C-beta and tope presentation, and multiple vs single lectin domain, which C-alpha positions is a long-standing problem that can now can affect the outcome of the recognition process, precluding easily be resolved with the wide carbon bandwidth HCCH- extrapolations and generalizations from simple models (small TOCSY. Even more demanding is to connect the aliphatic ligands and single protein domains).1 carbons directly to the backbone carbonyl, because of the large difference between the chemical shifts of C-alpha and 1 A. Ardá, P. Blasco, D. V. Silva, V. Schubert, S. André, M. 13 Bruix, F. J. Cañada, H.-J. Gabius, C. Unverzagt, J. Jiménez- carbonyl carbon. The C-detected all-carbon HCC-TOCSY Barbero. Manuscript submitted. yields all carbon connectivities comprising both the side chains and the backbone. TU002: NMR study of receptor-chemokine interac- HCCCONH is an ever so useful experiment while it yields tions using linked extracellular domains: Mapping the correlations from the side chain directly to the backbone RANTES surfaces interacting with CCR5 amides. The experiment suffers, however, of low sensitiv- 1∗ 1 1 1 ity for medium size proteins (>10kDa). By applying a suf- Jacob Anglister, Einat Schnur, Naama Kessler, Tali ficiently strong carbon spinlock to directly transfer carbon Scherf, 2Fa-Xiang Ding, 2Boris Arshava, 2Fred Naider, 3 3 4 magnetization from the side chain resonances to the carbonyl, Victoria Kurbatska, Ainars Leonciks, Alexander Tsima- a shortened experiment that omits the INEPT step between nis C-alpha and carbonyl, is obtained. This experiment is fa- 1Weizmann Institute, 2College of Staten Island, City Univer- vorable for larger proteins prone to relaxation losses. An sity of New York, 3Latvian Biomedical Research and Study additional consequence of the spinlock that covers all car- Centre, 4Biona Ltd bon frequencies is that the proposed HCCCONH experiment also connects the aromatic carbons directly to the backbone Chemokines constitute a large family of small proteins that amides. regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors which belong to the Spectra are presented at 600 and 700 MHz magnetic fields GPCR superfamily. The N-terminal (Nt) segment of the on 13C, 15N-labeled ubiquitin (7kDa) and kRas (21kDa) and receptor was found to be a major binding determinant in IVL-methyl protonated, 13C, 15N, 2H-labeled MBP (43kDa) many chemokine-receptor systems and interactions between to demonstrate the usefulness of the above mentioned ex- Nt-peptides and chemokines have been studied extensively periments. In addition, theoretical calculations of the trans- using NMR spectroscopy. Due to the lower affinities of pep- fer efficiency were carried out for the aromatics to C-alpha tides representing the three extracellular loops (ECLs) of and C-beta transfer in phenylalanine and for methyl to car- chemokine receptors to their respective chemokine ligand, bonyl transfer in isoleusine, valine and leusine. The theoret- information concerning these interactions is scarce. To over- ical transfer efficiencies were compared to experimental data come the low affinity of ECL peptides to chemokines, we using 16.7kHz spinlocks at 600MHz and 17.9kHz spinlock at linked two or three CCR5 extracellular domains by either 700MHz and going up to 50ms mixing times. Practical con- biosynthesis in E. coli or by chemical synthesis. This ap- sequences for the optimal setup of the spinlock experiments proach enabled the study of CCR5 binding to RANTES using are discussed. 1H-15N-HSQC titrations. Nt-CCR5 and ECL2 were found to be the major contributors by binding to opposing faces MO004: A NMR-based platform for efficient struc- of RANTES, creating almost a closed ring around RANTES. tural characterization of families of small proteins A RANTES positively charged surface involved in Nt-CCR5 binding is reminiscent of a positively charged surface in HIV- 1Jonas Fredriksson, 2Viviane Silva de Paula, 2Ana Paula

69 Poster Abstracts – Biomolecular Solution NMR

Valente, 2Fabio C. L. Almeida, 1∗Martin Billeter AD patients with promising results [6] and was recently an- 1University of Gothenburg Sweden, 2Universidade Federal do nounced to enter a phase III clinical trial [7]. Rio de Janeiro Brasil Using an integrated approach combining solution-state NMR Defensins form an ancient family of antimicrobial proteins spectroscopy and other biophysical techniques, we reveal a that form an innate immune system in most higher organ- distinct mechanism of action of MB and its metabolites azure isms including plants and animals [1]. They are active against A and azure B in the inhibition of Tau aggregation.[8] We both bacteria and fungi. Their strong antimicrobial activity show that the mechanism of Tau aggregation inhibition is makes them potent drug candidates. based on the interplay of reduction/oxidation of the native cysteine residues of Tau. This prevents the formation of fila- NMR projection techniques combined with analysis based on ments by retaining Tau in a monomeric disordered conforma- decompositions offer rapid experiments, high dimensionality, tion. Our data demonstrate that MB and its N-demethylated full resolution and a very high degree of automation; they are derivatives azure A and azure B modify the two native cys- well suited for systematic studies of entire protein families, teines of Tau to sulfenic, sulfinic and sulfonic acid. The which may also include unstable molecules [2-3]. modification of protein cysteine residues through reversible We are developing a platform for complete and fast structural oxidation of cysteine sulfhydryl groups and the formation of and functional characterization of families of small proteins sulfenic acids are a crucial regulatory event in biological sys- e.g. defensins. Once fully implemented, this platform will en- tems. compass high-throughput expression, fast NMR experiments, In summary we provide mechanistic insights into the inhi- automated spectral analysis, and systematic structural and bition of Tau aggregation by the family of phenothiazines. functional characterization. We are combining the locally de- Specific modification of the native cysteine residues retains veloped cell free expression protocol [4] with our projection- Tau in a monomeric conformation preventing the formation decomposition approach and with standard structure deter- of filaments and their toxic precursors. Demethylation of mination. methylene blue establishes new interactions with Tau and As an application, a complete characterization of human beta enables additional means for modulation of Tau aggregation. defensin 6 based on projection experiments analyzed via si- [1] J. Hardy, D. J. Selkoe, Science 2002, 297, 353-356. multaneous decompositions of all spectra is presented and discussed. NMR measurements consist of five experiments [2] D. G. Drubin, M. W. Kirschner, J Cell Biol 1986, 103, recorded in less than 3 days, two for backbone assignment 2739-2746. (5D and 4D), two for side chain assignments (4D, different TOCSY mixing times), and one 4D NOESY. A standard [3] B. Bulic, M. Pickhardt, B. Schmidt, E. M. Mandelkow, H. structure determination approach, using secondary structure Waldmann, E. Mandelkow, Angew Chem Int Ed Engl 2009, analysis, sequence comparisons, and 3D structure calculation 48, 1740-1752. yields identification of a flexible tail and structures with ac- [4] R. H. Schirmer, H. Adler, M. Pickhardt, E. Mandelkow, curacies of 0.6 Å RMSD (excluding the tail). Neurobiol Aging 2011, 32, 2325 e2327-2316. [1] H Ulm, M Wilmes, Y Shai, HG Sahl, Frontiers in Im- [5] C. M. Wischik, P. C. Edwards, R. Y. Lai, M. Roth, C. R. munology 3, 1-4 (2012). Harrington, Proc Natl Acad Sci U S A 1996, 93, 11213-11218. [2] J Fredriksson, W Bermel, DK Staykova, M Billeter, J. [6] C. Wischik, R. Staff, J Nutr Health Aging 2009, 13, 367- Biomol. NMR 54, 43-51 (2012). 369. [3] J Fredriksson, W Bermel, M Billeter, J. Magn. Reson. [7] C. Wischik, TauRX Therapeutics: Sept 10-th, 2012 Press 217, 48-52 (2012). release announcing the initiation of a global Phase 3 clini- [4] A Pedersen, K Hellberg, J Enberg, BG Karlsson, N. cal trial in a type of Frontotemporal Dementia (FTD) also Biotechnol. 28, 218–224 (2010). known as Pick’s Disease. [8] E. Akoury, M. Pickhardt, M. Gajda, J. Biernat, E. Man- TU005: Mechanistic Basis of Phenothiazine-driven delkow, M. Zweckstetter, Angew Chem Int Ed Engl 2013, Inhibition of Tau Aggregation DOI:10.1002/anie.201208290 1,3Elias Akoury, 1,2∗Markus Zweckstetter 1Max Planck Institute for Biophysical Chemistry, 2German TH006: Inhibiting the S100B-p53 protein protein in- Center for Neurodegenerative Disease (DZNE), 3GGNB doc- teraction toral program Biomolecules: Structure – Function – Dynam- 1,2∗ David J. Weber ics, University of Göttingen 1University of Maryland School of Medicine, 2Center for Alzheimer’s disease (AD) is the most widespread dementia Biomolecular Therapeutics syndrome showing progressive presence of abundant deposits of extracellular senile β–amyloid polypeptide (Aβ) plaques Inhibiting S100B-target interactions in the p53 tumor sup- and intracellular neurofibrillary tangles (NFTs) consisting of pressor degradation pathway is underway as a possible means Tau protein.[1] Tau protein is an intrinsically disordered pro- for treating malignant melanoma and other human cancers tein that is abundant in neuronal axons where it promotes with elevated S100B. and stabilizes microtubule assembly.[2] With progression of However, specifically inhibiting protein-protein interactions AD, Tau aggregates and accumulates into NFTs. As there (PPIs) is often not straightforward, and requires a plethora is still no causative treatment or cure for AD and other of structural and dynamic data for achieving this goal. In tauopathies, Tau-based research aims to reveal the patho- this presentation, methods used for inhibiting protein-protein logical consequences of amyloid formation and to implement interactions with small molecule inhibitors will be included new therapeutic strategies. In this effort identification of with a focus on how NMR and information pertaining to pro- inhibitors of tau aggregation as potential disease-modifying tein dynamics is increasingly becoming a necessity. Likewise, drugs and investigation of their mode of action play an im- a "PPI final folding model" will be presented as it pertains portant role.[3] to blocking the S100B-p53 interaction with small molecule Methylene Blue (MB), a tricyclic phenothiazine also known inhibitors of S100B (SBiXs). as methylthionine hydrochloride, has been shown to prevent Tau aggregation in vitro[4] and to reduce the amount of MO007: Insights into the Intramolecular Coupling Tau aggregates in a C. elegans model of Tau pathology.[5] between the N- and C-Domains of Troponin C De- Moreover, MB progressed to phase II clinical trial in human rived from High-Pressure, Fluorescence, Nuclear

70 Poster Abstracts – Biomolecular Solution NMR

Magnetic Resonance, and Small-Angle X-ray Scat- with a similar S2 profile and a slight increase in flexibility in tering Studies loop L3.

1Guilherme A. P. de Oliveira, 2Cristiane B. Rocha, Conformational exchange processes that occur on micro-to- 1Mayra de A. Marques, 3Yraima Cordeiro, 1Martha M. millisecond time scales, were probed by using CPMG relax- Sorenson, 1Débora Foguel, 1Jerson L. Silva, 1,4∗Marisa C. ation dispersion methods.[5,6] We observed that in the wt en- Suarez zyme there is only eight residues in conformational exchange. During the analysis of mutation N70S, which is deleterious 1Programa de Biologia Estrutural, Instituto de Bioquímica in the wild type background, there is no residues with con- Médica, Instituto Nacional de Biologia Es, 2UNIRIO- formational dynamics. Interestingly, the G262S mutation, Universidade Federal do Estado do Rio de Janeiro, CCBS- which is responsible of moving the Zn2+ ion in a more sol- Centro de Ciências Biológicas e da S, 3Faculdade de Farmá- vent exposed position through a direct interaction with C221, cia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, displays an enhanced relaxation profile for several residues, Brazil, 4Programa de Biologia Estrutural, Instituto de Bio- most of which are located in and near loop L10. In addition, química Médica-Polo Xerém, Universidade Federal the N70S/G262S double mutant showed residues located in 2+ Troponin C (TnC), the Ca -binding component of the tro- and near both loops L3 and L10 with an enhanced relaxation ponin complex of vertebrate skeletal muscle, consists of two dispersion profile. This active site’s conformational flexibility structurally homologous domains, the N- and C-domains; suggest a possible scenario for MBLs evolution. these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been con- References: structed using site-directed mutagenesis to replace different [1] Crowder, M. W., Spencer, J., and Vila, A. J. Acc. Chem. Phe residues with Trp. Previous studies utilizing these mu- Res. 39, 721-728, 2006 tants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and [2] Tomatis, P. E., et al. Proc. Natl. Acad. Sci. U. S. A that the N-domain has no effect on the stability of the C- 102:13761–13766, 2005 domain [Rocha, C. B., (2008) Biochemistry 47, 5047–5058]. [3] Tomatis, P. E., et al. Proc. Natl. Acad. Sci. U. S. A 105, Here, we analyzed the stability of full-length F29W TnC us- 20605-20610, 2008 ing structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a flu- [4] Mandel, A. M., Akke, M. and Palmer, A. G. 3rd J. Mol. orescent mutant, in which Phe 29, located in the N-domain, Biol. 246, 144-163, 1995 was replaced with Trp. High pressure nuclear magnetic res- [5] Loria, J. P., Rance, M., Palmer, A. G. 3rd J. Am. Chem. onance was used to monitor TnC unfolding. From these ex- Soc. 121, 2331-2332, 1999 periments, we calculated the thermodynamic parameters (δV and δG◦atm) that govern the folding of the intact F29W TnC [6] Boehr, D. D., Dyson, H. J., and Wright, P. E. Chem. Rev. in the absence or presence of Ca2+. We found that the C- 106, 3055-3079, 2006 domain has only a small effect on the structure of the N- Acknowledgements: ANPCyT, NIH, CONICET domain in the absence of Ca2+. However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca2+-bound state (i.e., when TH009: Structural Insights into Eosinophil Cationic Ca2+ was also bound to sites III and IV of the C-domain). Protein’s Cytotoxicity: Molecular Recognition of An accompanying decrease in the thermodynamic stability Cell Surface Glycosaminoglycans of the N-domain generated a reduction in δδG◦atm in abso- 1 2 3 2+ 2+ María Flor García-Mayoral, Ángeles Canales, Javier lute terms, and Ca binding affects the Ca affinity of the López-Prados, 3Pedro N. Nieto, 2Jesús Jiménez Barbero, N-domain in full-length TnC. Cross-talk between the C- and 1∗Marta Bruix N-domains may be mediated by the central helix, which has a 1Instituto de Química Física Rocasolano, CSIC, Madrid, smaller volume and likely greater rigidity and stability follow- Spain, 2Centro de Investigaciones Biológicas, CSIC, Madrid, ing binding of Ca2+ to the EF-hand sites, as determined by Spain, 3Instituto de Investigaciones Químicas, CSIC- our construction of low-resolution three-dimensional models Universidad de Sevilla, Sevilla, Spain from the small-angle X-ray scattering data. Protein-glycosaminoglycan interactions are essential in many TU008: Millisecond Loop Dynamics in Metallo-Beta- biological processes and human diseases. Eosinophil Cationic Lactamases Gained During Evolution Protein (ECP) is a cytotoxic RNase found in large amounts in the secondary granules of eosinophils. The protein is se- Mariano M. Gonzalez, Luciano A. Abriata, Pablo E. creted from activated eosinophils during infection and plays ∗ Tomatis, Alejandro J. Vila a role in the human innate immune defence, with reported Instituto de Biologia Molecular y Celular de Rosario (IBR) toxicity against bacteria, viruses, and parasites. Cationic - CONICET and aromatic residues have been found to be essential for ECP’s interaction with membranes[1] and glycosaminogly- Metallo-Beta-Lactamases (MBLs) represent one of the most cans (GAGs)[2], suggesting that recognition of heparan sul- relevant bacterial resistance mechanisms.[1] phates exposed at the mammalian cellular surfaces may drive The broad substrate spectrum of MBLs is attributed to the and modulate its cytotoxicity[3]. To get deeper insight into particular topology of the active site, a shallow groove formed the cytotoxic process, we have explored the interactions of and flanked by several loops (L3, L10 and L12). We have ECP with a GAG mimetic. By using NMR spectroscopy shown that mutations N70S and G262S, which alter the and MD simulations we have determined the 3D structure of hydrogen-bond network connecting loops L3 and L12, give ECP in complex with a representative trisaccharide heparin- rise to evolved enzymes with an extended substrate spec- derivative and dissected the structural requirements for this trum.[2,3] In order to explore the role of these mutations in interaction. Using NMR-monitored titrations we have also the loops flexibility and (within a broader perspective) the estimated its binding affinity, which is in the µM range. role of flexibility in protein evolution, we decided to study The structure of the complex shows that the carbohydrate the backbone dynamics of wild type BcII (BcII wt) and in binds at the catalytic site, revealing the mechanism of inhibi- vitro optimized mutants. tion of ECP’s ribonucleolytic activity. The charged sulphate We have measured backbone R1 and R2 relaxation rates, and carboxylate groups of the carbohydrate cluster with and heteronuclear NOEs for BcII wt, N70S, G262S and well-defined orientations interacting with charged and polar N70S/G262S optimized mutants, which were analyzed based residues of the protein. The conserved W10 is also involved in on the model-free approach.[4] The enzymes exhibit a rela- the recognition. The pyranose ring of IdoA adopts the skew- 2 tively rigid backbone in the pico-to-nanosecond time scale, boat S0 conformation as observed in other complexes[4]. We

71 Poster Abstracts – Biomolecular Solution NMR propose a molecular model for the membrane interaction pro- ligase and a mono-Ub stabilizer. Down-regulation and exten- cess in which this recognition event may constitute the first sive oxidative modifications of UCH-L1 have been observed step of the ECP’s cytotoxic mechanism of action by facili- in the brains of Alzheimer’s disease and Parkinson’s disease tating contacts with the membrane that would subsequently (PD) patients. Of importance, I93M and S18Y point muta- trigger membrane destabilization and cell death. This model tions in the UCH-L1 gene have been reported to be linked to can be potentially used for the design of inhibitors to block susceptibility to and protection from PD respectively. Hence, ECP’s toxicity in the treatment of eosinophil pathologies. the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable [1] E. Carreras, E. Boix, HF. Rosenberg, CM. Cuchillo, and interest. MV. Nogués Biochemistry, 2003, 42, 6636-6644. Our circular dichroism studies suggest that the S18Y point [2] TC. Fan, SL. Fang, CS. Hwang, CY. Hsu, XA. Lu, SC. mutation only slightly perturbs the structure while a signifi- Hung, SC. Lin and MDT. Chang J. Biol. Chem., 2008, 283, cant decrease in the α-helical content is observed in the I93M 25468-25474. mutant. We have determined the solution structure of S18Y [3] MF. García-Mayoral, M. Moussaoui, BG. de la Torre, D. and mapping its interaction with ubiquitin by chemical shift Andreu, E. Boix, MV. Nogués, M. Rico, DV. Laurents, and perturbation approach. The electrostatic surface potential M. Bruix Biophys. J., 2010, 11, 2702-2711. analysis reveals that the interaction between ubiquitin and UCH-L1-S18Y is primarily electrostatic in nature, with neg- [4] L. Nieto, A. Canales, G. Giménez-Gallego, PM. Nieto, and atively charged residues on the surface of UCH-L1-S18Y in- J. Jiménez-Barbero Chem. Eur. J., 2011, 17, 11204-11209. teracting with the positively charged residues on the basic This work has been carried out with financial aid of the face of ubiquitin. Although the active site and the L8 loop in MINECO (Project number CTQ2011-22514). UCH-L1-S18Y adopts conformations similar to that observed in the crystal structure of UCH-L1-WT, both the altered hy- MO010: Gene Regulation by Ligand-Mediated drogen bond network and surface charge distributions have Macromolecular Condensation demonstrated that the S18Y substitution could lead to pro- found structural changes. In particular, the difference in the 1Elihu Ihms, 1Ian R. Kleckner, 1Craig A. McElroy, 2Paul dimeric interfaces of the wild-type and the S18Y mutant has Gollnick, 1∗Mark P. Foster shown that mutation can significantly affect the distribution 1The Ohio State University, 2University at Buffalo of the surface-exposed residues involved in the dimeric in- terface. Such observed difference might weaken the stability Ligand-mediated allostery plays a central role in the regu- of the UCH-L1 dimer and hence may explain the reduced lation of a wide variety of biological processes. Thermody- dimerization-dependent ligase activity of UCH-L1-S18Y in namic linkage between allosteric ligands and competing con- comparison to UCH-L1-WT. formational states (minimally two: on, off) provides a mech- anism for thermodynamically modulating cellular responses. ACKNOLEDGEMENTS: Research Grant Council of Hong NMR studies will be presented of ligand-mediated regula- Kong (GRF 776509M and 765312M) tion of gene transcription by the 91 kDa un-decameric (11- mer) ring-shaped Bacillus TRAP protein, and its inhibition TH012: An irreversible protein rearrangement by the trimeric (3-mer) protein Anti-TRAP, AT. Trp bind- linked to cellular function ing to TRAP leads to altered protein dynamics and pro- motes high-affinity binding to RNA thereby inhibiting Trp Stefan Frost, Oanh Ho, Frederic Login, Christoph Weise, ∗ production via transcriptional attenuation and translational Hans Wolf-Watz, Magnus Wolf-Watz repression. However, AT can compete with the Trp-TRAP- Umeå University RNA interaction despite orders of magnitude weaker binding and low relative molar ratios in the cell. By application of While a firm coupling between equilibrium structural dynam- NMR and other complementary biophysical measurements, ics and protein function has been found for many systems, we show that AT binding leads to condensation of multiple less is known about irreversible protein rearrangements and TRAP rings into a high molecular weight complex. This their potential coupling to functionality. Here, we provide in vitro in vivio condensation process may explain the in vivo observation of (NMR) and based evidence for a coupling effective inhibition in the face of sub-stoichiometric abun- between the irreversible dissociation of a "hetero-dimeric" dance and relatively weak binding. Integration of TROSY protein and functionality at a cellular level. Pathogenicity of Yersinia pseudotuberculosis NMR data with those from other spectroscopies, hydrody- is dependent on secretion of vir- namic and thermodynamic measurements, mass spectrome- ulence effector proteins into eukaryotic host cells. Secretion try, will be presented. The studies suggest a new mechanism is controlled by a multi-protein machinery denoted the type Yersinia pseudotuberculo- by which small signals can be propagated to produce a robust III secretion system (T3SS). The sis cellular response. protein YscU plays a central role in regulation of effec- tor protein secretion. YscU is composed of an N-terminal trans-membrane domain (YscUN ) and a soluble cytoplas- TU011: Insights Into Ubiquitin C-Terminal Hydro- mic domain (YscUC ). The cytoplasmic domain undergoes lase L1 (UCH-L1) Mutation’s Association with the auto-proteolysis (self-cleavage) at a conserved NPTH motif Risk of Parkinson’s Disease and the resulting structure is a hetero-dimer. Dissociation of 1∗Kong Hung SZE, 2Ho Sum Tse, 3Hong-Yu Hu YscUC into its two polypeptides was found to be irreversible and triggered in vitro by increased temperature. Surpris- 1Microbiology Department The University of Hong Kong, ingly, we discovered that dissociation of YscUC occurs also Pokfulam Road, Hong Kong SAR, China, 2Chemistry De- in vivo since the C-terminal YscUC polypeptide is secreted partment The University of Hong Kong, Pokfulam Road, from the bacterium by the T3SS machinery. To test if disso- Hong Kong SAR, China, 3Shanghai Institutes for Biology ciation is functionally relevant we analyzed destabilized gain Sciences, Chinese Academy of Sciences, China of function YscUC mutations. These point mutants displayed Protein ubiquitination and deubiquitination, play important accelerated dissociation kinetics, and whereas the wild-type roles in many aspects of cellular mechanisms. Its defective protein is completely resistant to dissociation at 30 ◦C mu- regulation results in diseases that range from developmen- tated variants dissociate at this temperature. The in vitro ob- tal abnormalities to neurodegenerative diseases and cancer. servations were manifested in vivo; whereas wild-type strain Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a pro- secrete effector proteins at 37 ◦C but not at 30 ◦C, strains tein of 223 amino acids, which is highly abundant in brain, with mutated YscUC secret effector proteins efficiently at 30 ◦ constituting up to 2% of total brain proteins. Although it C. Thus, dissociation of YscUC is coupled to effector secre- was originally characterized as a deubiquitinating enzyme, tion and in turn pathogenicity of Yersinia pseudotuberculosis. recent studies indicate that it also functions as a ubiquitin These findings may be general since the T3SS machinery is

72 Poster Abstracts – Biomolecular Solution NMR utilized by many different pathogenic bacteria. In summary 1Yao He, 1Chenwei Zhang, 2Fangming Wu, 1,2∗Changlin our data provide a strong coupling between an irreversible Tian protein rearrangement and biological function at a cellular 1School of Life sciences, University of Science and Tech- level. nology of China, 2High Magnetic Field Laboratory, Chinese Reference: Frost, S. et. al. PLOS ONE, 2012, 7(11):e49349 Academy of Sciences Rv3882c has been reported to be a conserved component of MO013: The role of hydrophobic and aromatic inter- the Type VII Secretion System of mycobacteria. It was de- actions on the amyloid assembly of alpha-synuclein: scribed as a protein anchored on the inner membrane with An NMR view. two trans-membrane helixes. Here the recombinant trans- membrane domain of its homolog protein in M. smegmatis, 1,2M. E. Llases, 1,2M. L. Orcellet, 1,2G. R. Lamberto, 3C. 4 1,2∗ which named MS0082-TMH, is overexpressed in Escherichia W. Bertoncini, Christian Griesinger, Claudio O. Fernán- coli. Then the isotope labeled MS0082-TMH was purified dez in detergent solution. With the NMR spectroscopy data we 1Instituto de Biología Molecular y Celular de Rosario (IBR- finished the backbone assignment and the relaxation anal- 2 CONICET), Universidad Nacional de Rosario, Max Planck ysis (T1, T2, NOE). Moreover, with the titration data of Laboratory of Structural Biology, Chemistry and Molecular Mn2+EDDA2− we confirm the starting point and ending Biophysics of Rosario (MPLPbioR), 3Institute for Research point of this trans-membrane region. in Biomedicine Barcelona, Barcelona, Spain, 4Department of NMR-based Structural Biology, Max Planck Institute for TH015: Characterization of intermediates states of Biophysical Chemistry Sugarcane defensin 5 by relaxation dispersion NMR: Protein amyloidosis are considered the main cause of several thermodynamics and stability neurodegenerative disorders with fatal consequences, such 1∗ 2 as Parkinson’s disease (PD) and Alzheimer’s disease (AD). Luciana Elena S. F. Machado, Yulia Pustovalova, 1Viviane Silva de Paula, 2Irina Beszonova, 1Ana Paula Va- Parkinson’s disease is the second most common neurodegen- 2 1 erative disorder after AD, and is characterized by loss of lente, Dmitry M. Korzhnev, Fabio C. L. Almeida dopaminergic neurons in the substantia nigra and formation 1National Center of NMR, Institute of Medical Bio- of filamentous intraneuronal inclusions of the protein alpha- chemistry, Federal University of Rio de Janeiro, Bra, synuclein (AS). 2Department of Molecular, Microbial and Structural Biol- ogy, University of Connecticut Health Center 2 The general objective of our group is to study the structural and toxic mechanisms related to amyloid formation[1] and The sugarcane defensin 5 (Sd5) is a small protein with an- to understand the structural and molecular basis behind the tifungal activity. Sd5 has a long unfolded C terminal region aggregation inhibitory effects of small molecules[2,3] in order which is unique within the defensin protein family. Also, all to advance in the design of a therapeutic strategy based on the Sd5 secondary structure elements are in conformational amyloid inhibitors. exchange, which are those responsible to bind membranes. Based on these features, we aimed here to identify and to By using small compounds as structural probes, we have characterize the pre-existing state (invisible state) of Sd5, contributed recently to the development of this area of re- which would be important for protein function using relax- search. Through a multidisciplinary strategy we elucidated ation dispersion experiments by Nuclear Magnetic Resonance the structural and molecular basis of the interaction be- (NMR). The 15N relaxation dispersion of Sd5 could be fitted tween AS and one of the most studied aggregation inhibitors, using a two site exchange model (native and intermediate). the cyclic tetrapyrrole Phthalocyanine tetrasulfonate (PcTS). The population of Sd5 intermediate increased as a function of The residue-specific structural characterization of the AS- temperature as well as the exchange rate (kex). Interestingly, PcTS complex provided the basis for the rational design of all these events were mostly enthalpy-driven. In the presence non-amyloidogenic species of AS, highlighting the role of aro- of urea, we observed opposite results, since the population matic and hydrophobic interactions in driving AS amyloid of intermediate decreased as a function of urea concentra- assembly. tion while kex was kept constant. The urea dependence of δ In this work we studied the impact of conservative and non- G (m value) is negative, indicating that the excited state conservative mutations on the amyloidogenic potential of hu- of Sd5 is more compact. This suggestion is strengthen by i) man and mouse AS. The information derived from our analy- evidence demonstrating that: the hydrophobic residues of ii) sis is key to understand the structural basis behind the amy- defensins are usually exposed to solvent, and four pairs of loid aggregation of AS and to advance in the rational design cysteines are key to maintain defensin fold and two of them α β of more efficient amyloid inhibitors. that connect the -helix to the -sheet are in conformational exchange. Concluding, we demonstrate that Sd5 has an inter- References: mediate conformational state that is enthalpically-driven and disturbance of its structure indicates that the charge residues [1] Bertoncini et al, Proc Natl Acad Sci U S A, 2005, 102: are in conformational exchange and could favor its contact for 1430-1435. guarantee the fold of Sd5 and preservation of its structure. [2] G.R. Lamberto, A. Binolfi, M.L. Orcellet, C.W. Bertoncini, M. Zweckstetter, C. Griesinger and C.O. Fernán- Acknowledgments: CAPES, FAPERJ, Ciências sem dez, Proc Nat Acad Sci (USA). 106, (2009) 21057-21062. Fronteiras-CNPq, INBEB [3] G.R. Lamberto, V. Torres-Monserrat, C.W. Bertoncini, MO016: NMR with Multiple Receivers X. Salvatella, M. Zweckstetter, C. Griesinger and C.O. Fer- nandez, J Biol Chem. 1∗Eriks¯ Kupče, 2Ray Freeman, 3Kevin J. Donovan, 3Lucio Frydman, 4Lewis E. Kay 286 (2011) 32036-32044. 1Bruker Ltd, Coventry, United Kingdom, 2Jesus College, Acknowledgements: Cambridge, United Kingdom, 3Department of Chemical Financial support from ANPCyT, CONICET, Laboratorios Physics, Weizmann Institute of Science, 76100 Rehovot, Is- 4 Pförtner, Fundación Bunge y Born, Max Planck Society and rael, Departments of Molecular Genetics, Biochemistry and Alexander von Humboldt Foundation are acknowledged. Chemistry, The University of Toronto, Toronto, O The advent of multiple receivers that record several data sets TU014: Preliminary solution NMR studies of a in a single experiment increases the efficiency of spectrometer Membrane Protein in Type VII Secretion System of use. At the same time, development of cryogenically cooled M. tuberculosis probes has significantly increased the sensitivity of NMR ex-

73 Poster Abstracts – Biomolecular Solution NMR periments. This opens an exciting possibility of changing the Shannon Naughton, ∗Sandra Chimon-Peszek traditional paradigm of experiment design. DePaul University We show two main approaches for recording free induction In previous studies, the orthomolecular species melatonin has decays (FIDs) in multi-receiver experiments - parallel and been found to have a tremendous impact on the β-amyloid sequential acquisition. In both cases the magnetization is peptide that causes Alzheimer’s disease. Melatonin has been typically split into different pathways with the resultant sig- shown to inhibit oxidative stress and the death of neurons and nals from each of the paths recorded using different receivers. neuroblastoma cells exposed to the peptide. The purpose of The sequential acquisition method has been exploited in a research on an orthomolecular species such as melatonin is to number of applications to date involving a range of differ- determine how the chemical substance reacts with a disease ent molecules, from small molecule to bio-molecular applica- or abnormality by restoring proper levels of the chemical sub- tions. Notably, one application, PANACEA has been used stance in the brain. The first step in the process of research- for structure determination of small molecules from a sin- ing the effects of melatonin on the Alzheimer’s β-amyloid gle measurement. In labelled bio-molecules, e.g. proteins the peptide is to make melatonin water soluble. Studies sug- 13 gest that melatonin powder can become water soluble when weak signal that remains after C detected experiments (the ◦ ◦ 13C "afterglow") is measured with high sensitivity by proton mixed with water at 20 C or 50 C. Studies have also shown detection. This is illustrated by the combined dual-receiver that melatonin is soluble in ethanol. This study utilized all 2D (HA)CACO / 3D (HA)CA(CO)NNH experiment. three techniques to create melatonin solutions. Melatonin in a fine powder was added to water at 20 ◦C , water at 50 The utility of the parallel acquisition is established through ◦C , and ethanol at 20 ◦C. All three of the solutions were 1 13 the introduction of the H and CO detected 2D HSQC and centrifuged in order to gather only the water soluble aspects 3D HNCA experiments in which pairs of 2D and 3D spec- of the melatonin solutions. The melatonin concentration of tra are recorded simultaneously. The potential of combining each solution was determined by using a calibration curve. parallel-receiving, multi-nuclear technologies with ultra-fast The calibration curve was created by using a UV/vis ma- 1 1 spatial encoding methods is demonstrated by the 2D H- H chine to measure the absorbances of serially diluted solutions 1 19 31 and H-X (X = F, P) correlation spectra acquired within with known concentrations made from pure melatonin. The a single scan. The experiment brings new opportunities for presence of melatonin in each solution was confirmed using high-throughput analyses, chemical kinetics, and fast exper- ATR-IR spectroscopy by comparing the spectra of each so- iments on metastable hyperpolarized solutions. lution to a melatonin reference spectra. Each solution was β With the anticipated further increases in cryogenic probe sen- then combined with the -amyloid peptide and Congo red sitivity it is expected that multiple receiver experiments will and measured in the UV/vis machine for a week. Each solu- β become an important approach for efficient recording of NMR tion was combined with the -amyloid peptide and measured data. with ATR-IR for a week. The data was then analyzed and compared to solutions with β-amyloid peptide and no mela- tonin. TU017: Lipid binding by the Unique and SH3 do- mains suggests a new regulation mechanism for c- Src MO019: Mapping the residues crucial for protein sta- 1 1,2 3 2 bility - low-temperature studies of p53 tetrameriza- Mariano Maffei, Irene Amata, Yolanda Perez, Ana tion domain. Igea, 4Pau Bernadó, 2Angel R. Nebreda, 1∗Miquel Pons 1 Lukasz Jaremko, Mariusz Jaremko, Stefan Becker, Biomolecular NMR laboratory, University of Barcelona, ∗ Spain, 2Signaling and Cell Cycle laboratory, IRB Barcelona, Markus Zweckstetter Spain, 3IQAC-CSIC, NMR facility Barcelona, Spain, Department for NMR-based Structural Biology, Max Planck 4Centre de Biochimie Structurale, Montpellier, France Institute for Biophysical Chemistry The non-receptor protein kinase c-Src was the first proto- The tumour suppressor protein p53 plays an important role oncogene to be discovered and plays a central role in many in preventing cancer. Its active conformation is tetrameric signaling pathways that regulate cell growth, differentiation, and one domain - the tetramerization domain permits the migration, proliferation and survival. c-Src is constituted by oligomerization of this protein. a series of folded domains (SH2, SH3 and kinase) as well as a 85 residue long intrinsically disordered segment that is Cold denaturation of proteins is a physical phenomenon that divided in two distinct regions: the SH4 domain, which is can be tracked by NMR spectroscopy to study structural myristoylated and acts as a primary attachment point to the changes induced by temperature at atomic level [1]. Pre- membrane surface, and the Unique domain (USrc) connect- viously several point mutations among nine hydrophibic core ing the SH4 and SH3 domains. residues were reported to be crucial for the p53 tetrameriza- tion domain stability [2]. The p53 wild-type (WT) tetrameri- In this study, by combining NMR and biochemical tech- sation domain together with selected mutants were studied niques, we found that a short peptide motif within the Unique in several different temperatures ranging from 298K down domain (ULBR - Unique Lipid Binding Region) acts as a to 259K by solution NMR spectroscopy techniques. This al- secondary lipid binding region of c-Src. Moreover, we show, lowed us to track the structural stability of the tetramer- for the first time, that the SH3 domain of c-Src contains also a ization domain of p53 and to identify residues affected by lipid binding region in the opposite side of its peptide binding temperature changes. Low temperature-induced changes of site. We have further characterized the intra-molecular inter- 1H, 13C and 15N chemical shifts occurred among hydropho- action between the USrc and SH3 domains and its allosteric bic residues, which were previously identified to be the key modulation by a SH3-binding peptide or by Calcium-loaded determinants of the thermodynamic stability of p53 tetramer- calmodulin. These NMR results were integrated with in vivo ization domain [2]. experiments, in which the functional relevance of this novel lipid binding region of c-Src was confirmed in the X. laevis [1] Jaremko, M., Jaremko, L., Kim, H.-Y., Cho, M.-K., oocytes model system. Together, these results uncover an im- Schwieters, C. D., Giller, K., Becker, S., Zweckstetter, M. portant functional role for the intrinsically disordered Unique (2013) Cold denaturation of a protein dimer monitored at domain and suggest the existence of a novel regulation layer atomic resolution, Nat. Chem. Biol. Accepted in c-Src. [2] Mateu, M. G., Fersht, A. R. (1998) Nine hydrophobic TH018: Can Melatonin Help Prevent Alzheimer’s side chains are key determinants of the thermodynamic sta- Disease? bility and oligomerization status of tumour suppressor p53 tetramerization domain, EMBO J. 17, 2748-2758. Katherine Clausen, Lianna Di Maso, Kalah Bermudez,

74 Poster Abstracts – Biomolecular Solution NMR

TU020: A Grid-enabled web portal for NMR struc- The WeNMR project (European FP7 e-Infrastructure grant, ture refinement with AMBER contract no. 261572, www.wenmr.eu), supported by the Eu- ropean Grid Initiative (EGI) through the national GRID 1Lucio Ferella, 1Andrea Giachetti, 2David A. Case, 1∗ Initiatives of Belgium, France, Italy, Germany, the Nether- Antonio Rosato lands (via the Dutch BiG Grid project), Portugal, Spain, UK, 1University of Florence, 2Rutgers University South Africa, Taiwan and the Latin America GRID infras- tructure via the Gisela project is acknowledged for the use of The typical workflow for NMR structure determination in- web portals, computing and storage facilities. volves collecting thousands of conformational restraints, cal- culating a bundle of 20-40 conformers in agreement with them and refining the energetics of these conformers. The struc- MO022: Structural studies of protein disulfide iso- ture calculation step employs simulated annealing based on merase P5 molecular dynamics (MD) simulations with very simplified ∗ force fields. The value of refining the calculated conform- Guennadi Kozlov, Roohi Vinaik, Kalle Gehring ers using restrained MD (rMD) simulations with state-of-art McGill University, Montreal, Canada force fields is documented. This refinement however presents Protein disulfide isomerases (PDIs) constitute a class of en- various subtleties, from the proper formatting of conforma- zymes that catalyze the oxidative folding of proteins in the tional restraints to the definition of suitable protocols. endoplasmic reticulum (ER). Human PDI P5, also known as Here we describe a web interface to set up and run calcu- PDI A6, is a 47 kDa protein which contains three thioredoxin- lations with the AMBER package, which we called AMPS- like domains: two active domains possessing catalytic CGHC NMR (AMBER–based Portal Server for NMR structures). motifs and a non thiol-reactive C-terminal domain. P5 asso- The interface allows the refinement of NMR structures ciates with BiP, an Hsp70 ER homologue. BiP binds to heavy through rMD. Some predefined protocols are provided for chains of immunoglobulins as they enter the ER and the lu- this purpose, which can be personalized; it is also possible minal domains of receptors as part of the unfolded protein to create an entirely new protocol. AMPS-NMR can handle response, and prevents the accumulation of misfolded pro- various restraint types. Standard rMD refinement in explicit teins in the ER. We obtained high-resolution structures of all water of the structures of an all-helical and an all-beta protein three domains of P5 and are currently using small-angle X- are shown as examples. AMPS-NMR additionally includes a ray scattering to determine the global shape of the full-length workspace for the user to store different calculations. As an protein in solution. The structures provide novel insights into ancillary service, a web interface to AnteChamber is avail- mechanism of P5 regulation. We use pulldown assays, NMR able, enabling the calculation of force field parameters for and site-directed mutagenesis in order to characterize the in- organic molecules such as ligands in protein-ligand adducts. teraction between P5 and BiP and identify domains in both proteins that are required for the binding. These studies will AMPS-NMR is available at http://py-enmr.cerm.unifi.it/access provide novel insights into involvement of these proteins in /index/amps-nmr ; the ER protein folding.

Reference: I. Bertini et al., Bioinformatics, 2012, 27:2384- ACKNOWLEDGMENTS: NSERC 2390. TU023: Interaction Studies of Human Laforin with Acknowledgement: This work was supported by the Euro- Oligosaccharides by NMR, MST and ITC pean Commission [contract no. 261572, WeNMR project ]. 1,2,3David M. Dias, 4Joana Furtado, 3Emeric Wasielewski, 3 1 3 5 TH021: The WeNMR e-Infrastructure for Structural Tiago Faria, Alessio Ciulli, Rui M. Brito, Philipp Baaske, 4 2,3∗ Biology Pedro Castanheira, Carlos F. G. C. Geraldes 1 2 3 ∗ University of Cambridge, University of Coimbra, Centre Antonio Rosato for Neurosciences and Cell Biology of Coimbra, 4Biocant - University of Florence Association for Technology Transfer, 5Nano Temper Tech- The WeNMR (http://www.wenmr.eu) project is a European nologies GmbH Union funded international effort to streamline and automate Laforin is a unique human dual-specificity phosphatase as analysis of Nuclear Magnetic Resonance (NMR) and Small it contains an amino terminal carbohydrate binding module Angle X-Ray scattering (SAXS) imaging data for atomic (CBM) and a carboxyl terminal phosphatase domain con- and near-atomic resolution molecular structures of proteins taining a HCXXGXXR(S/T) catalytic active site motif [1]. and nucleic acids [1]. Conventional calculation of biomacro- Laforin mutations have been associated with Lafora disease, molecular structure requires the use of various software pack- an early onset fatal progressive myoclonus epilepsy with au- ages, considerable user expertise and ample computational tosomal recessive inheritance [2]. The laforin CBM carbo- resources. To facilitate the use of NMR spectroscopy and hydrate binding activity has been poorly characterized. We SAXS in life sciences the WeNMR consortium has established describe here our studies of the interaction of recombinant standard computational workflows and services through easy- human Laforin [3] with a series of oligosaccharides using a to-use web interfaces, while still allowing expert users to tune variety of techniques. The Laforin oligosaccharide binding parameters and protocols to their liking. At present, a num- preferences were first screened by Microscale Thermophore- ber of programs often used in structural biology have been sis (MST) using soluble oligosaccharides of increasing length, made available through application portals. WeNMR is also which has shown an increased preference of laforin for longer committed to fostering the development of automated proce- oligosaccharides (both linear and circular), with the highest dures in the field of biomolecular NMR, mainly through the affinity at pH 7.5. The affinity constant of Laforin to linear CASD-NMR initiative [2]. A selection of the most widely oligosaccharides decreased systematically from Kd = 2700 used portals is described, addressing, among other things, µM for maltotriose to Kd = 173 µM , while for the cyclodex- the use of paramagnetic NMR data in NMR structural biol- trins Kd varied from 841 µM for α-CD to 114 µM for γ-CD ogy. The Laforin affinity constants obtained for γ-CD and malto- heptaose) by Isothermal Titration Calorimetry (ITC) titra- References tions compare favorably with the above results. The thermo- [1] Wassenaar et al. Journal of Grid Computing (2012), dynamic parameters determined by ITC were also consistent 10:743-767. with other protein-carbohydrate interactions.

[2] Rosato et al. Structure (2012), 20:227-236. Finally we assessed ligand binding of γ-CD and maltoheptose using STD [4] and CPMG NMR techniques [5]. Both com- Acknowledgement pounds were detected unambiguously on the two ligand-based

75 Poster Abstracts – Biomolecular Solution NMR

NMR experiments. Although various resonance overlaps are ACKNOWLEDGMENTS: CAPES, FAPEMIG, CNPq, observed for this kind of molecules, a group epitope mapping PRPq-UFMG, CNRMN (GEM) from the STD-NMR experiment discloses the most likely binding profile for these sugars. MO025: Structural and Thermodynamic Character- Acknowledgments ization of the Capsid Protein of Hepatitis C Virus and its Interaction with the Tumor Suppressor Pro- We are grateful for funding from FCT, Portugal tein p53 (project PTDC/BIA-PRO/11141/2009 and PhD grant SFRH/BD/81735/2011 awarded to D.M.D.) 1Fabiana Pestana Albernaz, 1Vanessa L. Azevedo Braga, 2Theo L. F. Souza, 1Thiago Rodrigues Pinto, 1Luciana References [1] Wang, J., Stuckey, J. A., Wishart, M. J., and 3 1 1 Dixon, J. E. (2002) J. Biol. Chem. 277, 2377-2380 Pereira Rangel, David Peobody, Jerson Lima, Andre Marco de Oliveira, 1Fabio C. L. Almeida, 1∗Andrea Cheble [2] Chan, E. M., Andrade, D. M., Franceschetti, S., and Mi- de Oliveira nassian, B. (2005) Adv. Neurol. 95, 1Programa de Biologia Estrutural and Centro Nacional de [3] Castanheira, P., Moreira, S, Gama M. and Faro C. (2010) Ressonância Magnética Nuclear de Macromolé, 2Faculdade Prot. Expr. Purif. 71, 195-199. de Farmácia - UFRJ, RJ, Brazil, 3Department of Molecular Genetics and Microbiology and Cancer Research and Treat- [4] Mayer, M. and Meyer, B. (1999) Angewandte Chemie- ment Center, Universi International Edition 38, 1784-1788. Hepatitis C virus (HCV) acts as a major agent of chronic [5] Hajduk P, Olejniczak E, Fesik S (1997) J. Am. Chem. hepatitis, cirrhosis, and hepatocellular carcinoma. The HCV Soc. 119, 12257–12261 core protein (HCVcp) is involved with the nucleocapsid as- sembly and with different cellular processes. The interac- TH024: NMR Structural Studies of the Lyetx-Ii Pep- tion of HCVcp with the tumor suppressor protein p53 has tide been described in hepatocellular carcinoma but the mecha- 1 1 2 nism of this interaction remains unknown. In this work we Bruno A. de Assunção, Natália G. S Pinheiro, Rodrigo M. use thermodynamic and spectroscopic techniques to achieve Verly, 1Daniel M. Santos, 1Maria E. de Lima, 1Dorila Piló- 1∗ the structural characterization of HCVcp and its interaction Veloso, Jarbas M. Resende with p53. Although HCVcp is an intrinsically unstructured 1Universidade Federal de Minas Gerais, 2Universidade Fed- protein, our fluorescence spectroscopy results demonstrated eral dos Vales do Jequitinhonha e Mucuri a significant decrease in the spectral center of mass of the The peptide LyeTx-II has recently been isolated from the protein in function of increasing concentrations of chemi- venom of the spider Lycosa erythrognatha, a species known cal denaturants, indicating the presence of structural ele- as wolf spider, which is commonly found in the Brazil South- ments. These data are corroborated by Circular Dichroism east Region. Antimicrobial assays indicate that LyeTx-II ex- (CD) and Nuclear Magnetic Resonance (NMR). HCVcp back- hibit activity against different pathogens. This peptide car- bone assignments strategy based on projection spectroscopy, ries three charged lysine residues and is naturally amidated a way of reducing the experiment time when recording high- at C-terminus. In order to investigate the structure as well dimensional NMR experiments and Triple resonance exper- iments are in course. To evaluate the interaction between as the interactions of this peptide with mimetic membranes, 1 15 we have performed CD and NMR experiments. HCVcp and p53, H/ N HSQC NMR spectra of labeled HCVcp were acquired in the absence and in the presence of LyeTx-II was prepared by solid-phase synthesis, by using unlabeled p53. Changes in the chemical shift of some res- Fmoc chemistry. The obtained peptide was purified by HPLC onances of the HCVcp were observed, showing changes in and its identity was confirmed by mass spectrometry. CD the chemical environment of these amino acid residues and spectra of the peptide were recorded at different TFE:H2O suggesting interaction with p53. Fluorescence polarization mixtures as well as in the presence of aqueous micellar so- assays of FITC-labeled p53 in the presence of HCVcp cor- lutions of DPC and SDS. A sample containing LyeTx-II at roborate the interaction in vitro between both proteins. Ad- 1 mM in TFE-d2:H2O (50:50, v/v) was prepared for the ditionally to these studies, we are also investigating the in- 1 13 1 NMR analyses. TOCSY, NOESY, H- C HSQC and H- teraction of the HCVcp with the p53 in HepG2 and H1299 15 ◦ N HMQC experiments were acquired at 20 C on a Bruker cells. We constructed vectors to express both HCVcp and p53 Avance-III 800 MHz spectrometer equipped with a triple res- fused to the proteins GFP and DsRed-Monomer, respectively, 1 13 15 onance ( H/ C/ N) 5 mm gradient probe. in order to perform Fluorescence Resonance Energy Transfer analyses and these studies are in progress. Our data reveal a The CD spectrum of the LyeTx-II in water shows a profile new approach to understand the HCVcp-p53 interaction. coherent with spectra of random coil peptides, however for solutions containing significant amounts of TFE and in the ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq and IN- presence of either DPC or SDS micelles, the spectra show BEB clear profile of well defined helical structures. This is a quite common feature of several antimicrobial peptides, which are usually unstructured in aqueous media but tend to adopt well TU026: Effect of an antihistaminic-containing syrup defined conformations when in contact with the membrane in salivary metabolites environment. 1Liana B. Freitas-Fernandes, 1Tatiana Kelly S. Fidalgo, The spin systems of all residues have been recognized on the 1Ivete R. P. de Souza, 2Fabio C. L. Almeida, 2∗Ana Paula TOCSY spectra. Many of the sequential connections were Valente completed mainly on the basis of the strong dNN connec- 1Department of Pediatric Dentistry and Orthodontics,School tions. The high number of cross peaks dαN(i,i+3), dαβ of Dentistry, 2National Center Nuclear Magnetic Resso- (i,i+3), and dαN (i,i+4) suggested that the peptide shows nance a significant helical content, which extends from the third amino acid residue to the C-terminal carboxyamide. In spite The effect of medicines-containing syrup on the tooth sur- of showing a high helical content, the distribution between face is largely studied in literature. On the other hand, few hydrophilic and hydrophobic residues within the helix is not studies evaluate salivary metabolites changes. coherent with an amphipathic structure. We have already synthesized this peptide with selective 15N and 2H labeling Objectives: The aim of this study was to evaluate in vitro in order to obtain information about the peptide topology and in vivo local effect of an antihistaminic-containing syrup through solid state NMR experiments of samples containing in salivary metabolites using high-resolution 1H NMR spec- LyeTx-II reconstituted into lipid bilayers. troscopy.

76 Poster Abstracts – Biomolecular Solution NMR

Methods: For in vitro interaction, unstimulated whole saliva MO028: Site-specific acid-base properties of ovoth- from 10 healthy volunteers was collected and its supernatant iol, the octafarious antioxidant, determined by 1H was analyzed (G1) and then it was in vitro mixture with and 15N NMR spectroscopy Claritin® antihistaminic-containing syrup (G2) and antihis- in vivo Arash Mirzahosseini, Sándor Hosztafi, Gábor Orgován, taminic pill suspension as a control (G3). For study, ∗ after sample collection, the same volunteers were instructed Béla Noszál to rinse the mouth with Claritin® for 20 seconds and the Semmelweis University, Faculty of Pharmacy, Department content was analyzed (G4). The in vitro and in vivo mixture of Pharmaceutical Chemistr,y, Budapest, Hunga were studied in 1H NMR spectra were acquired and processed Thiol-disulfide equilibria in amino acids, peptides and pro- on a Bruker 400 MHz Advance spectrometer. For statistical teins are essential in maintaining the redox balance in living analysis it was used principal analysis component (PCA) and systems, providing thus protection against oxidative stress. ANOVA and Tukey test (p < 0.05). Probably the smallest, yet most-faceted thiol-containing Results: PCA demonstrated similarity between the groups biomolecule is ovothiol, a mercaptohistidine derivative in ma- G1 and G4 as well as G2 and G3. Products characteristics rine invertebrate eggs. It is also assumed to be a highly po- from microorganism metabolism found in salivary metabo- tent antioxidant, due to its remarkably low thiolate basicity. lites demonstrated a reduction after antihistaminic contain- Because the four basic moieties of ovothiol are in close vicin- ing syrup assay (G2 and G3), such as propionate, acetate and ity, the microconstants can not be determined by the titration butyrate (Tukey, p < 0.05). In the same way, arginine, glu- of ovothiol alone. Nevertheless, the fortunate position of the tamine, valerate, and proline were statistically higher before imidazole 2H and αH results in the selective sensitivity of antihistaminic-containing syrup in vitro and in vivo interac- these NMR nuclei towards the imidazole nitrogen/thiolate, tion (Tukey, p < 0.05). Conclusion: Similarity in salivary and amino/carboxylate groups, respectively. metabolites were found both in vitro and in vivo antihis- taminic interaction. In addition, it was demonstrated that A deductive method was designed to elucidate the entire mi- in vitro assays could be an alternative method for medicines crospeciation of ovothiol, by examining three derivative com- and salivary interaction studies. pounds (ovothiol amide, S-methyl ovothiol, S-methyl ovothiol amide) that model the minor microspecies. By conducting 1H ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, UFRJ. NMR-pH titrations, the macroscopic protonation constants of the model compounds were determined, and were then in- TH027: NMR analysis and site specific protonation volved to calculate the microscopic protonation constants of constants of streptomycin ovothiol. To ensure accurate logK determination, an internal indicator system was used to measure the in situ pH. Gábor Orgován, ∗Béla Noszál Semmelweis University, Budapest, Hungary Our results include the entire microspeciation of ovothiol (32 site-specific protonation constants for 16 microspecies) de- Streptomycin is the most widely known member in the termined by the 1H-NMR data. Each proton-binding site of aminoglycoside antibiotic class of drugs, and also, the first ovothiol bears 8 different basicity values depending on the effective remedy for tuberculosis. The two guanidino and the protonation state of the surrounding moieties. The unusu- secondary amino sites make the molecule peculiar: it has long ally large interaction between the basic centers, especially been known to be the most basic antibiotic drug. the one between the thiolate and imidazole nitrogen, causes 1H NMR-pH and 1H–13C HSQC-pH titrations were carried that basicity extrema of the imidazole differ by more than out on streptomycin and streptidine, a symmetrical con- five orders of magnitude, the largest difference observed in stituent compound of reduced complexity to monitor the any biomolecule so far. The interactivity parameters between proton-binding processes of the basic sites. Accurate, undis- the proton-binding sites, along with the Sudmeier-Reilly pro- torted, electrodeless pH measurement was ensured by a new tonation shift constants could also be determined. These re- 15 set of in tube indicators, which made possible the accurate sults were confirmed with UV-pH titration and N-HMBC- determination of extremely high pH values [1,2]. pH titration data. The microscopic protonation constants of the two guanidino TU029: NMR Structure Determination of the Hypo- groups of streptomycin were calculated by evaluating the var- thetical Protein Q4DY78 Conserved in Kinetoplas- ious NMR-pH data and transferring the pair-interactivity pa- tids rameter from streptidine to streptomycin. Inherent guani- dino basicities fall in the range of 13.03 – 13.39 logk units, ∗Everton Dias D’Andréa, Gabriela Pinheiro Heredia, José which drop to 12.48 – 12.85 upon protonation of the other site Ricardo Murari Pires [3]. The basicity of the secondary amino group is, however, Universidade Federal do Rio de Janeiro 5 logk units lower. Chagas Disease, Sleeping Sickness and Leishmaniasis are be- Our results showed that at 1 M NaOH solution 25 % of the tween the so called Neglected Diseases, endemic in poor streptomycin molecules are still protonated. At physiological countries of South America and Africa, exposing million of pH (pH = 7.4) the vast majority of streptomycin is triproto- economic most disfavoured people at risk. Sequencing of nated. the genome of the kinetoplastids Trypanosoma cruzi, Try- panosoma brucei Leishmania major The constants determined also indicate the hydrogen- and open up new per- bonding acceptor propensity of streptomycin at the site- spectives for drug research against these diseases. In each specific level, an important property in the highly selective diploid genome ca. 10,000 gene pairs were identified and biochemical processes. The fact, that the molecule exists in around 50% code for proteins of unknown function. In the every aqueous biological medium in a tricationic form, im- present work using bioinformatic tools available we mined the plies that it presumably enters the central nervous system by Tritryp Databank to end up with a list of 197 proteins up means of transporter protein system(s). to 30 kDa, conserved in kinetoplastids, without orthologues in mammals, plant or fungi, without transmembrane regions [1] Orgován, G.; Noszál, B. Journal of Pharmaceutical and and without homologous sequences in the PDB that should Biomedical Analysis 2011, 54, 958. be suitable for structural studies by solution NMR. From these proteins, the Q4DY78, which is predicted to be an a/b [2] Szakács, Z.; Hägele, G.; Tyka, R. Analytica Chimica Acta protein, its gene was obtained commercially cloned into the 2004, 522, 247. plasmid pUC57 (GenScript Inc.). It was sub-cloned into the [3] Orgován, G.; Noszál, B. Journal of Pharmaceutical and expression plasmid pGEX-4T2, followed by expression and 1 Biomedical Analysis 2012, 59, 78. purification of the protein. The Q4DY78 protein H NMR spectrum is compatible to a small monomeric protein. A set

77 Poster Abstracts – Biomolecular Solution NMR of NMR experiments were obtained: with a unlabeled sample matography was carried out. 1D 1H and 15N-1H HSQC spec- 15 - 2D TOCSY, 2D NOESY (in H2O and D2O); with a N- tra of Q4D059 showed a well-behaved and structured protein labeled sample - 15N HSQC, 15N HSQC TOCSY, 15N HSQC in solution. The NMR spectra required for protein struc- NOESY, 15N HSQC series with different relaxation delays for ture determination were recorded. Assignment of most atoms 1 15 measurement of T1 and T2, and H, N-heteronuclear NOE; in the protein was achieved and structure calculation is in and with a 13C, 15N-double-labeled sample - 13C HMQC, progress. CBCANH, CBCACONH, HCCH-COSY, HBHACONH, 13C HSQC NOESY, plus spectra using Fast NMR methodol- 15 TU032: Conformation Changes in Somatostatin ogy, bCBCANH, bCBCACONH, and bHNCO bHNCOi. N Analogue analyzed by NMR HSQCs were made at different temperatures to verify hydro- gen bonds and the amino acid are in secondary structure. 1,2∗Luisa Mayumi Ribeiro Arake, 2,3Diego Arantes Teix- The protein backbone assignment is in progress. eira Pires, 2Maura Viana Prates, 2Carlos Bloch Jr. 1Instituto de Biologia, Universidade de Brasilia, DF, Brasil, TH030: Experimental and Simulated Yeast Thiore- 2Embrapa Recursos Genéticos e Biotecnologia, DF, Brasil, doxin 1 Dynamics: Exchange Path of a Tightly 3Instituto de Química, Universidade de Brasília, DF, Brasil Bound Water Molecule Essential for Biological Ac- Small peptides are attracting increasing interest in nuclear tivity oncology to target tumors and thus facilitate cancer ther- 1Natália Corrêa Pereira, 2Francisco Gomes Neto, apy. The high level expression of the somatostatin receptors 1 1 1∗ on various tumor cells has provided its successful use as tu- Luciana Elena M. S. Fraga, Ana Paula Valente, Fabio 99m C. L. Almeida mor tracer. The use of Technetium ( Tc) as the coupled radioactive element is much explored in this kind of study 1Biomolecular NMR Laboratory, National Center for Nu- and it utilizes the 6-Hydrazinonicotinamide (HYNIC) as the clear Magnetic Resonance, Institute of Medical Biochem- bifunctional complexing agent. In this work we analyzed con- istry, Federal University of Rio de Janeiro„ 2Toxinology formational changes in the tridimensional structure of the so- Lab., Oswaldo Cruz Institute -FioCruz, Rio de Janeiro, matostatin synthetic analogue Tyr3-Octreotide. This study Brazil is of relevant issue given that changes in the molecule struc- Thioredoxins (Trx1) are small ubiquitous proteins with disul- ture might prevent it from binding to the radioactive ele- fide reductase activity. The active site lies in a loop (Trp- ment. The TYR3-octreotide was synthesized, purified and 29-Cys30-Gly31-Pro32-Cys33) where the disulfide reduction characterized by Mass Spectrometry. During the purification of substrate involves a sequence of two nucleophilic attacks step, two fractions (P1 and P2) were identified which had performed by the thiolate of Cys33 and Cys30. yTrx1 ac- the same molecular mass and the same fragmentation pro- tivity depends on Asp24, a residue buried in the protein file, which implies differences in their conformational struc- hydrophobic core, positioned near but not adjacent to the tures. To elucidate their structure, P1 and P2 were analyzed active site, working as a proton acceptor in the oxidation by NMR performing H-NMR, TOCSY, ROESY and HSQC of cysteines. The proton transfer from Cys33 to Asp24 is experiments, using H2O/D2O as solvent. The structures in mediated by a tightly bound water molecule, forming a wa- question showed significant differences and P2 was not able ter cavity. The mutation of D24N abolishes protein activity. to complex to the 99mTc even in the presence of HYNIC. Our group showed by NMR relaxation experiments that the residues within the water cavity are in conformational ex- ACKNOWLEDGEMENTS: CAPES/Embrapa (001/2011), change, while mutation D24N quenches millisecond motion CNPq, CNRMN of these residues. TH033: Unraveling thioredoxin-interacting protein We studied by MD simulations the influence of the hydration (TXNIP) functions through structural studies state of Asp24/Asn24. We evaluated the hydrogen bond life- time involving Asp24/Asn24 and showed that the mutation Gisele Cardoso de Amorim, Ramon Pinheiro Aguiar, Ana D24N leaded to an important decrease of the water residence Paula Valente, ∗Fabio C. L. Almeida time in the water cavity and a decrease in the average size BioNMR Laboratory, National Center for Nuclear Magnetic of the cavity. These results corroborate the experimental Resonance, Federal University of Rio de Janeiro dynamics that shows stabilization of a conformational state. The total amount of water also decreases in the mutant. The thioredoxin-interacting protein (TXNIP), also known as VDUP1 (vitamin D up-regulated protein) for its strong regu- Supported by: CAPES, FAPERJ, CNPQ, INCT-INBEB lation by vitamin D, belongs to the α-arrestin protein family. TXNIP was shown to function as a cell growth and transcrip- MO031: NMR structural characterization of tion repressor, metabolic regulator, modulator of inflamma- Q4D059, a conserved hypothetical protein from Try- tory response, cardiac function and cell signaling and apop- panossoma cruzi tosis. It is also the only member of the α-arrestin family capable of binding to thioredoxin in a redox-dependent fash- Aracelys López Castilla, ∗José Ricardo Murari Pires ion. Redox-dependent and independent regulatory roles were Federal University of Rio de Janeiro attributed to TXNIP activity. It has been demonstrated to play a crucial role in several pathological conditions such as Q4D059 (Uniprot ID) is a hypothetical protein of 86 amino cancer and cardiovascular diseases, increasing the interest in acids from T. cruzi. A PSI-BLAST search using Q4D059 studying its structure, dynamics and interaction with cellular sequence as query only identified hypothetical proteins from targets. trypanosomatids (E-value above the threshold), so failed to provide insight into the protein function. Q4D059 is con- Very few structural information is available for this class of served in the kinetoplastid genomes and show low-sequence proteins, and for their interactions in the cell. One of our homology with mammal proteins; therefore it is considered goals is determining the 3D solution structure of TXNIP us- a potential target for drug development against trypanoso- ing different constructions for each structural domain, called matids parasites. Structural studies of this protein could give here D1 (17 kDa) and D2 (15 kDa). The native and cysteine- information about its function and facilitate drug screening. mutated 15N and 15N/13C D1 and D2 have been expressed In this work, the gene codifying for Q4D059 was cloned into and purified and the NMR spectra are being assigned. We a pGEX expression vector and 13C- and 15N- NMR protein also probed their interaction with both oxidized and reduced samples were overexpressed in Escherichia coli as a GST- human thioredoxin, and we have evidence that mostly D2 in- fusion protein. Purification of Q4D059 was performed by teracts with thioredoxin. We have also collected SAXS data GST-affinity chromatography. GST was removed by throm- with the aim to reconstruct the entire TXNIP structure based bine protease digestion and as last step a size-exclusion chro- on the domain structures determined by solution NMR.

78 Poster Abstracts – Biomolecular Solution NMR

These data might be crucial for understanding the participa- performed to evaluate the pH effect in the Pla folding and tion of TXNIP in many different cell pathways and the molec- in the PK2-Pla interaction. CD studies showed structural ular mechanisms involved in the different functions TXNIP changes of PK2 in the presence of Pla at pH 8.0, indicating can play. Pla-PK2 interaction in this condition. NMR spectra of PK2 in the presence of Pla were acquired for the sample at pH Acknowledgments: FAPERJ, CNPq, INBEB, CNPq-Science 5.0. It was observed an increase in the peaks intensities com- w/o Borders, LNLS pared with the PK2 free spectra, suggesting less conforma- tional variability of PK2, probably due PK2-Pla interaction. MO034: Screening for new anticancer agents through However, a slight chemical shift change was observed only for the inhibition of the thioredoxin system Ha of VV residues of PK2 in the presence of Pla, suggesting 1 2 2 that Pla-PK2 interaction is affecting specifically the RVV re- Thais Oliveira, Anne Caroline Candido, Ricardo M 1 1∗ gion. In the plasminogen activation process, Pla cleaves the Kuster, Fabio C. L. Almeida, Gisele Cardoso de R561-V562 bond in the sequence PGRVVGG. The absence Amorim of significant alteration on the NMR spectra suggests that 1 BioNMR Laboratory, National Center for Nuclear Mag- PK2-Pla interaction at pH 5.0 could be weak, probably be- netic Resonance, Federal University of Rio de Janeiro, cause Pla in this condition is in the dimeric form. These data 2 Laboratório de Plantas Medicinais, Instituto de Pesquisa show that Pla folding and aggregation state is sensitive to pH de Produtos Naturais, Federal University of Rio de Janeiro changes and interfere in the PK2-Pla interaction. Cancer is still one of the primary causes of death in the world. ACKNOLEDGEMENTS: FAPERJ, CNPq, INBEB Furthermore, many cancers are becoming resistant to conven- tional chemotherapeutic approaches. The discover of novel molecular targets and new anticancer agents are therefore of TH036: Solution-state NMR structure of a cy- greatest interest. tomegalovirus nuclear egress complex subunit and identification of the critical interaction surface with Thioredoxin (Trx) is a small ubiquitous oxidoreductase con- its heterodimer partner served from Archebacteria to humans. Trx can interact with a broad range of proteins by a redox-sensitive mechanism that Kendra Leigh, My D. Sam, Mayuri Sharma, Ming Lye, Ger- ∗ regulates reversible oxidation of two cysteine thiol groups to a hard Wagner, James M. Hogle, Donald M. Coen, Haribabu disulfide. In addition to its function as a scavenger of reactive Arthanari oxygen species, thioredoxin has been implicated in a great Harvard Medical School, Boston MA variety of cellular processes, including transcription factors modulation, cell growth and cell-survival regulation, through Murine cytomegalovirus (MCMV) makes use of a conserved, interaction with an extensive number of signaling molecules. essential heterodimeric nuclear egress complex (NEC) con- Trx expression is increased in a variety of human primary sisting of M50, a membrane protein, and M53, a soluble pro- cancers, where it is associated with aggressive cell growth tein. Genetic, electron microscopy, and immunohistochem- and inhibition of apoptosis. Several studies implicate Trx as ical studies have revealed the importance of these proteins a contributor to cancer progression and metastasis, represent- for viral replication, localization of the complex to the inner ing thus a molecular target for development of new anticancer nuclear membrane, and specific steps during nuclear egress of drugs. the viral nucleocapsid. We present here an NMR-determined solution-state structure of residues 1-168 of M50 (M50-NTD), In the search for new agents, flavonoids emerged as cancer which exhibits a novel protein fold. We mapped the binding chemopreventive molecules because of their antioxidant ac- of a highly conserved region of the M53 homologue, HCMV tivity. However, the precise mechanisms of flavonoids action UL53, required for heterodimerization and identified specific are not totally understood. It has been demonstrated that residues in a groove on the surface of the M50 NTD that this class of molecules can bind to and inhibit different pro- interacts with the UL53 peptide. Single amino acid sub- teins of the Trx system, including hTrx1. stitutions of the corresponding residues of the M50 human homologue, UL50, resulted in decreased UL53 binding in With the aim to find new molecules able to inhibit the Trx vitro as measured by isothermal titration calorimetry, elimi- system, we performed a screening using flavonoids extracted nated UL50-UL53 co-localization in immunofluorescence ex- from sugarcane. Using 15N-HSQC and saturation transfer periments, and caused lethal defects in the context of HCMV difference (STD) experiments we were able to identify a gly- infection in human foreskin fibroblasts. These findings com- coflavonoid that binds to the active site of hTrx1. A docking bined with essential nature of nuclear egress in CMV infec- model is being calculated based on the experimental NMR tions, and the novelty of the structure make the HCMV NEC data. Other extracts from sugarcane are being tested and an attractive potential drug target. showed a high potential to present inhibitor molecules.

Acknowledgments: FAPERJ, CNPq and INBEB MO037: Looking for participation of an intermediate state on the mechanism of chaperon-like activity of TU035: Interaction studies of Yersinia pestis Plas- yeast thioredoxin 1 min (Bubonic Plague Agent) and Human Plasmino- 1 1 gen by Circular Dichroism and NMR Mariana T. Q. Magalhães, Luciana Elena S. F. Machado, 1Adolfo H. Moraes, 2Dmitry M. Korzhnev, 1Ana ∗Carolina Galvão Sarzedas, Thaís Jerônimo Vidal, Jés- Paula Valente, 1∗Fabio C. L. Almeida sica Resende Barreto, Luzineide Wanderley Tinoco 1Biomolecular NMR Laboratory, National Center for Nu- Universidade Federal do Rio de Janeiro clear Magnetic Resonance, Institute of Medical Biochem- istry, Federal University of Rio de Janeiro, 2University of Yersinia pestis is the agent of bubonic plague classified in Connecticut Health Center category A of bioterrorism agents according with Center for Disease Control and Prevention, USA. Plasmin (Pla) of Y. Thioredoxins (Trx) are small ubiquitous proteins with disul- pestis is a membrane protein that cleaves/activates the hu- fide reductase activity. Trxs are able to reduce buried disul- man plasminogen in the cell invasion process, destabilizing fides more efficiently than small reductants. A chaperon–like the fibrinolitic cascade causing bleeding. Our goal was to activity was postulated to explain this behavior. However characterize the pH effect in the Pla folding and their inter- there is no accumulated knowledge on this particular activity. action with human plasminogen peptide (PK2). The struc- We showed the participation of the Asp24 in the modulation tural characterization of PK2-Pla interaction could be useful of the slow dynamics of residues involved in the formation of to plan mimetic peptides to inhibit such interaction. Gel the water cavity. Asp24 works as a proton acceptor in the filtration chromatography showed that Pla is dimeric at pH oxidation of Trx. The proton transfer from Cys33 to Asp24 6.5 and monomeric at pH 8.0. CD and NMR analyses were is mediated by a tightly bound water, which forms the con-

79 Poster Abstracts – Biomolecular Solution NMR served Trxs water cavity. Its dynamics might be linked to with low stability at the relatively high concentrations and water exchange and catalysis. In this work, we are aiming temperatures typically required for NMR measurements. To to explore if the excited state conformation is involved in the overcome these challenges, different buffer conditions were opening of this water cavity, which might explain chaperon screened in order to obtain samples stable enough to allow activity. In order to investigate this hypothesis, we use re- NMR data acquisition. The best buffer condition was in MES laxation dispersion NMR over a range of experimental condi- 50 mM pH 6.5 with 100 mM NaCl and glycerol 1%. In this tions, such as temperature and urea concentration, to identify condition, the protein (300 µM) was fairly stable for 3 days at a low-populated folding intermediate state. 288 K. Increasing the temperature to 298 K resulted in great protein precipitation. The 1H-15N HSQC spectra of Cdc25B Additionally, we are investigating the monomer-dimer equi- full-length catalytic domain displayed relatively good cross- librium, which also plays an important role in Trxs bio- peak dispersion, 198 peaks were observed out of 188 expected. logical activity. Our research group demonstrated that the Clustering of amide cross-peaks in the 1H chemical shift re- monomer-dimer equilibrium is in slow exchange regime, while gion of 8 ppm suggest the presence of disordered regions. the monomer to intermediate works in fast to intermediate Several peaks which were absent in the HSQC spectra of exchange. Significant information on this equilibrium can a truncated construct, where the C-terminus was removed, be obtained performing HSQC and HMQC experiments at were also clustered in the 8 ppm region of 1H chemical shift, different temperatures, concentrations and magnetic fields. pointing to the flexibility of this region. ACKNOLEDGE- Here we will show partial results on complex system charac- MENTS: FAPESP & CNPq terization by using global fitting of data provided from our experiments. MO040: NMR Structure Determination of a Meta Acknowledgements: CNPq-Science w/o Borders, CAPES, Domain Conserved in Kinetoplastids FAPERJ, INCT-INBEB Rachel Santos de Menezes, ∗José Ricardo Murari Pires TU038: NMR structure of human restriction factor Federal University of Rio de Janeiro APOBEC3A reveals substrate binding and enzyme Chagas disease, sleeping sickness, and leishmaniases are specificity among the neglected tropical diseases, which affect the eco- nomically most disfavored population. Recently the genome 1,2In-Ja L. Byeon, 1,2Jinwoo Ahn, 3Mithun Mitra, of kinetoplastids etiological agents of these diseases, Try- 1,2Chang-Hyeock Byeon, 3Kamil Hercík, 1Jozef Hritz, panosoma cruzi, Trypanosoma brucei and Leishmania ma- 1,2Lisa M. Charlton, 3Judith G. Levin, 1,2∗Angela M. Gro- jor were sequenced, so the perspectives of research for new nenborn drugs are being increased. META domain is a small domain 1 2 University of Pittsburgh Medical School, Pittsburgh Center family found in proteins of unknown function and occurs in 3 for HIV Protein Interactions, National Institutes of Health Leishmania spp as an essential gene. Over-expression of that Human APOBEC3A (A3A) is a member of the A3 fam- domain is related with virulence increase in Leishmania ama- ily of single-stranded DNA (ssDNA) deoxycytidine deami- zonensis, and is also implicated in motility in bacteria. The nases that play a role in the innate immune response to gene codifying META domain, that form part of the Q582E3 viral pathogens. A3A has a broad range of activities: it (Uniprot ID) hypothetical protein from Trypanosoma brucei, potently restricts LINE-1 retrotransposition, inhibits repli- was cloned into a pGEX-4T2 expression vector and protein cation of retroviruses (e.g., HIV-1 and HTLV-1) and certain sample was overexpressed in Escherichia coli BL21(DE3) as DNA viruses, degrades foreign DNA, and can function as a a GST-fusion protein. Purification was performed by GST- genomic DNA mutator. Here, we report the NMR solution affinity chromatography and then GST-tag was removed by structure of A3A, define the critical interface for interaction cleavage by thrombine. A second step of purification, a size- 1 with its ssDNA substrates and model complex structures. exclusion chromatography, was carried out. H spectra re- The overall structure of A3A is very similar to that of the vealed a well-behaved and structured protein in solution. The A3G catalytic C-terminal domain, although structural details NMR spectra required for protein structure determination are different. Oligonucleotides containing the A3A-specific are being recorded. recognition site TTCA bind A3A 10-fold more tightly (Kd µ 60 M) than dCTP alone and the interaction surface includes TU041: Human beta-defensin 6/Glycosaminoglycan residues that extend beyond the catalytic center. A3A also interaction by NMR: a model for extracellular ma- µ binds with similar affinity ( 90 M) to oligonucleotides con- trix complex taining the A3G-specific CCCA sequence. Real-time mon- itoring of the deamination reaction by NMR on A3A- and Jessica Oliveira, Viviane Silva de Paula, Vitor Hugo A3G-specific ssDNA substrates was conducted to determine Pomin, Fabio C. L. Almeida, ∗Ana Paula Valente the catalytic constants for A3A. Based on the structural and Centro Nacional de Ressonância Magnética Nuclear, Insti- biochemical results, we propose a molecular mechanism that tuto de Bioquímica Médica, Universidade Federal do Rio de provides new insights into A3A functional activities. Janeiro Defensins are small cationic, cysteine-rich, antimicrobial TH039: Improving the stability of Cdc25B for NMR peptides that play a crucial role in host defense against measurements pathogens. Human β-defensins (hBD) plays important Raphael Santa Rosa Sayegh, Fabio Kendi Tamaki, San- roles in the innate and adaptive immune system when a dro Roberto Marana, Roberto Kopke Salinas, ∗Guilherme threat is present. Defensins present other activities such as Menegon Arantes chemoattraction of a range of different cell through specific chemokine receptors and this chemoattraction could be reg- University of São Paulo ulated by glycosaminoglycan (GAG). In analogy to hBD2 Cdc25B phosphatase plays an essential role on cell cycle and chemokine/(GAG) interactions, we decided to charac- checkpoints and is overexpressed in several kinds of tumors. terize the interaction between human β-defensin 6 (hBD6) Therefore, it has became a potential target for the develop- and GAG. In the present study we use NMR spectroscopy of ment of new anticancer drugs. Computer simulations, bioin- 15N hBD6 to map the binding site for a synthetic pentasac- formatic analyses and crystallographic data indicate that the charide fondaparinux (Arixtra ®), a highly sulfated heparin C-terminus of this protein is highly flexible and might in- mimetic. Chemical shift mapping of the interaction reveals a fluence the binding affinity of inhibitors. In order to ob- contiguous binding surface on hBD6, which comprises amino tain a better structural representation of Cdc25B in solu- acid residues of the α-helix and loop between β2-β3. The tion, we plan to evaluate different NMR data (e.g., chemical dissociation constant of the hBD6/GAG complex was deter- shifts and RDC’s). However, Cdc25B is a 25 kDa protein mined by NMR spectroscopy to be 4±mM. The formation

80 Poster Abstracts – Biomolecular Solution NMR of the hBD6/GAG complex was also confirmed by the over- in slow exchange at the NMR time scale. Spectral changes 15 15 15 all decrease in NR1 concomitant with an increase in N observed in the N-HSQC spectra of Xac VirB9 in the ab- R2 indicating an increase in the overall tumbling time of the sence and presence of a peptide corresponding to residues protein. 24-46 of Xac VirB7 (VirB7NT ), suggest that at least part of Xac-VirB9CT undergoes a disorder-order transition upon TH042: Structural and dynamics properties of the binding [4]. In order to obtain further high-resolution struc- glycoprotein E – domain III of dengue virus free and tural information on Xac’s T4SS, we initiated a study of the three-dimensional structure of the complex formed by in complex with specific single chain fragments (scFv) CT NT analyzed by NMR spectroscopy Xac-VirB9 and Xac- VirB7 . As much as 96% of the backbone NMR resonances were assigned. Experiments for 1Geovana Vargas, 1Adolfo H. Moraes, 1Fabio C. L. backbone and side-chain NMR resonance assignment were Almeida, 2Luca Varani, 1∗Ana Paula Valente recorded and are being analyzed. Preliminary 13C chemical 1Centro Nacional de Ressonância Magnética Nuclear, IBqM- shift analysis indicates the presence of 8 beta-strands, which UFRJ, RJ, Brazil, 2Medical Institute for Research in is consistent with the available structures for E. coli VirB9 Biomedicine, Bellinzona, Switzerland C-terminal domain (PDB 3JQO and 2OFQ). The dengue virus belongs to the family of flavivirus and is re- ACKNOLEDGEMENTS: FAPESP, CNPq sponsible for more than 100 million cases annually. The viral [1] Chandran, V., Fronzes, R., Duquerroy, S., Cronin, N., envelope glycoprotein E is the major constituent of dengue Navaza, J., Waksman, G. (2009). Structure of the outer virus surface and is the main target for antibody response membrane complex of a type IV secretion system. Nature against DENV. The crystal structures of the virus demon- 462, 1011–1015. strate that glycoprotein E is a dimer, where each monomer is composed by three domains: DI, DII and DIII. The develop- [2] Zechner, E.L., Lang, S., and Schildbach, J.F. (2012). As- ment of a dengue vaccine has been hampered by, the poorly sembly and mechanisms of bacterial type IV secretion ma- understood process: antibody depended enhancement (ADE) chines. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 367, in which antibodies generated against a dengue serotype fa- 1073–1087. cilitate the infection by a different one. This process can [3] Da Silva, A.C.R., Ferro, J.A., Reinach, F.C., Farah, C.S., lead to dengue hemorrhagic fever, a lethal form of the dis- Furlan, L.R., Quaggio, R.B., Monteiro-Vitorello, C.B., Van ease. In this work, we characterized the dynamics and the Sluys, M.A., Almeida, N.F., Alves, L.M.C., et al. (2002). structural properties of the DIII of serotypes 1 and 4 using Comparison of the genomes of two Xanthomonas pathogens NMR spectroscopy. We mapped residues that participate in with differing host specificities. Nature 417, 459–463. the interaction with specific scFv using chemical shift pertur- bation. The molecular dynamics on ps-ns time scale, which is [4] Souza, Diorge P., Andrade, Maxuel O., Alvarez-Martinez, related to thermal flexibility, and movements on ms-ms time Cristina E., Arantes, Guilherme M., Farah, Chuck S., Salinas, scale, which are associated to conformation exchange events, Roberto K. (2011). A Component of the Xanthomonadaceae 15 were studied using N relaxation experiments. Quantita- Type IV Secretion System Combines a VirB7 Motif with a tive analyses were obtained using relaxation dispersion exper- N0 Domain Found in Outer Membrane Transport Proteins. iments based on CPMG pulse sequences. We will present the PLoS Pathogens, v. 7, p. e1002031. conformational dynamics characterization of DIII in the free state and in complex with scFv. These data can be crucial for TU044: Structural studies on Trypanosoma cruzi a better understanding of epitope-antibody interaction and P21 protein as a strategy for Chagas disease treat- can help in the development of new dengue vaccine. ment

ACKNOLEDGEMENTS: FAPERJ, CNPq, CAPES, IN- Francesco Brugnera Teixeira, Eduardo Horjales, BEB. ∗Claudia Elisabeth Munte Physics Institute of São Carlos, University of São Paulo, MO043: Solution structure of the VirB9Ct-VirB7Nt Brazil complex from the Xanthomonas Type IV Secretion System Cell invasion by the different infective forms of Trypanosoma cruzi, the causative parasite of Chagas disease, involves sev- Luciana Coutinho de Oliveira, Diorge Paulo de Souza, eral mechanisms which culminate in entry of T. cruzi into Denize Cristina Favaro, Angie Lizeth Davalos, Chuck S. host cells. Numerous studies have been performed in order to ∗ Farah, Roberto Kopke Salinas identify surface and secreted proteins related to cell invasion, Department of Biochemistry, Institute of Chemistry, Uni- mostly from metacyclic and tissue culture trypomastigote versity of São Paulo forms. However, little attention has been given to the ex- tracellular amastigote, also capable to invade mammalian Type IV secretion systems (T4SS) are supramolecular com- cells in an alternative invasion mechanism. The molecules plexes used by bacteria to secrete proteins or nucleoprotein involved in this alternative process are still poorly known or complexes to the extracellular environment, or to deliver studied and may be significant to T. cruzi survival under them inside a host cell [1]. T4SSs are generally composed high cytotoxic conditions afforded by the host cells. In this of 12 proteins, VirB1 to VirB11 and VirD4, as found in the context, a new protein named P21 has been recently iden- prototype T4SS of Agrobacterium tumenfaciens [2]. VirB7, tified. Its participation in the invasion mechanism cannot VirB9 and VirB10 form the channel used to translocate sub- yet be explained at molecular level, but experiments suggest strates across the bacterial cell envelope. This channel con- that P21 interacts with the CXCR4 chemokine receptor, ac- sists of an inner layer and an outer layer [1]. A high-resolution tivating phagocytosis in macrophages and promoting actin crystal structure of the outer layer from the T4SS of a con- polymerization in mammalian cells. jugative plasmid of E. coli, pKM101, shows fourteen repeti- tions of a complex formed by VirB7 and the C-terminal (CT) As the first step towards P21 NMR structural studies, we domains of VirB9 and VirB10. Xanthomonas axonopodis pv. developed a purification/refolding protocol and performed citri (Xac), a phytopathogen that infects citrus, has one T4SS the 1H, 13C and 15N sequential assignment using triple res- encoded by the chromosome [3]. Previously we showed that onance NMR spectra (Bruker 600 MHz). The secondary a Xac VirB7-VirB9-VirB10 complex assembles in vivo. We structure prediction, based on chemical shifts (TALOS+), also showed that Xac VirB7 has a C-terminal globular do- showed five α-helix regions and an unstructured N-terminal main (residues 52-133) that is absent in the VirB7 of other region (residues 1-42). We also identified several regions of organisms [4]. This observation suggests that there could the protein with multiple conformations, indicating a large be a structural variation in the T4SS of Xanthomonadaceae. structural flexibility. Future structural and interaction ex- Isolated Xac VirB9CT and Xac-VirB7 form a tight complex, periments with the N-terminal peptide of CXCR4, essential

81 Poster Abstracts – Biomolecular Solution NMR for the protein activation, may lead to a new therapy based are being carried out in order to characterize the structure on peptides, which may change the current worldwide sce- and dynamics of CBD12-1.1 in solution. nario on the Chagas disease treatment. ACKNOLEDGEMENTS: FAPESP, CAPES.

TH045: Excited States of the Alzheimer beta- TU047: Structure and Dynamics of Allergen Gad m Amyloid Peptide can be Detected by High Pressure 1 free and in complex with single chain variable frag- NMR Spectroscopy ment by NMR Spectroscopy

1,2Claudia Elisabeth Munte, 2Markus Beck Erlach, 1Adolfo H. Moraes, 2Daniela Ackerbauer, 2Maria Kostadi- 2Werner Kremer, 2Joerg Koehler, 2∗Hans Robert Kalbitzer nova, 2Merima Bublin, 3Fatima Ferreira, 1Fabio C. L. 2 1∗ 1Physics Institute of São Carlos, University of São Paulo, Almeida, Heimo Breiteneder, Ana Paula Valente 13566-590 São Carlos, SP, Brazil, 2Institute of Biophysics 1Centro Nacional de Ressonância Magnética Nuclear, IBqM- and physical Biochemistry, University of Regensburg, D- UFRJ, RJ, Brazil, 2Medical University of Vienna, Vienna, 93053 Regensburg, Germany Austria, 3University of Salzburg, Salzburg, Austria Amyloid fibrils in the brain consisting of the amyloid peptide Allergenic proteins are able to stimulate an inappropriate IgE (Aβ) are a lead marker of Alzheimer’s disease. A deeper un- production in atopic individuals, which results in manifesta- derstanding of the mechanism of fibril formation may help to tions of clinical symptoms such as asthma, rhinitis and atopic design drugs for preventing the deposition of amyloid, there- dermatitis. Gad m 1 is the major allergen from Atlantic cod fore the existence of specific conformations of monomeric Aβ and belongs to β-parvalbumin protein family, which are the is potentially important. Aβ-monomers in aqueous environ- most important fish allergens and their high cross-reactivity ment are usually assumed to occur in a disordered state. In is the cause of the observed polysensitization in allergic pa- contrast, by high pressure NMR spectroscopy we detect two tients. Despite extensive efforts, the complete elucidation of main conformational states at atmospheric pressure, a com- β-parvalbumin-IgE complexes has not been achieved yet. In pactly folded state 1 and a partly unfolded state 2, with rel- this work, we solved the solution structure of Gad m 1 using ative populations of 0.7 and 0.3, respectively. Pure random NMR spectroscopy. Concomitantly, scFv interaction stud- coil like structures were not detected. The pressure response ies were performed, using chemical shift perturbation assays. indicates an ordered structure between amino acids 16 to 24 We compared our results with other sites mapped in homol- and 30 to 37 in state 1. Aβ-fibrils depolymerise at high pres- ogous parvalbumins: Cyp c 1, Sco j 1 and Gad c 1. Residues sure, the dissociation constant of monomers from the fibrils around positions 30 to 40 matched epitopes mapped for the increases by two orders of magnitude at 200 MPa and 283 K. three allergens. Residues around position 50 to 60 were only The partial molar volume of the monomer unit changes by observed for Gad m 1 and Gad c 1, while residue around 101 ml/mol with binding. The thermodynamic data suggests 80 was mapped on Cyp c 1. On the other hand, the N- that state 1 is responsible for fibril elongation. terminal was only mapped in Gad m 1 and our results did not showed the participation of residues around position 90. The dynamic properties of Gad m 1 in the free state and in MO046: NMR study of the calcium-binding do- the complex with scFv were obtained from relaxation exper- + 2+ mains of the Na /Ca exchanger of Drosophila iments. Residues 31-33 and 75-78 exhibit increased R2/R1 melanogaster by solution NMR ratios. These residues are located in two regions mapped by CSP. The big ratios observed may be due to chemical ex- 1 1 1 Layara A. Abiko, Priscila Hauk, Denize C. Favaro, change between bound and free forms. This work will present 1 2 2 Luciana Coutinho de Oliveira, Lei Bruschweiler-Li, Rafael for the first time the solution structure of a β-parvalbumin as 1∗ Brüschweiler, Roberto Kopke Salinas well as the characterization of Gad m 1-scFv interaction as 1Institute of Chemistry, University of São Paulo, a first and crucial step in the development of hypoallergenic 2Department of Chemistry and Biochemistry, Florida State vaccine and to an understanding of the molecular interactions University between allergens and IgE’s. + 2+ Drosophila melanogaster’s Na /Ca exchanger (CALX) ACKNOLEDGEMENTS: FAPERJ, CNPq, CAPES, and IN- + 2+ catalyzes the co-transport of Na and Ca across the BEB plasma membrane and is essential for the maintenance of Ca2+ homeostasis in Drosophila’s sensory neuronal cells. TH048: The Putative Hepatitis C Virus Fusion Pep- The exchanger consists of a transmembrane domain, which is tide Interacts with Model Membranes and Gains He- responsible for ion translocation across the membrane, and an lix Content intracellular loop that contains two calcium binding domains 2+ (CBDs). Besides transporting Ca , the exchanger is also 1∗Nathalia S. Alves, 1Ygara S. Mendes, 2Theo L. F. Souza, regulated by this ion. There are two CALX isoforms, CALX- 1M. Lucia Bianconi, 3Cristiane D. AnoBom, 1Ana Paula Va- 1.1 and CALX-1.2, which only differ in a segment of five lente, 1Jerson L. Silva, 1Andre M. O. Gomes, 1Andrea Cheble amino acid residues located in the second calcium-binding do- de Oliveira main, CBD2. Binding of four calcium ions to the first intra- 1IBqM, UFRJ, RJ, Brazil, 2Faculdade de Farmácia, UFRJ, cellular Ca2+-binding domain, CBD1, of CALX-1.1 inhibits RJ, Brazil, 3IQ, UFRJ, RJ, Brazil the exchanger. In contrast, the isoform CALX-1.2 is insensi- tive to the binding of Ca2+ to CBD1. In order to understand Introduction: The Hepatitis C virus (HCV) is an enveloped how the binding of Ca2+ to CBD1 could be involved in the RNA virus, and the most common cause of chronic liver dis- regulation of CALX, we initiated a study of the structure and ease. Although much information has been gathered in recent dynamics of a covalent construct containing the two CBD years, the exact mechanisms that lead to membrane fusion domains (CBD12) by high-resolution NMR. 1H-15N-TROSY are poorly understood. The envelope glycoprotein, E1 or E2, spectra of CBD12-1.1 and CBD12-1.2 acquired in the ab- directly interacts with biological membranes, throughout the sence or presence of Ca2+ are consistent with well-folded fusion peptide (FP). As the localization of FP remains un- domains. The translational diffusion coefficient (Dt) was known, we aim to characterize the interaction of a putative 2+ measured for CBD12-1.1 in the apo and Ca -bound states HCV FP, corresponding to residues 421-445 (HCV421−445), by Diffusion-Ordered NMR Spectroscopy. The Dt values ob- present in E2, with different membrane models. Methods: tained for CBD12-1.1 (7.68×10−11 and 7.38×10−11m2s−1, We used spectrophotometry, fluorescence spectroscopy, circu- for the unliganded and Ca2+-bound forms, respectively) are lar dichroism (CD), isothermal titration calorimetry (ITC), consistent with predicted data using SOMO (8.04×10−11 differential scanning calorimetry (DSC), dynamic light scat- m2s−1) or HYDROPRO (7.92×10−11m2s−1), and indicate tering (DLS) and nuclear magnetic resonance (NMR) to that the proteins are monomeric in solution. Further studies evaluate peptide-membrane interaction and conformational

82 Poster Abstracts – Biomolecular Solution NMR changes due to this interaction. Results: ITC and fluo- ing of membrane surface interaction process, which is proba- rescence polarization data showed that HCV421−445 can in- bly followed by cell lysis, is of crucial importance. Although teract with large unillamelar vesicles (LUV). However, DSC the outlines of the mechanism are well consolidated, its full revealed that the peptide cannot change the phase transition pathway is not yet completely unveiled, hence the need to temperature of multilamellar vesicles. Photometry analyses perform different studies to elucidate it better. revealed that the peptide can induce LUV aggregation, but DLS data revealed that the peptide can either disrupt or In this work, solution NMR spectra were acquired with a sam- aggregate the same vesicles. Since the interaction of fusion ple containing the peptide in micellar medium and, through peptides with membranes can lead to an important structural the resulting geometric restraints, a model containing the 20 least energetic structures was determined. The solid- gain, we also aim to solve HCV421−445 structure. NMR data state NMR-derived orientational restraints, calculated from of the peptide free in solution indicated low structure content 15 2 but addition of SDS micelles leaded to a structural gain, con- the N chemical shift and H quadrupolar splitting with static samples containing the peptide locally labelled with firmed by CD data. The preliminary structure calculation 15 2 showed that the C-terminal of the peptide in the presence of N and H3-Ala and reconstituted in phospholipid bilayers, micelles presents a tendency to form an α-helix, which could were then applied to all of the structures that compose the be stabilized by a helix stabilization motif, GXXXG. Con- solution-NMR model. With this approach, it was possible to determine the topologies adopted by the peptide during clusions: Altogether our results indicate that HCV421−445 peptide is able to perturb lipid membranes, and becomes its interaction with membranes and also to cover a broader structured. These studies are important to better under- orientational landscape, by considering a higher number of stand the fusion mechanisms of HCV, which could help in geometries which are coherent with NMR data. the development of new antiviral therapies. This work has been carried out with financial aid of CNPq, CAPES, FAPEMIG, PRPq-UFMG and CNRS Support: Capes, CNPq, FAPERJ, PRONEX, INBEB

TH051: Dynamics and Structural Studies of an In- MO049: Structure and Dynamics of D24A mutant of ternal Fusion Peptide from SARS-CoV Spike Glyco- yeast thioredoxin reveals a less stable conformation protein in Lipid Model Membranes with increased flexibility. 1Luis Guilherme M. Basso, 2Tácio Vinício Amorim Anwar Iqbal, Luciana Elena S. F. Machado, Catarina A. Fernandes, 1José Fernando de Lima, 1Claudia Elisabeth Miyamoto, Ana Paula Valente, ∗Fabio C. L. Almeida Munte, 1Edson Crusca Júnior, 3Eduardo Festozo Vicente, Biomolecular NMR Laboratory, National Center of Nuclear 3Eduardo Maffud Cilli, 2Pedro Geraldo Pascutti, 1∗Antonio Magnetic Resonance, Institute of Medical Biochemistry, Fed- José Costa-Filho eral University of Rio de Janeiro, B 1University of Sao Paulo, 2Federal University of Rio de Thioredoxins are ubiquitous proteins that play a key role in Janeiro, 3Sao Paulo State University - UNESP cell regulation. They interact with multiple targets in the The S2 domain of the spike glycoprotein from SARS-CoV cell. Thioredoxin exhibits chaperonin like activity, allowing is responsible for driving viral and host cell membrane fu- local unfolding of the target proteins, thereby gaining access sion. An internal fusion peptide (SARSIFP ), located near to the internal disulfide bonds, making it a very efficient en- the S2 domain N-terminus, plays a crucial role in the fusion zyme. Our group determined the structure of S. cerevisiae process. Although much information has been obtained in Trx1 and its dynamics in different timescales (pico to sec- recent years on membrane fusion, many aspects of the molec- onds). The dynamics is modulated by Asp24 that is buried ular mechanism behind virus-host cell membrane fusion are in the core of the protein, facing a water cavity. We have stud- still not totally understood. For instance, major questions ied the mutant D24A to examine if the presence of a small regarding the structure and function of fusion peptides still hydrophobic residue would stabilize a closed conformational remain to be answered. In this regard, we report here mag- state without a water cavity. The presence of an alanine netic resonance and computational studies on the structure at position 24 induced conformational exchange in residues and dynamics of SARSIFP in different membrane mimetic facing the water cavity, such as C33. This is an indication environments. The observation of strong dNN -(i,i+1) NOEs of the role of this cavity in the stabilization of the protein. along with the presence of many medium-range dαN (i,i+3) We also compared the temperature coefficients obtained from NOEs and upfield Hα shifts from random coil values sug- the chemical shift variation of HN and observed an increased gests an α-helical conformation for the peptide in DPC mi- thermal susceptibility for several residues, indicating a de- celles, which agrees qualitatively well with CD experiments. crease in protein stability. Lipari-Szabo order parameters The absence of HN-Hα cross peaks in the 2D TOCSY NMR were the same as for the wild type protein. The uniformly spectrum from residues pertaining to the peptide central re- 13C and 15N labeled sample were used to acquire the exper- gion is likely due to a slow dynamics experienced by this iments to calculate the structure of D24A and methyl order peptide segment in micelles. On the other hand, CW-ESR parameters. spectra of both N- and C-terminally attached spin labeled Supported by: FAPERJ, CNPq and INBEB. analogues, TOAC-SARSIFP and SARSIFP -Cys-MTSSL, in- dicate a great flexibility of the water-exposed peptide ends. GSA and MD simulations were also used to gain insights on TU050: Structural and Orientational Topology of the the peptide tertiary structure. The minimum-energy confor- Antimicrobial Peptide Hylaseptin P2 from Conjoint mation obtained from the fully stretched peptide generated Use of Solution and Solid-State NMR by GSA was further refined by long timescale MD simulations 1,2∗Victor H. O. Munhoz, 1,3Rodrigo M. Verly, 1Jarbas in aqueous, DMSO, and TFE/water solutions using different M. Resende, 2Burkhard Bechinger, 1Dorila Piló-Veloso force fields. Cluster analysis reveals that both Amber99SB- ILDN and CHARMM27/CMAP force fields were able to cor- 1Universidade Federal de Minas Gerais, 2Université de rectly capture an α-helix structure for the peptide. The dis- Strasbourg, 3Universidade Federal dos Vales do Jequitin- tance distribution between the N- and C-termini obtained honha e do Mucuri from MD was well reproduced by the recovered distance his- The Hylaseptin P2 (HSP-2) is an antimicrobial peptide found tograms from four-pulse DEER experiments on the double- on the skin secretions of Hypsiboas punctatus anurans, an labeled derivative TOAC-SARSIFP -Cys-MTSSL. The av- species mainly found at the Amazon rainforest. This peptide erage nitroxide-nitroxide distances in different membrane has shown considerable activity against pathogens such as mimetic environments are compatible with a partially folded bacteria and fungi. Like most of the antimicrobial peptides, α-helix. The broad distance distribution encountered might it is believed that HSP-2 mechanism of action is guided by reflect the great flexibility of the N-terminus and/or the dif- its affinity for bacterial membranes. Therefore, understand- ferent MTSSL rotamers. Taken together, our results suggest

83 Poster Abstracts – Biomolecular Solution NMR

that SARSIFP is located at the interface of the model mem- NMR structure of a sweet orange Poly(A)-Binding Protein branes in an α-helical conformation with both ends exposed (CsPABP) identified as an interacting partner of PthA. to the water phase. To our knowledge, this is the first re- port that provides structural and topological information of NMR experiments for structure determination were per- SARSIFP in model membranes at atomic resolution, which formed at 293 K using an Agilent Inova 600 MHz spectrometer equipped with a cryogenic probe. The may shed much light on the mechanism by which the peptide 15 fold and insert into lipid bilayers. following experiments were recorded: N-HSQC, HN- CACB, CBCA(CO)NH, HNCO and HN(CA)CO, 15N- ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq. NOESYHSQC; 15N-TOCSYHSQC; 13C-HCCH-TOCSY; 13C-CCH-TOCSY and 13C-NOESYHSQC. MO052: 199Hg NMR Parameters in thymine-Hg- The structure of CsPABP is composed by flexible residues at thymine metal mediated base pair the N-terminal region that include a α-helix portion following 1T. Daraku, 2K. Furuita, 3J. šebera, 1D. Yamanaka, to intercalated β-strand and α-helix leading to two β-sheets 1Y. Kondo, 4A. Ono, 2R. Katahira, 5F. M. Bickelhaupt, at one side and two α-helix at the other side of the fold. 3 , 2C. Kojima, 1∗Yoshiyuki Tanaka Vladimir Sychrovsky Titration of CsPABP with Poly(A) results in shifts on a group 1Laboratory of Molecular Transformation, Tohoku Univer- of cross-peaks in the 15N-HSQC (heteronuclear single quan- sity, 2Institute for Protein Research, Osaka University, tum correlation) spectrum. Considerable shifts were observed 3Institute of Organic Chemistry and Biochemistry, Academy for the first β-strand, that comprises residues I77, V79, and of Sciences of Czech Republic, 4Department of Material & the loop that links it to the first α-helix and contains residues Life Chemistry, Faculty of Engineering, Kangawa Univer- N81, Y84 and C86; L105, at the end of second β-strand and sity, 5Department of Theoretical Chemistry, VU University K105 and G106 in the beginning of the third β-strand; and Amsterdam also at the C-terminal region including residues R150, T151 The thymine-Hg-thymine is one of the most thoroughly stud- and N152. Most of these contacts are close to residues re- ied metal-mediated base pairs owing to its possible usability sponsible for the dimerization of the protein. II in future nanotechnologies. The base pairing of T-Hg -T Supported by: FAPESP, CNPq and LNBio. was unambiguously determined by 15N NMR spectroscopy 2 15 II 15 with the J15N,15N J-coupling across N-Hg - N link- age.[1] The mercury bonding to imino nitrogen of thymine TH054: A Redox 2-Cys Mechanism Regulates the was recently characterized as covalent with significant ionic Catalytic Activity of Divergent Cyclophilins II character.[2] The mercury atoms in two consecutive T-Hg - 1∗ 1 1 Bruna M. C. Ramos, Mauricio Luis Sforça, Andre T base pairs attract each other owing to the metallophilic 1 2 Luis Berteli Ambrosio, Mariane N. Domingues, Sara B-M. attraction despite their cationic nature.[3] Here we report 1 1 199 1 Whittaker, Mario T. Murakami, Ana Carolina de Mattos the Hg NMR chemical shift and the J199 15 coupling 1 Hg, N Zeri, Celso E. Benedetti that was both measured and calculated in thymine-HgII - 1Laboratório Nacional de Biociências (LNBio), Centro Na- thymine base pair. The theoretical calculations provided de- cional de Pesquisa em Energia e Materiais (CNPEM), tail information on dependence of the NMR parameters on CP6192, CEP 13083-970, Campinas, SP, Brazil, 2The nature and chemical bonding in metal-mediated base pair. Henry Wellcome Building for Biomolecular NMR Spec- [1] Tanaka, Oda, Yamaguchi, Kondo, Kojima, Ono, J. Am. troscopy, School of Cancer Sciences, University of Birming- Chem. Soc. 2007, 129, 244-245. ham, Birmingham B15 2TT, United Kingdom Citrus sinensis Xan- [2] Uchiyama, Miura, Takeuchi, Dairaku, Komuro, Kawa- The cyclophilin CsCyp is a target of the thomonas citri mura, Kondo, Benda, Sychrovsky, Bour, Okamoto, Ono, TAL effector PthA, required to elicit cankers Tanaka, Nucleic Acids Res. 2012, 40, 5766-5774. on citrus. CsCyp binds the citrus thioredoxin CsTdx and C-terminal domain of RNA Polymerase II (CTD). CsCyp is [3] Benda, Straka, Tanaka, Sychrovsky, Physical Chemistry a divergent cyclophilin that carries the additional loop KS- Chemical Physics 2011, 13, 100-103. GKPLH, invariable cysteines C40 and C168, and conserved glutamate E83. Despite the suggested roles in ATP and metal Acknowledgement: Grant Agency of the Czech Republic binding, the function of these unique structural elements re- P205/10/0228. mains unknown. Here we show that the conserved cysteines form a disulfide bond that inactivates the enzyme, whereas TU053: Structural analysis of Citrus sinensis E83, which belongs to the catalytic loop and is also criti- Poly(A)-Binding Protein PABP cal for enzyme activity, is anchored to the divergent loop to 1∗ 1 maintain the active site open. In addition, we demonstrate Mauricio Luis Sforça, Mariane N. Domingues, that CsCyp binds the citrus CTD YSPSAP repeat. Our 3Tatiana A.C.B. Souza, 2Tiago A. Souza, 1Adriana S. So- 1 1 data support the model where formation of the C40-C168 prano, Ana Carolina de Mattos Zeri, Celso E. Benedetti disulfide bond induces a conformational change that disrupts 1Laboratório Nacional de Biociências (LNBio), Centro Na- the interaction of the divergent and catalytic loops, via E83, cional de Pesquisas em Energia e Materiais (CNPEM), causing the active site to close. This suggests a new type Campinas-SP - Brasil., 2Universidade de São Paulo, Insti- of allosteric regulation in divergent cyclophilins, involving a tuto de Ciências Biomédicas, Centro de Facilidades de Apoio disulfide bond formation and loop displacement mechanism. a Pesquisa (CEFAP-USP), São Paulo,SP - Brasil, 3Insituto Carlos Chagas- Fiocruz/PR, ICC., Curitiba,PR - Brasil ACKNOLEDGEMENTS: FAPESP, CNPq, CNPEM (LNBio), UNICAMP Citrus canker, which affects most commercial citrus vari- eties and is a treat to the Brazilian citriculture, is caused MO055: Solution structures and interaction studies by Xanthomonas citri, a bacterial pathogen that induces the of two natural inhibitors of bacterial cell division formation of eruptions and pustule-like lesions on the sur- face of leaves, stems and fruits. Development of canker le- 1,2Patricia Castellen, 1Mauricio Luis Sforça, 1Ana Car- sions depends on the action of PthA, the major transcription olina de Mattos Zeri, 2∗Frederico José Gueiros-Filho activator-like (TAL) effector of X. citri. We have shown that 1Laboratório Nacional de Biociências, CNPEM, 2Instituto to act as a transcriptional activator, PthA interacts with com- de Química, Universidade de São Paulo ponents of the host basal transcriptional machinery. How- ever, in addition to proteins directly involved in transcrip- Bacterial cell division is a highly regulated process. This tion regulation, some of the PthA-interacting proteins play process is initiated by the assembly of the tubulin like FtsZ roles in mRNA stabilization. Here we present the solution protein giving rise to a cytokinetic ring called Z-ring. The Z

84 Poster Abstracts – Biomolecular Solid-State NMR ring must be placed at the middle of the cell to ensure the equally to this work. Acknowledgments: FAPESP for finan- production of equally-sized daughter cells. cial support and LNBio for accessibility to all the facilities.

The Min system is a well-described negative regulatory sys- TH057: Evaluation of Sample Processing Protocols tem involved in the spatial regulation of the Z ring posi- for Metabolomic Studies of Coffee Plants tioning. This system is highly conserved and consists of MinC and MinD proteins that together act to prevent di- 1Daniel de Menezes Darbello, 1∗Oliveiro Guerreiro vision at the cell poles. In the absence of min genes, division Filho, 2Carolina de Mattos Zeri at cell poles produces nucleoid-free minicells and elongated 1Agronomy Institute of Campinas, 2National Biosciences cells that contain two or more nucleoids. Another inhibitor Laboratory of cell division was discovered in Bacillus subtilis, in a two- hybrid screen using FtsZ as bait. This 40 amino-acid peptide A powerful tool to the execution of the early selection in plant was called MciZ (for mother cell inhibitor of FtsZ). Synthetic breeding programs, is the utilization of biomarkers, that can MciZ interacts with FtsZ inhibiting its GTPase activity and be identified by the metabolomic associated to the Proton 1 polymerization. MciZ is produced during sporulation. In nuclear magnetic resonance ( H NMR) in seedlings. The the absence of MciZ, a Z-ring forms in the sporangium at a object of this study is related to the standardization of the time in development when cytokinetic events normally have metabolic extraction technique of foliar tissue coffee trees re- ceased. sistant and susceptible to the leaf coffee miner, in order to define and easy execution protocol, less onerous and that al- The structure of B. subtilis MinC is unknown and previously lows the separations between theses classes. This way, the little was known about the structure of MciZ. Here we present adult coffee trees were classifieds in relation to the resistance the solution structure of the N-terminal domain of MinC and Leucoptera coffeella resistance level, according to the scale the structure of the MciZ peptide. In addition, we determined stablished by Ramiro et al (2004). Thirty plants were uti- the interaction interface for FtsZ in the MinC N-terminal do- lized to the composition of the insect-resistance groups, being main through NHSQC titration experiments and the binding 15 resistant plants (R) and 15 susceptible plants (S). Differ- site in MciZ through STD experiments. ent techniques were tested in relation to the aqueous extract quality, which are: lyophilized (L/AQ), aqueous extraction of Acknowledgements: FAPESP, USP and LNBio. fresh leaves (Fr/Aq), microwave dried leaves (Mw/Aq), fresh leaves extraction in perchloric acid (Fr/Pa). The efficiency TU056: Functional diversification of cerato-platanins of the method were evaluated through the obtained separa- in Moniliophthora perniciosa as seen by differential tion between the R and S classes, by partial least squares expression and protein function specialization regression (PLS-DA) and chemometric standards, like stan- 1 1,2 dard error of calibration (SEC) and correlation coefficient Juliana Ferreira de Oliveira, Mario Ramos de for calibration (rCal). The metabolic extraction technique Oliveira Barsottini, 3Douglas Adamoski Meira, 2Paulo 1,2 Fr/Aq performed as the most efficiency protocol to classify José Pereira Lima Teixeira, Paula Favoretti Vital do the R and S groups to the leaf coffee miner, allowing the sepa- Prado, 1Henrique Oliveira Tiezzi, 1Mauricio Luis Sforça, 1 1 ration of the classes and providing satisfactory data rCal and Paulo Sérgio Lopes de Oliveira, Ana Carolina de Mattos SEC. It is also the technique with the less number of steps Zeri, 1Sandra Martha Gomes Dias, 1,2Gonçalo Amarante 1∗ and lower execution costs, comparing to the other studied Guimarães Pereira, Andre Luis Berteli Ambrosio methods. 1Laboratório Nacional de Biociências, CNPEM, 2Departamento de Genética e Evolução, UNICAMP, MO058: Biomarkers of Resistance to the Leaf Miner 3Departamento de Genética, Universidade Federal do in Coffee Plants by NMR Paraná 1Daniel de Menezes Darbello, 1Oliveiro Guerreiro Filho, Cerato-platanins (CP) are small cysteine-rich fungal-secreted 2∗Ana Carolina de Mattos Zeri proteins involved in various stages of the host-fungus inter- 1 2 action process, acting as phytotoxins, elicitors and allergens. Agronomy Institute, Campinas, National Biosciences Lab- It has been suggested that this protein family has impor- oratory tant physiological functions, including cell wall interaction As the coffee a perennial species with a long reproductive and manipulation of the host’s defense system. However, cycle, the time and resources applied in the selection of new the precise molecular function remains elusive. Its members cultivars are relatively substantial. One of the methods to have in common low molecular weights, a high percentage of shorten the selection time and reduce the costs with agro- hydrophobic residues and four conserved cysteine residues in- nomic trial is to make an earlier selection. In this way, the volved in the formation of two intramolecular disulfide bonds. object of this study was to identify secondary metabolites Genomic-scale studies have identified twelve sequences en- by proton spectroscopy (1H NMR), which characterize resis- coding putative proteins similar to cerato-platanin proteins tance plants (R) and susceptible (S) to the leaf miner coffee. (termed MpCP1 to 12) in the genome of Moniliophthora For this, the progeny segregating coffee trees H14954-46 be- perniciosa, the causal agent of witches’ broom disease in longing to the generation F2RC5, were classified in relation cacao plants. In this work, we show that the twelve MpCP to the level of resistance to the insect, according the point proteins present distinct expression profiles throughout fun- scale established by Ramiro et al (2004) e subsequently se- gus development and infection. We determined the X-ray lected to metabolic analysis by 1H NMR from aqueous ex- crystal structures of MpCP1, 2, 3 and 5, representative of tracts obtained by the adapted Gomes-Cadenas et al, 2002, different branches of a phylogenetic tree and expressed at technique. By means of variance analyses performed with different stages of the disease. The X-ray crystal structures the identified metabolites, it was observed that just the myo- guided the investigation of their capacity of binding and inositol metabolite, differed signicantly among the groups R hydrolyzing fungus and plant cell wall components and to and S, performing at higher concentration in the susceptible self-aggregate. NMR analysis showed that MpCP3 and 5 plants, which can be considered a biomarker candidate to the distinctly bind to N-acetylglucosamine tetramers (NAG4), identification of the resistant plants e susceptible non-infested allowing the description of an unforeseen binding interface by using the 1H NMR analysis. in MpCP5. MpCP2 and 3 are the most responsive to self- aggregation into amyloidogenic fibrils, which might be impor- tant to hyphae growth and development. The structure and Biomolecular Solid-State NMR information provided in this work will be a useful platform for compound design which can block the protein activity and help to compose a combat approach against the disease. TU059: Structure and orientation of bovine lacto- *Juliana F. Oliveira and Mario R.O. Barsottini contributed ferrampin in the mimetic bacterial membrane as re-

85 Poster Abstracts – Biomolecular Solid-State NMR vealed by Solid-State NMR and molecular dynamics of short-lived coherences (e.g. CA, CB) while evolving on simulation longer-lived spins (e.g. CO) [3]. Overall, we show how this 1∗ 1 1 approach provides complete resonance assignment of back- Akira Naito, Atsushi Tsutsumi, Namsrai Javkhlantugs, bone and sidechains CB resonances. The whole assignment 1Atsushi Kira, 1Masako Umeyama, 1Izuru Kawamura, 2 1 procedure is i) highly sensitive, using only 2 mg of sample, Katsuyuki Nishimura, Kazuyoshi Ueda and less than a week of experimental time; and ii) very ro- 1Yokohama National University, 2Institute for Molecular bust, since sequential assignment results from the alignment Science of three common carbon (CO, CA and CB) chemical shifts for each pair of 1H and 15N nuclei. Bovine lactoferrampin (LFampinB) is a new antimicrobial peptide found in the N1-domain of bovine lactoferrin (268- [1] Klovins J, Overbeek GP, van den Worm SH, Ackermann 284), and consists of 17 amino acid residues. It is important HW and van Duin J (2002). Nucleotide sequence of a ssRNA to determine the orientation and structure of LFampinB in phage from Acinetobacter: kinship to coliphages. J. Gen. bacterial membranes to reveal the antimicrobial mechanism. Virol. 83:1523-1533. We therefore performed 13C and 31P NMR, 13C-31P RE- DOR, potassium ion selective electrode (ISE) and quartz [2] Marchetti A, Jehle S, Felletti M, Knight MJ, Wang Y, crystal microbalance (QCM) measurements for LFampinB Xu ZQ, Park AY, Otting G, Lesage A, Emsley L, Dixon NE with mimetic bacterial membrane and molecular dynamics and Pintacuda G (2012). Backbone assignment of fully pro- 1 (MD) simulation in the acidic membrane [1]. 31P NMR re- tonated solid proteins by H detection and ultrafast magic- sults indicated that LFampinB caused defect in mimetic bac- angle-spinning NMR spectroscopy. Angew. Chem. Int. Ed. terial membranes. ISE measurements showed that ion leak- 51:10756-10759. age occurred for the mimetic bacterial membrane containing [3] Barbet-Massin E, Pell AJ, Jaudzems K, Franks WT, cardiolipin (CL). QCM measurements revealed that LFamp- Retel JS, Akopjana I, Tars K, Emsley L, Oschkinat H, inB had greater affinity to acidic phospholipids than that to Lesage A and Pintacuda G (2013). "Out-and-back 13C-13C neutral phospholipids. 13C DD-MAS and static NMR spec- scalar transfers for protein resonance assignment by proton- tra showed that LFampinB formed a-helix in the N-terminus detected solid-state NMR under ultra-fast MAS", submitted. region and tilted 45◦ to the bilayer normal and center part to C-terminus region takes random coil structure. REDOR dephasing patterns between carbonyl carbon in the helix and MO061: Structural Characterization of Ca- phosphate group were measured by 13C-31P REDOR and ATPase/Phospholamban Complex by Solid-State the results revealed that LFampinB is located in the interfa- NMR Spectroscopy cial region of the membrane. The results of MD simulation ∗Gianluigi Veglia, Nathaniel J Traaseth, Dan Mullen, Raf- showed that the tilt angle was evaluated to be 42◦ and the faello Verardi, Martin Gustavsson rotation angle to be 92.5◦ for Leu3 which are in excellent agreement with the experimental values determined by solid University of Minnesota state NMR. A detailed understanding of protein function requires struc- tural and dynamic analyses of functional complexes rather [1] A. Tsutsumi et al. Biophys. J. 2012, 103, 1735-1743. than that of the isolated binding partners. I will present our latest results on the structural characterization of the TH060: Proton-detected solid-state NMR of viral sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phos- nucleocapsids pholamban (PLN) reconstituted both in synthetic and native hybrid 1∗Emeline Barbet-Massin, 2Kristaps Jaudzems, 1Andrew lipid membranes using a NMR approach. This method 1 3 1 combines distance and angular restraints from magic angle J. Pell, Michael J. Knight, Kaspars Tars, Anne Lesage, 1 15 1 1 spinning (MAS) NMR with orientational restraints ( H- N Lyndon Emsley, Guido Pintacuda 15 1 dipolar couplings and anisotropic N chemical shift) from Centre de RMN à Très Hauts Champs, University of Lyon oriented solid-state NMR (OSSNMR) for the simultaneous (CNRS / ENS Lyon / UCB Lyon1), Villeurbanne„ 2Latvian 3 determination of structure and topology of membrane protein Institute of Organic Synthesis, Riga, Latvia, Biomedical complexes. In addition, I will demonstrate how paramagnetic Research and Study Center, Riga, Latvia relaxation enhancements (PREs) can be used to determine Acinetobacter phage 205 (AP205) is a single-stranded RNA long-range protein-protein interactions. The structure of the coliphage belonging to the family of Leviviridae [1]. In SERCA/PLN complex reveals that the membrane detached, AP205, 180 copies of a dimeric coat protein (2x130 residues) unfolded state of PLN (excited state) binds the cytoplasmic assemble into an icosahedral capsid of about 30 nm in diam- domain of SERCA forming a non-inhibitory complex; while eter. Atomic-level characterization of this system is a chal- the membrane associate helical state of PLN (ground state) lenge since only low-resolution structures from cryo-EM are is the inhibitory form and interacts with the transmembrane 2+ available, and crystals of the viral particles do not diffract. domains of SERCA, inhibiting Ca flux. However, these crystals or hydrated sedimented precipitates are amenable to solid-state NMR, for which the repetitive TU062: Solid-state NMR study of the mechanism positioning of the monomers inside the complex superstruc- of action of novel amphipathic cationic peptides in tures provides a high degree of local order and consequently model membranes highly resolved spectra. ∗Michele Auger, Matthieu Fillion, Aurelien Lorin, Math- Due to the size and complexity of the system, traditional ieu Noel, Marie-Eve Provencher, Normand Voyer strategies based on 13C acquisition for assignment and struc- Université Laval, Department of Chemistry, PROTEO, tural determination are extremely inefficient. CERMA, Quebec, Canada, G1V 0A6 1 We show here that i) high resolution H-detected finger- A wide variety of organisms produce antimicrobial peptides prints of the capsid protein can be obtained by combining as part of their first line of defense. These short cationic very high field and MAS at ultra-fast rates, on both deuter- peptides are being considered as a new generation of an- ated/fully back-exchanged and fully-protonated samples, as tibiotics and represent great hopes against multiresistant- already observed for less complex assemblies [2]. Moreover, ii) 1 resistant bacteria which are an important clinical problem. we present a suite of six 3D H-detected experiments, which Despite their diversity, the main target of antimicrobial pep- yield a rapid and robust assignment of protein backbone tides is the membrane(s) of pathogens. 1H, 15N, 13CO and 13CA and side-chain 13CB resonances. These experiments combine heteronuclear cross-polarization We have previously shown that a non-natural peptide com- (CP) steps with homonuclear scalar-based CC out-and-back posed of 14 residues (10 leucines and 4 phenylalanines mod- transfer blocks, which allow us to record the chemical shifts ified with a crown ether) is able to disrupt lipid bilayers but

86 Poster Abstracts – Solid State NMR is not selective towards bacterial membranes. To gain speci- (ss) NMR techniques to obtain structural information diffi- ficity against negatively charged membranes, several leucines cult to gather using any of these techniques separately. [1] of this 14-mer have been substituted by positively charged 3,5-dimethylpyrazole (DMPZ) was the first reported com- residues (lysine, arginine and histidine). Biological tests in- pound to present a dynamic process in the solid-state dicate that some peptides are active against E. coli but inef- that consists in the transfer of several NH···N protons fective against human erythrocytes. Solid-state NMR exper- from different DMPZ molecules forming a trimer. When iments performed in model membranes were used to better DMPZ is mixed through mechanical grinding with 4,5- characterize the mode of action of the charged peptides. The dimethylimidazole (DMIM) - in a 1:1 molar proportion - the results suggest that the peptides arrange themselves prefer- aforementioned dynamic behavior is suppressed, the DMPZ entially near the bilayer interface perturbing the membrane trimer breaks down and a new co-crystal is formed. [2] by the formation of pores. The structure and orientation of labelled peptides has also been investigated in bilayers ori- Our approach combines powder X-ray diffraction data (re- ented between glass plates as well as in biphenyl bicelles. vealing topological details), solid-state 13C, 15N and 1H Simulations of pore formation in bilayers oriented between NMR data (yielding atomic connections, internuclear prox- glass plates will also be presented. imities and orientation relations on local and intermediate length scales), molecular modeling and ab-initio DFT calcu- TH063: Structural Characterization of MTTR Fib- lations (to create meaningful structures). Multinuclear high- 1 1 1 13 rils by Solid-State NMR resolution 2D H- H double-quantum, H- C (PRESTO and LG-CP) HETCOR and 1H-15N (CP and LG-CP) HET- 1 1 Juliana Santos Santana, Ricardo Oliveira Sant’Anna, COR NMR methods combined with CRAMPS (Combining 1 3 3 Flavia Guedes Andrade, Daniel H A Correa, Lisandra Rotation And Multiple Pulse Sequences) decoupling [3] were 2 1 Marques Gava, Annette Diehl, Carolina Alvares Braga, performed and the resulting structural information was com- 3 2 1 Carlos Henrique Ramos, Hartmut Oschkinat, Débora bined with symmetry restraints derived from powder X-ray 1∗ Foguel, Monica Santos de Freitas diffraction and included in a crystal structure generation evo- 1Universidade Federal do Rio de Janeiro, 2Leibniz-Institute lutionary algorithm to unveil structural features of the co- für Molekulare Pharmakologie, 3Universidade Estadual de crystal formed between DMPZ and DMIM molecules. Campinas We acknowledge FCT for financial support Amyloidosis is a clinical dysfunction caused by extracellular to the Portuguese National NMR Network, accumulation of proteins that are normally soluble in their project PTDC/QUIQUI/100998/2008 and post-doc original structure, but suffered structural modifications gen- grants for MS (SFRH/BPD/65978/2009) and SMS erating insoluble and abnormal fibrils that impair the proper (SFRH/BPD/64752/2009). This work was financed functioning of tissues. Although many challenges have been by FEDER, via ”COMPETE”, and by FCT, PEst- overcoming in the field of amyloidosis many questions are C/CTM/LA0011/2011. still waiting for answers. In this work we are interested [1] NMR crystallography (EMR Books) 2009, R. K. Harris, to evaluate the pathway involved in fibril formation, fol- R. E. Wasylishen and M. J. Duer (editors), Wiley; lowing structural features that could indicates how soluble proteins undergo conformational changes that result in ag- [2] C. López et al., Cent. Eur. J. Chem. 2004, 2(4), 660-671; gregation. The diffraction pattern did not indicate typical [3] M. Sardo et al. J. Phys. Chem. A 2012,116, 6711–6719. value for interstrand distance, suggesting a variation on fib- 13 15 rillar architecture. u- C N MTTR fibrils displayed a well TU065: Structure and nucleation mechanisms in alu- resolved HN-HSQC with linewidth comparable to solution- minum metaphosphate glasses. state NMR, suggesting a high degree of mobility. On the other hand, poor resolution was obtained for experiments 1,2∗Jefferson Esquina Tsuchida, 2Edgar Dutra Zanotto, probing dipolar-dipolar interactions, such as Proton Driv- 1José Fabian Schneider ing Spin Diffusion (PDSD). In order to have the sequen- 1Instituto de Física, Universidade de São Paulo, São Car- tial assignment, a 10% back hydrogen exchanged sample was los, 2Departamento de Engenharia de Materiais, Universi- analyzed to improve spectral resolution by decreasing line dade Feederal de São Carlos broadening. The data indicates a fibrillar core composed of around 30 amino acids. The solid-state data taking together Phosphates glasses are materials with great technological po- solution-state NMR has been cooperative tools to improve tential (optics, bio-materials, glass-metal seals). Study the our knowledgement concerning the misfolding pathway. structure of these systems has basic and technological inter- est, due to the close correlation with macroscopic properties ACKNOLEDGEMENTS: ALV, CAPES, FAPERJ, INBEB, desired in applications and the structure of the vitreous net- UFRJ-ALV work. The low chemical stability of phosphate glasses can be solved by Al incorporation. At this writing no study has shown that phosphate glasses exhibit internal homogeneous Solid State NMR nucleation when subjected to a devitrification process. How- ever in recent preparation of Al(PO3)3 glass, was observed a small internal homogeneous nucleation. MO064: Structure elucidation of azole-based co- In this study will submit the Al(PO3)3 glass to different ther- crystals using high-resolution solid-state NMR, X- mal treatments with the purpose to promote the internal ho- ray diffraction and molecular mechanics mogeneous nucleation process. After the glass melting, the 1∗Mariana Sardo, 1Sérgio M. Santos, 2Concepción López, samples are analyzed by X-ray diffraction to identify the pos- 3Ibon Alkorta, 3José Elguero, 2Rosa M. Claramunt, 1Luís sible presence of crystals formed during cooling. The char- Mafra acteristics temperature such as glass transition (Tg), crystal- lization (Tc) and melting (Tm) are determined by differential 1CICECO – Chemistry Department, University of Aveiro, scanning calorimetry analysis (DSC), in order to plan appro- P-3810-193 Aveiro, Portugal, 2Dep. de Química Orgánica priate thermal treatments purpose to promote the internal y Bio-Orgánica, Facultad de Ciencias - UNED, E-28040 homogeneous nucleation process. After the accomplishment Madrid, Spain, 3Instituto de Química Médica (CSIC), E- of the thermal treatments the samples obtained are character- 28006 Madrid, Spain ized by optical microscopy and scanning electron microscopy In the last few years, there has been an increasing recognition in order to evaluate the microstructure formed and identify of the power of NMR Crystallography, a combined multidis- the aspect ratio of the crystals formed. The structural envi- ciplinary approach integrating powder diffraction, computer- ronments of the forming and modifying species of the network based structure determination and experimental solid-state were analyzed through of 31P and 27Al NMR, quantifying the

87 Poster Abstracts – Solid State NMR distribution of phosphates groups and determining the coor- ppm. Specially, we have investigated the hydride proton dination number of Al. shielding tensors of three Ir(III) complexes, hydridodichloro- bis(tricyclohexylphosphine)irridium(III), HIrX2(PCy3) (X = The challenges in the development of phosphate glasses that Cl, Br, and I). have internal homogeneous nucleation can help considerably in understanding the nucleation mechanisms in a general ox- The principal components of the hydride proton chemical ide glasses. shift tensors of these three complexes were measured by an- alyzing magic-angle spinning H-1 NMR spectra acquired at ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq several spinning speeds at Canada’s National Ultrahigh Field Facility for Solids in Ottawa using a 900 MHz Bruker Avance TH066: Time Reversal Dynamics in Scaled Dipolar II spectrometer. The computer program WSOLIDS was used Interactions by means of Continuous Radiofrequency to irrelatively fit the experimental intensities of spinning- Irradiation sidebands to those calculated as outlined by Herzfeld and Berger [3]. This presentation will also focus on the devel- ∗Axel D. Dente, Lisandro Buljubasich, Patricia R. Levstein, opment of techniques designed to suppress the probe back- Horacio M. Pastawski, Ana Karina Chattah ground as well as peaks of protons from non-hydride protons. IFEG, CONICET-Córdoba, Argentina and FAMAF, UN of Córdoba, Argetnina Finally, the principal components of our experimental pro- ton shielding tensors will be compared with recent quantum The creation of quantum computing devices requires the pre- chemistry calculations by Kaupp and co-workers [4] as well cise control in the evolution of quantum states [1]. In this as DFT calculations carried out in our laboratory. sense, the uncontrolled processes in many-body systems per- turb coherent dynamics leading to decoherence. Thus, the [1]. Kaesz H.D., Saillant, R.B., Chem. Rev., 1972, 72, 231- understanding and the control of these uncontrolled process 281. constitutes a challenge nowadays. In the last decades, the [2]. Masters C., Shaw B. L., Stainbank R. E., J. Chem. Soc. development of NMR techniques, allowed the study of deco- Dalton Trans., 1972, 664-667. herence in liquid and solid state. [3]. Herzfeld J., Berger A. E., J. Chem. Phys., 1980, 73, In this work, we combine the Loschmidt Echo with the solid 6021-6030. state NMR technique, to study the decoherence process in scaled dipolar evolutions using as a specific model system: [4]. Hrobárik P., Hrobáriková V., Meier F., Repiský M., Ko- the polycrystalline Adamantane. In other words, we have morovský S., and Kaupp M., J. Phys. Chem. A, 2011, 115, made used the Loschmidt Echo to quantify the amount of 5654-5659. decoherence produced by the non-controlled internal interac- tions, the environment and the experimental conditions [2]. Acknowledgements: In 1970, Rhim et al. , introduced the Magic Echo pulse se- This project was partly co-operated within the Foundation quence, which enable the reversion of the secular terms in the for Polish Science MPD Program co-financed by the Euro- dipolar Hamiltonian [3]. This was, somehow, the first pulse pean Union (EU) European Regional Development Fund. Ac- sequence to observe the Loschmidt Echo in multi-spin dy- cess to the 900 MHz NMR spectrometer was provided by the namics. Inspired in the Magic Echo pulse sequence, we have Canadian National Ultrahigh-Field NMR Facility for Solids designed new experiments having symmetric conditions, in (http://nmr900.ca). REW acknowledges financial support of the sense that the backward and forward evolutions require the Canada Research Chairs program and NSERC. the same time. In this new pulse sequence we have been able to scale the secular terms of the dipolar evolutions in the TU068: The NQR of highly abundant arsenic min- range between 0 and 1⁄2 (the 0 value coincides with the Lee- erals Goldburg condition [4]). Our experimental results are sup- ∗ ported by detailed numerical simulations in a Hilbert space J. A. Lehmann-Horn, T. Bastow, D. G. Miljak of 19-coupled spins, achieved with the aid of GPU cards. Commonwealth Scientific and Industrial Research Organisa- tion [1] Zurek, Wojciech Hubert, Rev. Mod. Phys. 75, 715 (2003). Arsenic-containing minerals of the form MxAsySz occur in [2] Sánchez, C. M. and Levstein, P. R. and Acosta, R. H. and certain types of gold, copper and nickel mineral deposits and Chattah, A. K., Phys. Rev. A. 80, 12328 (2009). are the primary source for natural and anthropogenic arsenic [3] Rhim, W.-K. and Pines, A. and Waugh, J. S., Phys. Rev. contaminations in groundwater aquifers. The detection of Lett. 25, 218 (1970). arsenic phases on mining sites would be of significant bene- fit to mitigate the release of arsenic into mine waters. Nu- [4] Lee, Moses and Goldburg, Walter I., Phys. Rev. 140, clear quadrupolar resonance (NQR) is a potentially useful A1261 (1965). method to characterise minerals in bulk volumes. We re- port new NQR measurements of arsenic-containing minerals, MO067: Solid-state NMR studies of the influence of commonly occurring in the earth crust and important inter- halide substitution on an extremely shielded hydride metallics in their own right. We determined the NQR fre- proton of iridium complexes quencies from high-field NMR powder patterns and via zero- field frequency sweeps. A brief discussion is given on how 1 1 2 Piotr Garbacz, Mariusz Kędziorek, Victor Terskikh, NQR techniques may be employed in mining operations. 3Guy Bernard, 3∗Roderick E. Wasylishen 1University of Warsaw, Faculty of Chemistry, War- TH069: Improving Delivery Systems through saw, Poland, 2University of Ottawa, Ottawa, Canada, Supramolecular Complexes of Maltodextrin or 3University of Alberta, Department of Chemistry, Edmon- Clyclodextrin and Furosemide Polymorphs ton, Canada 1∗Ana Karina Chattah, 2Marcela Longhi, 2Claudia Gar- Hydrogen nuclei in organic compounds typically have chem- nero ical shifts in range, 0 to 15 ppm. In contrast, most 1Facultad de Matemática, Astronomía y Física and transition-metal hydrides are characterized by a strongly IFEG (CONICET), Universidad Nacional de Córdoba, shielded proton that has a chemical shift in the range, -3 2Departamento de Farmacia, Facultad de Ciencias Quími- to -20 ppm [1]. Notable exceptions include hydridodichloro- cas, Universidad Nacional de Córdoba bis(trialkylphosphine)irridium(III) complexes [2], the focus of this report, which contain an extremely shielded pro- Two critical factors responsible for the poor and highly vari- ton with an isotropic chemical shift of approximately –50 able human bioavailability of Furosemide polymorphs (a loop

88 Poster Abstracts – Solid State NMR diuretic agent) are their low permeability and solubility. On the HPW polyanions yields blue-colored species, a property the other side, selecting a proper polymorph and to control that makes these materials suitable for UV sensors or per- polymorphic transformations has a great impact in pharma- sonal dosimeters. The strength and duration of the pho- ceutical and regulation issues. With all these facts in mind, tochromic response depend on the nature of the organic we have focused our work on preparing and characterizing groups interacting with the polyanion, and on the hydra- new supramolecular systems of Furosemide polymorphs I and tion level of the sample. The understanding of the physic- II with maltodextrin and cyclodextrins. ochemical mechanisms of the photochromic effect and the structure of these materials are fundamental issues to opti- Several techniques have been accomplished to study these mize formulations and preparation conditions. Several solid- complexes in the solution and solid state: spectroscopic tech- state NMR techniques were used to get chemical and struc- niques as solution and solid state NMR, FT-IR and XRPD, tural information: high-resolution 1H, 29Si and 31P-NMR, and also thermal analysis, and scanning electron microscopy. 1 29 1 31 1 H- Si HETCOR and H- P SEDOR. Identification of dif- Relaxation times measurements ( H-T1) have been decisive ferent proton species, far/near to the polyanion, was attained to confirm the formation of the complexes and to assess dif- analyzing the proton spin-lattice relaxation time in the ro- ferences between them. In order to investigate the potential 1 tating frame (T1ρ) with direct and indirect methods ( H- application of the supramolecular complexes as new delivery 31P cross-polarization, TORQUE). The model compound systems enhancing the bioavailability of the drug, solubility HPW·6H2O and photochromic xerogels with varying com- analysis and dissolution tests were used to correlate the per- position and/or preparation atmosphere were studied. The formance between the polymorphic forms and the complexes. results show four sets of H species in the xerogels: free H2O, + ACKNOWLEDGMENTS: CONICET, SECyT-UNC loosely bound H (H2O)n, species strongly interacting with + + HPW: H2O/H3O /H5O2 and species from the ormosil MO070: Solid-state NMR studies on methane activa- (SiOH/CH). The first two species are critically dependent tion and conversion over Zn modified ZSM-5 Zeolites on the atmosphere control during preparation and on ther- mal treatments, and their amount is determinant to the P-H ∗Feng Deng environments and the relative mobility hydrogen-polyanion. Wuhan Institute of Physics and Mathematics, Chinese EPR experiments carried out in irradiated samples showed a Academy of Sciences thermally activated hopping process of an unpaired electron, possibly transferred from organic functionalities. The EPR Methane, the cheapest and most abundant natural resource, spectra also show dynamical differences dependent on the is the least reactive hydrocarbon molecule due to its strong composition of the hybrid material, which could be traced C–H bond strength (104 kcal/mol). Now, the commercial to differences in the photochromic response. The xerogels utilization of methane is still largely dependent on the multi- prepared in low humidity conditions showed weaker EPR in- step syn-gas (CO + H2) strategy, which incurs energy costs tensities than the xerogels prepared in ambient conditions, and lacks selectivity. Environmentally benign heterogeneous indicating that the hydration species on the HPW environ- catalysts in which transition metal (Ga, Zn, Mo etc.) modi- ment have positive influence in the photochromic effect. fied zeolites catalyze the co-conversion of methane with alka- nes and alcohol to hydrocarbons (C2 C12) at relatively low temperatures (< 873K) have attracted significant attention. TH072: In Situ Monitoring of Lithium Ion Con- However, nearly all those catalysts keep silent for conversion ductive Pathways in Lithium-Ion Rechargeable Bat- of methane to more valuable chemicals when the reaction teries using Stray Field Imaging (STRAFI) temperature is below 523 K. 1Joel A. Tang, 1Sneha Dugar, 2Guiming Zhong, 2Yong Yang, In this contribution, low-temperature activation and conver- 1∗Riqiang Fu sion of methane over a new Zn modified H-ZSM-5 zeolite cat- 1National High Magnetic Field Laboratory, Florida State alyst (denoted as ZnZSM-5) was investigated by solid-state University, 2Xiamen University, China NMR spectroscopy in combination with other spectroscopic methods and DFT theoretical calculations. We found for the Electrochemical processes involve ion conductive pathways to first time that the activation of methane resulted in prefer- achieve inter-conversion between electric energy and chemi- ential formation of surface methoxy intermediates at room cal energy for energy storage[1-2]. During charge/discharge temperature, which mediated the formation of methanol and cycles, many inter-exchangeable structural phases are devel- its further conversion to hydrocarbons. Experimental and oped in the electrodes. Some of them become irreversible, theoretical calculation results demonstrated that an oxygen- leading to capacity decay, energy inefficiency, and a short cy- containing dizinc cluster centre in open shell was responsible cle life. A variety of diagnostic tools, such as Infrared spec- for homolytic cleavage of the C–H bond of methane at room troscopy, optical microscope, scanning electron microscope temperature, leading to formation of methyl radicals. The ze- (SEM), electron diffraction and transmission electron micro- olite matrix readily trapped the methyl radicals by forming scope (TEM), X-ray diffraction, and nuclear magnetic reso- the surface methoxy intermediates for further selective con- nance (NMR), have been used to identify the main causes version. In addition, carbonylation of methane with carbon responsible for capacity degradation and cycle life limitation monoxide to directly produce acetic acid over the ZnZSM-5 of the electrodes. However, the electrodes being investigated catalyst under mild condition (573 623K) was also investi- are typically extracted from preparative electrochemical cells ex situ gated by in-situ solid-state NMR technique and two different charged at different potentials, i.e., through processes. ex situ intermediate-dependent reaction pathways were unambigu- During such processes, the equilibrium composition ously identified. of the samples may be altered. It is always desirable to ob- tain the local structures of the electrodes at the atomic level under in situ conditions to understand the electrochemical TU071: Hybrid organic-inorganic photochromic xe- processes. rogels analyzed by solid-state NMR and EPR spec- troscopy Here, we used the stray-field imaging (STRAFI)[3,4] tech- 1∗Marcos de Oliveira Junior, 1José Fabian Schneider, nique to monitor Li-ion transfers in situ. In STRAFI ex- 2Ubirajara P. Rodrigues Filho, 1Cláudio José Magon periments, samples are placed in the strongest static field gradient (> 50T/m) of a magnet. By translating the sam- 1Instituto de Física de São Carlos - Universidade de São ples across the static field gradient, spin populations within Paulo, São Carlos, SP, Brazil, 2Instituto de Química de São a thin slice can be detected. Resolutions as low as 39µm Carlos - Universidade de São Paulo, São Carlos, SP, Brazil are achievable with minimal influence of metal current col- Hybrid materials based in dodecatungstophosphoric acid lectors, making it capable of imaging thin materials in situ (HPW) show reversible photochromic properties in the such as rechargeable batteries[5]. Here, we assembled half cell 200nm-390nm UV range. The photochemical reduction of batteries with Li foil as the anode and Graphite/LiFePO4 as

89 Poster Abstracts – Solid State NMR the cathode and observed in situ Li-ion transfers between the PAS (Per-Arnt-Sim) domain (PASp) and the input signal is electrodes during charge-discharge cycles. We further discuss then relayed to a second, cytosolic PAS domain (PASc) be- the structural changes of these electrodes. These images aid fore leading to auto-phosphorylation in the conserved kinase in the visualization for the formation of Li microstructures, core. To gain mechanistic insights, individual domains of the the primary cause of poor battery performance[6], and will dimerising 60 kDa-protein are characterised with liquid-state eventually assist in designing better electrode materials to NMR. The liquid-state assignments are then used to interpret improve battery performance. solid-state NMR spectra of CitA constructs incorporated into liposomes. [1] M. Armand and J.-M. Tarascon, Nature 451: 652-657 (2008). Although this approach has been used previously for the HK DcuS[3], for which crystal structures[4] and liquid-state NMR [2] Kang, et.al., Science 311: 977 (2006). data[5,6] are available only for PASp, we hereby present a [3] P. J. McDonald, Prog. Nuclear Magn. Reson. Spectrosc. study of a HK by solid-state NMR spectroscopy that is able 30: 69 (1997). to prepare the activated and inactivated state for compari- son and relies on experimental structures for the globular do- [4] Randall, et.al., Solid State Nucl. Magn. Reson. 14: 173- mains. By analysing spectral differences between the citrate- 179 (1999). free and citrate-bound states, it is possible to gain knowledge [5] Tang et. al, J. Magn. Reson. 225: 93-101 (2012). on residues involved in signal transduction. So far, we have been able to determine the alteration of the signal-receiving [6] Chandrashekar, et.al., Nat. Mater. 11: 311-315 (2012). PASp-domain in context of membrane-embedded CitA, show- ing a significant reduction in plasticity upon citrate binding. MO073: 17O NMR Study of High-Tc Cuprate In the near future, the role of PASc in signal transduction (CaLa)(BaLa)CuO will be addressed.

1∗Tonci Cvitanic, 1Miroslav Požek, 2Amit Keren, 2Eran Ultimately, the aim of this study is to acquire an atomic Amit model for signal transduction across the plasma membrane 1Department of Physics, Faculty of Science, University of through HKs. Strategies to reach this goal will be discussed. 2 Zagreb, Department of Physics, Technion-Israel Institute HKs are the most abundant signal receptors in bacteria and of Technology crucial for virulence of several pathogenic species. Thus, un- We investigate the high temperature superconductor cuprate derstanding the function of CitA will have implications not (CaxLa1−x)(Ba1.75−xLa0.25+x)Cu3Oy(CLBLCO) [1], with only for research of transmembrane receptors in general, but 17 YBa2Cu3Oy like structure by O NMR. Measurements were could potentially also lead to new approaches for treating made on underdoped and optimally doped powder samples certain bacterial infections such as Helicobacter pylori - or from two families (x = 0.1 and 0.4). Maximum Tc varies by Staphylococcus aureus - related diseases. 30% with minimal structural changes between families [1]. [1] Kaspar et al.: Mol. Microbiol. 1999, 33(4): 858-72. The spectrum differentiates two distinctive oxygen sites - pla- [2] Sevvana et al.: J. Mol. Biol. 2008, 377: 512-23. nar and apical. Spin-lattice relaxation times on these two sites differ by more than an order of magnitude as oxygen in [3] Etzkorn et al.: Nat. Struct. Mol. Biol. 2008, 15(10): conductive plane relaxes much faster. NMR lines shift with 1031-39. respect to temperature occurs only on planar site, while the apical site shift is temperature independent. The temper- [4] Cheung et al.: J. Biol. Chem. 2008, 283(44): 30256-65. ature dependence of the planar oxygen Knight shift of un- [5] Pappalardo et al.: J. Biol. Chem. 2003, 278(40): 39185- derdoped and doped samples, shows opening of gap at tem- 88. peratures high above Tc, (as previously indicated by magne- tization measurements [2]). Unlike YBCO, where optimally [6] Kneuper et al.: J. Biol. Chem. 2005, 280(21): 20596-603. doped samples show no evidence of pseudogap forming [3], we observe opening of the gap even in optimally doped samples. TH075: Multinuclear solid state NMR investigation of structural development upon heat treatment and [1] O. Chmaissem, Y. Eckstein, and C. G. Kuper, Phys. Rev. dissolution mechanism of sol-gel prepared calcium sil- B 63, 174510 (2001). icate based biomaterials [2] Y. Lubashevsky and A. Keren, Phys. Rev. B 78, 020505 ∗ (2008). Zhongjie Lin, John V. Hanna University of Warwick [3] M. Takigawa et al., Phys. Rev. B 43, 247 (1991) Glassy sol-gel prepared calcium silicate materials show sig- nificant bioactivity depending strongly on composition. A TU074: Transmembrane Signalling: combining systematic multinuclear solid-state MAS NMR study involv- structural biology methods to tackle multi-domain ing nuclei such as 17O, 29Si, 31P, 43Ca and 1H has been membrane proteins undertaken on a range of sol-gel prepared silicate-based bio- 1Benjamin Schomburg, 2Gottfried Unden, 1Stefan glass with three different compositions (CaO)x(SiO2)1−x Becker, 1Adam Lange, 1∗Christian Griesinger (x = 0.2, 0.3, 0.5) to investigate the structural effects in- 1Max-Planck-Institute for biophysical chemistry, 2Institut duced by heat treatment and the reaction with simulated body fluid (SBF) and formation of bone-like hydroxyapatite für Mikrobiologie und Weinforschung, University of Mainz 17 Ca10(PO4)6(OH)2. O MAS and 3QMAS NMR measure- NMR spectroscopy has become one of the principal methods ments on enriched samples have attempted to identify and for investigating the structure and function of proteins. In quantify the bridging oxygen (bo) and non-bridging oxy- spite of recent developments in the field, the study of mem- gen species (nbo) which are characterised by very different brane proteins remains challenging, especially with respect isotropic chemical shift (δiso) and electric field gradient ten- to multi-unit complexes and larger proteins. sor parameters (CQ and ηQ) parameters; this analysis has been combined with 29Si MAS NMR measurements in or- We therefore employ a combination of liquid-state and solid- der to quantify the Qn Si speciation and thus monitor the state NMR spectroscopy to shed light on the mechanism of structural evolution of these systems upon heat treatment. bacterial transmembrane signalling through receptor histi- These solid state 17O NMR results show that the silicate dine kinases (HKs). network is defined by a dominance of bo at lower temper- The Citrate Receptor A (CitA) is used as a model system atures (120◦C), however a gradual introduction of nbo into for HKs[1,2]. Free citrate is recognised by a periplasmic the network is observed at higher temperatures (350-600◦C)

90 Poster Abstracts – Solid State NMR as more Ca becomes incorporated and assumes a key network tation, 3University of São Paulo-Campus São Carlos modifier role. The intensity of the nbo resonance increases Solid state NMR has been a powerful tool for conformation with both increasing CaO molar ratio and annealing temper- analysis of water insoluble proteins, found in cereal grains. atures. Therefore, the objective of this work was to study the kafirin These multinuclear NMR results also demonstrate that the conformations extracted by different methods from grain bioreactivity with respect to SBF reaction of sol-gel prepared sorghum. kafirin is found in the endosperm of the sorghum, calcium silicates depend strongly on the surface OH and cal- corresponding to approximately 70% of its total proteins. cium content. The presence of calcium aids hydroxyapatite kafirin is insolubility in water and its structure is not known. (HA) formation via the promotion of surface hydration and The analyses were performed in Varian Inova 400 spectrom- 13 increasing supersaturation of Ca2+ ions. The 17O MAS and eter. The solid-state C NMR spectra were obtained with 3QMAS NMR results show that the rapid loss of nbo as Ca CPMAS and high-power decoupling, π /2 pulse of 4 ms, 1ms is leached from the silicate network results in changes in sil- contact time, 1024 points, repetition time of 3s, 5mm ZrO ro- icate framework connectivity and the formation of apatite. tor, 8 kHz spinning, 1000 transients and lb = 20. The content The new insights from 31P and 17O MAS NMR of the disso- of α-helix, β-sheet, and disordered structures at 172, 174 and lution mechanism of these materials indicate the importance 176 ppm were determined by fitting the carbonyl peaks area 13 of achieving the right balance of bo/nbo ratio for optimal with lorentzian function. The C NMR spectrum of kafirin biochemical and mechanical properties, and the importance extracted without bisulfite indicated that this protein has of an atomic scale probe such as solid state NMR for de- 84% of α-helix, 14% of disordered and 2% of β-sheet struc- 13 tailed understanding of the structure, bioactivity, dissolution ture. The C NMR spectrum of the kafirin extracted with mechanism and their relationships in such materials. bisulfite indicated that this protein has 89% of α-helix and 11% of β-sheet and no disordered structure. Therefore, both 13 MO076: Host-guest interactions by solid state NMR C NMR spectra showed that this protein these proteins is predominantly α-helix, but the extraction with bisulfite, that 1∗Ljubica Tasic, 1Camila P. Silveira, 2Juliana Fattori, reduced the disulfide bond, increased the proportion β-sheet 3Mariangela B. M. de Azevedo, 4Leonardo Rojas, 4Mavis and reduced the content of disordered structures to a unde- 13 L. Monteiro tected level. In conclusion the solid-state C NMR was the 1Organic Chemistry Department, University of Campinas good method to study of conformation of solid proteins. Al- (UNICAMP), Campinas, SP, Brazil, 2LNBio, CNPEM, though, the bisulfite extraction was more efficient it increase β Campinas, SP, Brazil, 3IPEN, Sao Paulo, SP, Brazil, the -sheet content. 4University of Costa Rica (UCR), San Jose, Costa Rica ACKNOWLEDGMENTS: FAPESP, FAPERJ, CAPES, Inter and intra molecular interactions are crucial for almost PETROBRAS, CNPq all processes in the world of chemistry, biology and biochem- istry, and one of the most powerful techniques in monitoring these interactions is NMR spectroscopy. Herein, we present TH078: Solid state NMR study of cement hydration solid-state NMR applied in monitoring the host-guest inter- to define chemical compositions actions in the development of new drug formulations using 1∗ 2 2 Abdul-Hamid Emwas, Rae Taylor, Paulo Monteiro beta-cyclodextrine as host, and in studies of surface modifi- 1 cations of a biomaterial, hydroxyapatite (HAP). Solid-state King Abdullah University of Science and Technology, 2 NMR spectra were recorded with an AVANCE II 400 MHz Thuwal, Kingdom of Saudi Arabia., Civil and Environmen- spectrometer using a probe equipped with a magic angle spin- tal Engineering, University of California Berkeley, USA 13 ning system having a rotor diameter of 4 mm. C MAS Calcium Silicate Hydrate (C-S-H) is the main binding phase NMR spectra were recorded at 100 MHz with a spinning in all cement based systems, making it the main binding 31 rate of 10 kHz. P NMR spectra were recorded at 161.9 phase of concrete; worldwide the most wildly used mate- MHz, the samples were rotated at a frequency of 15 kHz rial. Using NMR to study the different metal complexes in and an angle of 54’27” to the external magnetic field. Inclu- C-S-H and other cement phases helps to determine the cor- sion complexes of two natural products: auraptene (AUR) relation between molecular structure and macro properties, and 4’-geranyloxyferulic acid (GOFA), known as very power- such as compressive strength, permeability and ultimately ful cancer chemopreventers, with beta-cyclodextrine as host durability of the concrete. Gaining an understanding of how were prepared and their interactions were monitored us- molecular structure correlates with macro properties is most 13 ing C solid-state NMR. Comparison among host, guest useful when considering cement replacement material, such 13 and host-guest C MAS NMR spectra enabled us to pro- as ground granulated blast-furnace slag (GGBS) or fly-ash pose models for host-guest interactions and to calculate (pfa). Materials which are commonly used alone side cement complexation-induced chemical shifts (CICS). Hydroxyap- to either reduce the amount of cement used or to improve atite Ca10(PO4)6(OH)2 (HAP) surface contains two active selected properties of the cement. Different silicon coordina- sites: calcium hydroxide groups and phosphate groups, and tion models can be correlated with the corresponding 29Si can adsorb large quantities of ions or molecules present in NMR chemical shifts, affording an effective tool to differenti- solution. These properties make it a very attractive material ate between different silicon coordinations modles. The 27Al for the synthesis of hybrid materials. Succinamic acid, suc- isotropic chemical shifts can be used to differentiate tetrahe- cinic anhydride, succinimide, 2-aminoethyl dihydrogen phos- dral Al(tetr) and octahedral coordination’s, where the range 13 31 phate and stearic acid were used as ligands. C and P of Al(tetra) 100-50 ppm and Al(octa) in the 20 to -10ppm MAS NMR measurements indicated that the ligands inter- range [1]. In this study we employed 27Al and 29Si solid acted with hydroxyapatite (HAP) surface and a mechanism state NMR spectroscopy to investigate the effect of cement of surface modification was proposed based on the obtained replacement materials and variation of water/cement ratios results. on C-S-H with the aim of gaining further insight in to the for- FAPESP and Spinlab IQ-UNICAMP mation of C-S-H and the resulting effect on concretes macro structure. TU077: Analysis of kafirin conformation by 13C References NMR in solid state

1∗Tatiana Santana Ribeiro, 3Manoel Messias P. Miranda, [1] Skibsted, J., Jakobsen, H.J., and Hall, C., Direct observa- 2Juliana Scramin, 2Luiz Alberto Colnago, 2Lucimara Apare- tion of aluminium guest ions in the silicate phases of cement 27 cida Forato minerals by Al MAS NMR spectroscopy. Journal of the Chemical Society, Faraday Transactions, 1994. 90(14): p. 1Federal University of São Carlos - Campus Araras, 2095-98. 2Brazilian Agricultural Research Corporation - Instrumen-

91 Poster Abstracts – Solid State NMR

MO079: Multinuclear Solid State NMR Investiga- this context, the study presented here aims to investigate the tion into the Speciation and Structure of Aluminium application of theoretical calculations, as an auxiliary tool and Gallium Doped Phosphate Bioactive Glasses on the unequivocal assignment of the 13C SS-NMR signals of the lamivudine drug and the lamivudine salicylate cocrystal. Scott P. King, Steven P. Brown, ∗John V. Hanna University of Warwick, Department of Physics, England. The SS-NMR experiments of the lamivudine and the respec- tive cocrystal were obtained on a Bruker Avance III 500 Calcium phosphate glasses exhibit interesting properties and MHz spectrometer. Theoretical calculations were performed compatibility for use as bioactive materials by providing bone in Gaussian 09 program using B3LYP/cc-pVTZ model and support and by demonstrating tendencies to stimulate bone GIAO method for calculate the NMR shielding tensors. regeneration. Doping of these glass systems with different cations can induce more favourable properties; Al that can The SS-NMR data indicated that there is only one lamivu- strengthen the structure and thus result in greater control of dine in the asymmetric unit of the lamivudine salicylate dissolution rates from the glass network while Ga exhibits dis- cocrystal, confirming a previous X-ray analysis. A very tinct antibacterial properties.[1,2] A multinuclear solid state good corroboration, between experimental and theoretical MAS NMR study of two glass series with the nominal sto- SS-NMR data, was observed for the lamivudine drug and ichiometry x(Al2O3) (11-x)(Na2O) 44.5(CaO) 44.5(P2O5) the value of linear correlation coefficient (R) was 0.99. On (with x = 0, 3, 5, 8) and x(Ga2O3) (25- x)(Na2O) 30(CaO) other hand, comparing the data of the lamivudine salicylate 45(P2O5) (with x = 1, 3, 5, 10, 15) has been undertaken cocrystal, the correlation (R= 0.89) was not as good as for the in order to construct a picture of the glass networks, and lamivudine. This difference could be justify by the fact that to determine how the structure may influence the bioactive in the cocrystal there are intermolecular hydrogen bonds be- properties. tween the asymmetric units, and these effects were not taken into account by the calculation performed for the cocrystal. 27 71 Al MAS and Ga fast MAS NMR measurements demon- Theoretical calculations provided to be a good tool to help strate that for low Al2O3 and Ga2O3 contents in their re- the unequivocal assignment of SS-NMR signals. Calculations spective glasses, the Al and Ga speciation is found to be with specific conditions for mimic solid-state are currently in dominated by octahedrally coordinated species upon initial study. entry into their networks. However, both glass systems tend to favour higher proportions of tetrahedrally coordinated Al 27 TH081: 29Si and Al MAS NMR ratio calculation and Ga structural moieties as the Al2O3 and Ga2O3 contents in zeolites increase, thus showing that Al and Ga play similar structural roles. Information on the phosphorous Qn speciation present 2,3∗Edson de Souza Bento, 1Antônio Euzébio Goulart 31 within each glass is evidenced by P MAS NMR and the Sant’Ana, 1Henrique Fonseca Goulart, 3Geoffrey Ernest 31 P refocused INADEQUATE Spin Echo (REINE) technique Hawkes, 3Harold Toms, 4Ingrid Graça Ramos, 4Artur José 2 that reveals 2D correlations of the JPP couplings with the Santos Mascarenhas 31P chemical shifts of the coupled nuclei.[3,4] These measure- 1Laboratório de Pesquisa em Recursos Naturais, IQB, ments have been performed for the first time on a coherent Universidade Federal de Alagoas, Brasil, 2Laboratório de suite of samples thus enabling the evolving speciation and Ressonância Magnética Nuclear, IQB, Universidade Federal polymerisation of the phosphate network to be mapped out de Alagoas, Brasil, 3NMR Laboratory, SBCS, Queen Mary throughout the entire series. The overall trend of these NMR University of London, UK, 4Instituto de Química, Universi- data suggests that the Al and Ga are playing important sim- dade Federal da Bahia, Brasil ilar roles in the glass network, and may help in providing much greater insight to support their development as bioac- The objective of this work is to encapsulate pheromones in tive materials.[1,2] zeolites, using the nanometric cavities in the zeolite porous structure, so that, when the pheromone is used in the field, [1] Manupriya et al., Phys. Status Solidi A, 2009, 206, 1447– its release will be made gradually. The idea is to evaluate 1455. how different structural properties of various zeolites pre- [2] Valappil, S. P.; Ready, D.; Abou Neel, E. A.; Pickup, D. pared synthetically, will affect the encapsulated pheromone M.; Chrzanowski, W.; O’Dell, L. A. Newport, R. J.; Smith, release. It has been shown that by increasing the Si/Al ratio M. E.; Wilson, M.; J.C. Knowles, Adv. Funct. Mater. 2008, and the nature of the compensating cations, i.e., the higher 18, 732-741. the polarizability and the lower the polarity, the slower the re- lease of attractants.[1] 27Al MAS NMR studies used a Bruker [3] Cadars et al., Phys. Chem. Chem. Phys., 2007, 9, 92-103. Avance 600 spectrometer at resonance frequency of 156.375 MHz, with a 4 mm MAS rotor and sample spinning rate of 12 [4] Guerry et al., J. Am. Chem. Soc., 2009, 131, 11861– kHz, single-pulse excitation (2ms), and repetition time 0.5s. 11874. In the 27Al MAS NMR spectra, signals at ca. 60 ppm are due to tetrahedral aluminum species, AlIV , and the signals TU080: Application of Theoretical Calculations to 13 at ca. 0 ppm are due to octahedrally coordinated aluminum Unequivocal Assignment of Lamivudine C NMR species, AlVI . From integration of these separated signals, Signals in Solid-State and by knowing the total Si/Al ratio of these sample, the 1∗Vinicius Sousa Ferreira, 1Diego Alves Rodrigues, Si/Al ratio of the tetrahedral framework, (Si/Al)fr, could be 2Felipe Terra Martins, 1Luciano Morais Lião, 1Luiz Henrique calculated using the following equation:[2]

Keng Queiroz Júnior (Si/Al)fr = (Si/Al)total (IAl(tet) + IAl(oct))/IAl(tet) 1Universidade Federal de Goiás, Instituto de Química, Lab- oratório de Ressonância Magnética Nuclear, 2Universidade References Federal de Goiás, Instituto de Química, Laboratório de [1] J. Agric. Food Chem., 2001, 49 (10), pp 4801–4807. Cristalografia [2] High-Resolution Solid-State NMR of Silicates and Zeo- Cycle of HIV virus replication presents various events, and lites, John Wiley & Sons, 1987, p. 213. proteins play an important role on it. Such proteins become targets of pharmacological antivirals agents and one of these CNPq is the lamivudine, which has an important role in the treat- ment of AIDS. As the number of antivirals increase, there MO082: Structural Characterization of Oxyfluride is also a growing concern with the quality control, and an Glasses Using Solid State NMR important tool to evaluate the quality of drugs is the Solid- State Nuclear Magnetic Resonance (SS-NMR). However, the 1Raphaell Moreira, 1R. G. Fernandes, 2Jinjun Ren, 1A. S. assignment of the SS-NMR signals is not a trivial task. In S. de Camargo, 1,2∗Hellmut Eckert

92 Poster Abstracts – Solid State NMR

1Physics Institute of Sao Carlos, Sao Paulo University – Sao Leitão 2 Paulo, Brazil, Institut für Physikalische Chemie, WWU 1Instituto de Pesquisas de Produtos Naturais, Universi- Münster, Corrensstrasse 30 - Münster, Germany dade Federal do Rio de Janeiro, Brasil, 2Instituto de Oxyfluoride glasses combine the favourable optical properties Química, Universidade Federal do Rio de Janeiro, Brasil, of fluoride glasses with the superior thermal, mechanical and 3Departamento de Química, Universidade Federal de Juiz chemical stability of oxide glasses. Such glasses are attractive de Fora, Brasil candidates for many applications, for instance optical fibers, Different hydration schemes of pharmaceutical solids implies amplifiers, detectors and sensors.[1] also in distinct intermolecular crystal interactions. This af- The present contribution is understanding with detailed fects the total lattice energy and the solubility, and hence structural studies of various glass systems based on fluo- the active pharmaceutical ingredient (API) bioavalability. rophosphates and fluoroborate compositions, using solid state Cephalexin monohydrate and cefadroxil monohydrate are two nuclear magnetic resonance (NMR) methods.[2,3] Details of very similar β-lactam antibiotics which differ by one pheno- the information on the organization of the framework is ob- lic hydroxyl group. Cephalexin monohydate is described in tained by 31P, 19F, 27Al, and 11B single and double reso- the literature as an isomorphic desolvated solid, but the ce- nance NMR techniques under static and magic-angle sample fadroxil monohydrate crystal lattice dehydration behaviour spinning (MAS) conditions. A number of established (HET- has not been reported so far. In this communication, ther- 13 COR, REDOR) and new (DQ-DRENAR)[4] spectral editing mal, X-ray, C CPMAS NMR experiments and first prin- techniques are used in these systems based on the selective ciples calculations were combined to describe, at the atomic measurement of homo- and heteronuclear magnetic dipole- level the cefadroxil monohydrate dehydration process. dipole interactions. Furthermore, new NMR methodology Cefadroxil monohydrate was purchased from Sigma Aldrich will be introduced for quantifying the omnipresent problem Inc. and dryied in a Abderhalden’s drying apparatus with of fluorine losses during melting. ◦ refluxing toluene (110.6 C) under reduced pressure by 90 References min. The reflux solvent was chosen based on cefadroxil monohydrate thermogravimetric (TG) and differential scan- [1] Zuo, X.; Itoh, K.; Toratani, H.J. Non-Cryst. Solids 1997, ning calorimetry (DSC) analyses. Thermogravimetric analy- 11, 215. sis shows a 25% loss of H2O content, i.e., one water molecule [2] Zhang, L.; Araujo, C. C.; Eckert, H. J. Phys. Chem. B per unit cell before the drying treatment, even though the 2007, 111, 10402. DSC analysis do not shown any signal in the same tempera- ture range. 13C CPMAS NMR studies and X-ray measure- [3] Fernandes, R. G.; Ren, J.; Camargo, A. S. S.; Hernandes, ments were performed before and after the drying treatment. A. C.; Eckert, H. J. Phys. Chem. C 2012, 115, 6434. In order to provide evidence on the ionization state of the [4] Ren, J.; Eckert, H. Angew. Chem. Int. 2012, 51, 12888. amine and the carboxyl groups (Zwitterionic structure), in- frared spectroscopy (FT-IR) was used. First principles cal- TU083: Study of the thermal processes in molecular culations were also done using X-ray cell parameters and the crystals of peptides by means of NMR Crystallogra- heavy atoms positions, combined with FT-IR based protona- phy tion states, with pseudopotential and planewaves approach. The 13C CPMAS NMR spectra shows great changes, espe- ∗Tomasz Pawlak, Piotr Paluch, Katarzyna Trzeciak- cially at the β-lactam carbonyl and aromatic resonances. Karlikowska, Agata Jeziorna, Marek J. Potrzebowski 27 Centre of Molecular and Macromolecular Studies, Polish MO085: Solid-state Al NMR study of the thermal Academy of Sciences changes occurring in alumina-carbon composites 1D and 2D Very Fast Magic Angle Spinning (VF MAS) NMR Thierry R. Lopes, Gustavo R. Gonçalves, Ewerton de Bar- experiments with sample rotation up to 55 kHz were ap- cellos Jr., Miguel A. Schettino Jr., Alfredo G. Cunha, Fran- plied to study both the dihydrate form of Tyr-(D)Ala-Phe- cisco G. Emmerich, ∗Jair C. C. Freitas Gly (N-terminal sequence of opioid peptide dermorphin) and Laboratory of Carbon and Ceramic Materials, Department the anhydrous form, which was obtained by thermal treat- of Physics, UFES ment. Employing both homo-nuclear (1H-1H BABA, 13C- 27 13C SHANGHAI) and hetero-nuclear 2D correlations (1H- Solid-state Al nuclear magnetic resonance (NMR) spec- 13C and 1H-15N) with inverse detection, it was shown that troscopy with magic angle spinning (MAS) is long recog- removing water from the crystal lattice of this tetrapeptide nized as a powerful tool for the characterization of crystalline does not destroy its subtle pseudo-cyclic structure, and its or disordered aluminas. Porous carbon materials containing supra-molecular array is preserved. The GIPAW method was dispersed aluminum oxides are of interest for applications employed to compute the geometry of the peptides and cal- such as the removal of fluoride from water, besides their ad- 13 culate the C sii principal elements of the NMR shielding vantageous use as catalysts supports. In this work we have tensor parameters and 1H isotropic NMR shifts. The theo- investigated the structural and chemical transformations oc- 13 retical values of C sii were compared with the experimental curring in alumina-carbon composites upon heat-treatment, 13 27 C dii chemical shift tensor values obtained by a 2D PASS by using solid-state Al MAS NMR spectroscopy in com- 13 1 experiment. The correlations C sii versus dii and H siso bination with X-ray diffraction (XRD) and thermal analy- versus diso were used to evaluate the quality of the compu- ses. Two different carbon precursors were employed: a com- tational approach. With the new set of coordinates obtained mercial activated carbon (denoted as AC) and a char ob- by the GIPAW method, the crystal and molecular structure tained by carbonization of the endocarp of babassu coconut ◦ of the dehydrated Tyr-(D)Ala-Phe-Gly peptide that was ob- at 700 C (denoted as BC), with specific surface areas of 1300 2 tained by thermal treatment was constructed. Methodology and 340 m /g, respectively. The alumina-carbon composites used in this project combining NMR measurements, analy- were prepared by aqueous impregnation of AC or BC with sis of X-ray powder diffraction data and advanced quantum aluminum nitrate and, after filtering and drying, were sub- mechanical calculations is known as NMR crystallography. mitted to heat treatments under argon flow at temperatures up to 1500 ◦C. The aluminum compounds present in the as-synthesized AC_Al samples were identified by XRD and TH084: Probing cefadroxil isomorphic desolvate be- 27 haviour by 13C CPMAS NMR and first principles solid-state Al MAS NMR as nanocrystalline aluminum oxy- calculations hydroxides or hydroxides, depending on the detailed synthe- sis conditions. On the other hand, all aluminum-containing 1Daniel Lima Marques de Aguiar, 1,2∗Rosane Aguiar da phases were X-ray amorphous in the as-synthesized BC_Al 2 2 Silva San Gil, Ricardo Bicca de Alencastro, Eugênio Fur- samples, with the presence of a distribution of AlO6 (octa- 3 3 tado de Souza, Viviane da Silva Vaiss, Alexandre Amaral hedral Al site), AlO5 and AlO4 (tetrahedral Al site) units

93 Poster Abstracts – Solid State NMR revealed by solid-state 27Al NMR. The quadrupole coupling advantage of being sensitive to internal magnetic fields and parameters, isotropic chemical shifts and relative fractions of electric field gradients (EFG), without the need of strong each component were determined by fitting the spectra to a static external magnetic fields. In the case of NMRz, used for set of second-order quadrupolar lineshapes broadened by a studying magnetic materials, the spectra result from strong distribution of quadrupole couplings and chemical shifts as- Zeeman coupling interaction perturbed by quadrupolar in- sociated with disorder in the 27Al environments. The results teraction. Using two arbitrary radio-frequency pulses, it is showed that the nature of the carbon material used to pre- possible to observe multiple-quantum (MQ) echoes, where pare the composite was of great relevance for the definition each echo spectrum presents a specific spectral line broader- of the type and crystallinity of the alumina phases formed ing. To explain such behavior, it was proposed a model in after heat treatments. which the magnetic sample presents inhomogeneity distribu- tions of Zeeman and quadrupolar coupling. Our experiments ACKNOWLEDGEMENTS: CAPES, CNPq, FAPES, show that the magnetic materials have two different regions FINEP, Petrobras, NCQP/UFES. where: (i) the Zeeman coupling distribution is larger than the quadrupolar one and (ii) the dispersions of both distri- TU086: Solid-state 13C and 31P NMR studies of butions are similar. Under these two conditions, it was pos- biochars prepared by chemical and thermal treat- sible to observe one echo for each coherence of nth order. To ment of peat observe individually each echo and better explore the phys- ical information contained in each of them, MQ coherence 1 2 2 Hercílio D. A. Honorato, Daniel F. Cipriano, Gustavo selection was performed by applying specific phase cyclings 2 1,2∗ R. Gonçalves, Miguel A. Schettino Jr., Jair C. C. Fre- and time-averaging. These methods offered complementary itas physical information about the nuclear spin interactions and 1Centre of Competence on Petroleum Chemistry (NCQP), the magnetic materials properties. In the case of NQR, the Department of Chemistry, Federal University of E, quadrupolar coupling is the most intense interaction. A weak 2Laboratory of Carbon and Ceramic Materials (LMC), De- external static magnetic field is applied just to remove the partment of Physics, UFES nuclear quadrupolar states degeneracy. The transition fre- quencies depend on the relative orientation between the weak Biochar is a carbonaceous material produced by pyrolysis external static magnetic field and the EFG main axis. This of biomass, which attracts great interest due to its applica- feature allows performing some new experiments, being our tions as soil additive and in carbon sequestration. The phys- main goal to employ NQR for designing quantum information ical and chemical properties of biochars can be changed by procedures, e.g., the creation of pseudopure quantum states subjecting the material to different types of chemical and/or and logic gates. thermal treatments. This work was devoted to a study of the occurrence of phosphorus in carbon materials obtained AKNOWLEDGEMENTS: IFSC, USP, FAPESP, CNPq, and by heat treatments of peat impregnated with H3PO4. Solid- CAPES. state nuclear magnetic resonance (NMR) was used as the primary characterization technique. Peat samples were sub- MO088: Comparison of Efavirenz Data Obtained by jected to heat treatments in the presence of H3PO4, leading Solid State NMR Spectra and Molecular Modeling to the incorporation of phosphorus into the structure of the produced biochars and in some cases to an increase in poros- 1Eduardo Gomes Rodrigues de Sousa, 1∗Erika Mar- ity. The resulting material was then analyzed by solid-state tins de Carvalho, 1Osvaldo Andrade Santos-Filho, 2Rosane 31 13 P and C NMR under high magnetic field, among other Aguiar da Silva San Gil characterization methods, in order to investigate the nature 1Instituto de Tecnologia em Fármacos/ FIOCRUZ, Departa- of the phosphorus-containing species in the structure of the mento de Síntese, RJ, Brazil, 2Universidade Federal do Rio biochars. The results showed that phosphorus was gener- de Janeiro, IQ, Lab. RMN de Sólidos, RJ, Brazil ally incorporated into the biochars as phosphate groups, in most cases bonded to aliphatic groups. The formation of Nowadays, in order to evaluate a drug in the solid state it is polyphosphate chains was observed at relatively low heat- very important to ensure its quality. An extremely powerful treatment temperatures (ca. 350 ◦C), but these groups were tool, which has been increasing their importance and applica- decomposed with the increase in the heat-treatment temper- bility for the characterization of pharmaceutical formulations, ature. By X-ray diffraction, it was also possible to observe is the solid state nuclear magnetic resonance spectroscopy (ss- a beneficial effect of the phosphorus presence for the pro- NMR). Many regulators recognize the importance of the tech- cess of structural organization of graphene-like planes within nique of NMR in the process of drug development and qual- the turbostratic microcrystallites. With increase in the heat- ity control thereof by the pharmaceutical industry. On the treatment temperature, a general trend of upfield shifting of other hand, molecular modeling (M.M.) is an important tool the main 31P NMR peaks was observed, which was associ- for predicting the chemical shifts expected for each carbon ated with the increase in the diamagnetic susceptibility of the of a particular molecule. The aim of this work was to com- graphene-like planes. pare experimental chemical shifts (ppm) data of Efavirenz (EFZ) obtained by ssNMR and the calculated data obtained ACKNOWLEDGEMENTS: CAPES, CNPq, FAPES, by M.M. and verify the ability of the empirical method in FINEP, Petrobras, NCQP/UFES. predicting the experimental chemical shifts. The structure of monomeric EFZ was fully optimized with TH087: New Zero-field NMR and NQR Experiments density functional theory –DFT– (B3LYP/6-311+G(2d,p)) for Studying Materials and Quantum Information methodology. A conformational analysis was carried out on Processing the optimized structure using an adaptation of the method 1Christian Rivera Ascona, 1Rodrigo de Oliveira Silva, described by Mahapatra et al. (2010). Crystallography 1Roberson Saraiva Polli, 1Daniel César Braz, 1Diogo O. data showed that EFZ unitary cell contains two different Soares Pinto, 2João Teles de Carvalho, 3José Roberto To- molecules, associated by two hydrogen bonds (Cuffini, 2009), zoni, 1∗Tito José Bonagamba and thus the molecular modeling of dimeric form was also performed. The X-ray geometry of EFZ dimeric form was 1Instituto de Física de São Carlos - Universidade de São used as a starting point for calculations. There are no signif- Paulo, 2Departamento de Ciências da Natureza, Matemática icant differences between the crystal and optimized structure. e Educação - Universidade Federal de São Carlos, 3Instituto The results have shown that the dimeric form is much more de Física - Universidade Federal de Uberlândia stable than the monomer. The largest deviations could be Zero-field Nuclear Magnetic Resonance (NMRz) and Nuclear observed between the calculated and experimental 13C NMR Quadrupole Resonance (NQR) are techniques that offer im- chemical shifts data obtained for the two forms in deuter- portant information about nuclear environments, having the ated chloroform (CDCl3). However when the experimental

94 Poster Abstracts – Solid State NMR data of dimeric form obtained by ssNMR were compared to TH090: Combining Solid-State NMR, X-Ray those obtained by molecular modeling (in vacuum), it could Diffraction and DFT Calculations to Characterize be seen minimum deviations between experimental and cal- Carvedilol Polymorphs culated data. 1∗Carlos A. Rezende, 1Katia Z. Leal, 2Rosane Aguiar da References Silva San Gil, 2Daniel Lima Marques de Aguiar, 3Viviane da Silva Vaiss, 1Jackson A. L. C. Resende, 2Ricardo Bicca de MAHAPATRA, S. et al. New Solid State Forms of the Anti- Alencastro, 3Alexandre Amaral Leitão HIV Drug Efavirenz. Conformational Flexibility and High Z’ 1Universidade Federal Fluminense, 2Universidade Federal Issues. Crystal Growth & Design. v. 7, n. 10, p. 3191-3202, do Rio de Janeiro, 3Universidade Federal de Juiz de Fora 2010. Drugs can exist in different crystal structures, a phenomenon CUFFINI, S. et al. (S)-6-Chloro-4-cyclopropylethynyl-4- known as polymorphism. Polymorphism can alter drug’s 1 trifluoromethyl- H-3,1-benzoxazin-2(4H)-one. Acta Crys- physico-chemical properties which are essential to ensure talolographica Section E: Structure Reports Online, v. 65, effectiveness, safety and quality of a product. Thus the p. 3170-3171, 2009. knowledge of the solid state properties is very important in the pharmaceutical field. Carvedilol belongs to beta- ACKNOWLEDGEMENTS: INSTITUTO DE TECNOLO- adrenolytics, a large group of cardiovascular drugs used clin- GIA EM FÁRMACOS/ FIOCRUZ, UFRJ. ically in the treatment of heart failure, hypertension, and certain types of cardiac arrhythmias. Regarding the poly- TU089: Influence of the Recrystallization Solvent on morphism of carvedilol, the crystal structures of three anhy- the Efavirenz Crystalline Structure Probed by Solid drous modifications were previously reported, including form State NMR I, form II (oral dosage form), form IV and hydrate form III. Solid-state NMR has become an essential technique for the 1Eduardo Gomes Rodrigues de Sousa, 1∗Erika Martins 2 3 solid-state characterization of pharmaceuticals. The tech- de Carvalho, Tereza Cristina dos Santos, Rosane Aguiar nique can not only differentiate between different solid-state da Silva San Gil forms of a material, but also intimately probe the structural 1Instituto de Tecnologia em Fármacos/Fiocruz, Departa- aspects of each solid-state form. This is especially impor- mento de Síntese Orgânica, Núcleo de RMN, RJ, 2Instituto tant for solid-state forms that can not be crystallized and de Tecnologia em Fármacos/Fiocruz, Coordenação de De- studied by single-crystal X-ray techniques. 13C Cross Po- senvolvimento Tecnológico, RJ, Bra, 3Universidade Federal larization Magic Angle Spinning experiments of Carvedilol do Rio de Janeiro, IQ, Lab. RMN de Sólidos, RJ, Brazil. form II were made and the number of peaks are exactly the In recent years, much attention has been focused on the pre- number of atoms present in the molecule. To help in reso- disposition of pharmaceutical solids to crystallize in several nance assignment, ab initio codes based on periodic bound- crystalline forms. The different crystalline forms of a drug ary conditions have been used regarding the study of small can exhibit distinct chemical and physical properties, the molecules. QUANTUM - Espresso is an integrated suite of important ones being stability, dissolution and bioavailabil- computer codes for electronic structure calculations and ma- ity. Some data on literature[1-3] report that efavirenz (EFZ) terials modeling at the nanoscale. It is based on Density crystalline structures are obtained, depending upon the fac- Functional Theory, planewaves, and pseudopotentials (both tors such as solvent, temperature, additives and preparation norm-conserving and ultrasoft). A very good agreement was methods. The aim of this work was to obtain crystalline found for the comparison between the global results of ex- perimental and calculated NMR chemical shifts for carvedilol EFZ through the use of different solvents (THF and hep- 2 tane), probed by solid state nuclear resonance magnetic (13C form II (R > 0.99). This work aims a comprehensive under- CPMAS) spectroscopy and X-ray diffraction. standing of carvedilol crystalline forms employing SSNMR and DFT calculations. Other results and discussion will be 13C NMR solution spectra of the samples thus obtained included in this communication. showed identical chemical shifts, and the 13C chemical shifts observed for solid samples were assigned on the basis of Acknowledgements: CAPES, CNPq and FAPERJ. liquid-state data. Some differences in the chemical shifts δδ = δliquid–δsolid observed and are indicative of rigid and confor- MO091: Multinuclear Solid-State NMR Characteri- mationally flexible fragments in the molecule (the latter are zation of Hydrotreating Catalysts and its Precursors expected to undergo larger changes). They could be used as 1,2L S. Chinelatto Júnior, 2,3H.R.X. Pimentel, 2∗Rosane a probe for ascertain intermolecular interactions as H-bond. 1 13 δ δ Aguiar da Silva San Gil, Sonia Maria Cabral de Menezes, Some C presented large differences liquid– solid and are 2 1 splitted into a doublet (C3,C6,C9,C12,C13 and C14) or Leandro B. Borré, S.S.X. Chiaro triplet (C10), indicating a different chemical environments 1PETROBRAS-CENPES, 2Universidade Federal do Rio de due to the crystallization process. Janeiro, 3Universidade Federal Fluminense

REFERENCES The characterization of hydrotreating (HDT) catalysts and its precursors with multifield, multinuclear and multidimen- [1] DESHMUKH, V. et al. Solubility Enhancement of sional solid-state Nuclear Magnetic Resonance (NMR) tech- Efavirenz Hydrochloride by Hot Melt Technique. Current niques is essential to achieve a detailed understanding of Pharma Research, v. 1, n. 4, p. 320-336, 2011. the large differences in the distribution and structure of the species formed upon impregnation, calcination and sul- [2] MAHAPATRA, S.; THAKUR, T. S.; JOSEPH, S.; fidation. In this work, commercial and synthesized pseu- VARUGHESE, S.; DESIRAJU, G. R. New Solid State Forms dobohemites and γ-aluminas prepared from these precur- of the Anti-HIV Drug Efavirenz. Conformational Flexibility sors, HDT catalysts prepared by impregnation of γ-alumina and High Z. Crystal Growth & Design, v. 7, n. 10, p. 3191- and hydrotalcite with Mo, Co, Ni, P and sulphidation and 3202, 2010. commercial HDT catalysts were characterized by solid-state NMR (MAS-NMR) of 27Al, 31P and 17O, using Bruker [3] CUFFINI, S. et al. (S)-6-Chloro-4-cyclopropylethynyl- Avance III (9.4 and 11.7T) and Bruker DRX300 (7.05T) 4-trifluoromethyl-1H-3,1-benzoxazin-2(4H)-one. Acta Crys- NMR equipments. The characterization of γ-aluminas by talolographica Section E: Structure Reports Online, v. 65, p. 17 using O selective solid echo sequence (π/2)s-τ1-(π)s- 3170-3171, 2009. 17 τ2-acquire was performed through O enrichment of 17 ACKNOWLEDGEMENTS: FARMANGUINHOS/ the samples with H2 O-40% before and after impregnation FIOCRUZ, UFRJ with the metals and sulphidation. The subscript s in the pulse sequence representation refers to a selective ±1/2 cen-

95 Poster Abstracts – Solid State NMR tral transition pulse. The 27Al MAS NMR spectra of those of Sugarcane Bagasse and Eucalyptus Bark γ -aluminas evidenced that the enrichment did not alter the 1∗ 1 alumina. The impregnation with the metals and P was done Oigres Daniel Bernardinelli, Marisa Aparecida Lima, 2Gabriela Bassetti Lavorente, 2Hana Karina Pereira da Silva, both in steps and by using co-impregnation. It was possi- 2 1 3 ble to establish the presence of P-O-Al bonds, by 2D NMR Juliano Bragatto, Camila Alves Rezende, Leonardo D. 27 27 31 Gomez, 3Simon J. McQueen-Mason, 2Carlos Alberto Labate, experiments ( Al MQMAS NMR and Al- P HETCOR), 1 1 especially in co-impregnated catalysts, unlike shown in the Igor Polikarpov, Eduardo Ribeiro de Azevêdo literature.[1] The presence of phases Mo-17O-Mo were also 1Instituto de Física de São Carlos, USP, 2Laboratório Max confirmed by 17O MAS (selective solid echo). The results Feffer de Genética de Plantas, USP, 3CNAP, Department showed that the support structure influences the formation of Biology, University of York of intermediate species during the early stages of impregna- Characterization of lignocellulosic biomass to produce multi- tion, calcinations and sulfidation. The natural abundance products such as ethanol and other biomaterial is challenging selective solid echo 95Mo NMR experiments performed on due to its structural complexity and heterogeneity. How- the HDT catalysts reveals a loss of symmetry upon calcina- ever, know the chemical structure of lignocellulosic biomass tion, probably due to polymerization of the surface species is essential to understand possible modifications in the mor- to form a MoO3-like species. phology and chemical composition of the materials submit- References ted to specific pretreatment. The pretreatment is responsible for the successful use of lignocellulosic biomass feedstock in [1] KRAUS, H.; PRINS, R. The Effect do Phosphoroys on Ox- ethanol production. Solid state nuclear magnetic resonance γ idic NiMo(CoMo)/ -Al2O3 Catalysts: A Solid State NMR (ssNMR) is regarded as one of the best tools for elucidat- Investigation J. Cat, 170: 20-28, 1997. ing structures of lignocellulosic biomass. The primary solid state ssNMR technique that has been used so far is the rou- 13 1 TU092: C Solid-Sate and H Time-domain NMR tine 13C cross polarization-magic angle spinning (CP-MAS) characterization of mobility and secondary structure technique. Although this technique has markedly advanced of wheat gluten based materials our understanding of lignocellulosic biomass, full potential of ssNMR for characterizing these samples has yet to be real- 1Fabiana Diuk Andrade, 1Oigres Daniel Bernardinelli, ized. Technical developments and applications of advanced 2Ramune Kuktaite, 3Tomás S. Plivelic, 1∗Eduardo Ribeiro solid state NMR have revealed the promise deeper insights de Azevêdo into structures of lignocellulosic biomass. In this work, we 1 Institute of Physics of São Carlos - University of São Paulo, present a ssNMR oriented characterization of the delignifi- 2 Department of Agriculture - Farming Systems, Technology cation process with increasing sodium hydroxide concentra- and Product Quality, The Swedish University of Agricultural tions, preceded or not by diluted acid, of two eucalyptus barks 3 Sciences, MAX-lab - Lund University clones, Eucalyptus grandis (EG) and the hybrid, E. grandis We report a combined DSC, 13C Solid-state and 1H Time- x urophylla (HGU), as well as in sugarcane bagasse. The en- domain NMR study in films of gliadin and glutenin rich zymatic digestibility and total cellulose conversion were mea- wheat gluten using different amount of glycerol (0%, 10%, sured and correlated with the modification in the composition 20%, 30% and 40%) as the plasticizer [1]. High field (400 of the solid and the liquor fractions, as seen by ssNMR and MHz) Magic Angle Solid-state 13C NMR experiments per- Fourier-transform infrared spectroscopy (FTIR). Besides, a formed with cross polarization, CP/MAS, and single pulse comparison between the crystalline fraction as obtained by excitation, SPE/MAS, allowed the identification of at least ssNMR and X-Ray diffraction was also performed, showing a three distinct components based on their mobility. While tendency of removing the amorphous contend by the chemical the more mobile components were attributed to the lipid and treatment. glycerol moieties, the more rigid segments, solid-like, as well ACKNOLEDGEMENTS: CNPq as those with intermediate mobility were attributed to the backbone and side-chains of the proteins. These identifica- tions allowed to analyse low field (20 MHz) FIDs acquired MO094: Solid State NMR and Low Field Re- after the application of a Magic-Sandwich Echo sequence laxometry Characterization of Brazilian Clay-EVA (MSE-FID) as well as Time-domain signals obtained with Nanocomposites a mixed magic-sandwich echo-Carr-Purcell-Meiboom-Gill se- 1Elisabeth G. van der Linden, 1∗Rosane Aguiar da quence (MSE-CPMG) in terms of three components, whose Silva San Gil, 1Leandro B. Borré, 1Luis G. V. Gelves, relative amounts and the average motion correlation times 1Vera L. P. Soares, 1Simone P. Silva, 2Maria Inês Bruno were obtained for all samples. Besides, the 13C CP/MAS Tavares, 2Roberto P. Cucinelli Neto, 3Sérgio S. Camargo Jr., spectra and DSC thermograms also showed that the relative 3Emanuel Santos Jr. fractions of protein segments with a-helix, b-sheets, and ran- 1 2 dom conformations varied according to glycerol content and UFRJ, Instituto de Quimica, UFRJ, Instituto de Macro- 3 relative amount of gliadin and glutenin in the films. The re- moleculas, UFRJ, COPPE, Eng.Metalurgica e Materiais sults showed that the segmental mobility and relative fraction Polymer nanocomposites have been used to design new ma- of segments with a-helix conformation tend to increase as a terials with improved properties. The lamellar structure function of the glycerol contend in gliadin rich samples, which present in montmorillonite furnishes the basic structure nec- occur together with a reduction in the conformation disorder essary for the synthesis of clay based nanocomposites. The of the a-helices. In contrast, for glutenins rich fractions the substitution of exchanging cations by ammonium quaternary results pointed to a promotion of b-sheet conformation for salts leads to organically modified montmorillonites. The increasing glycerol amounts. Since the predominance of seg- presence of these hybrid materials in a polymer matrix in- ments with a-helix or b-sheet conformations has strong effects creases its thermal stability, and also its mechanical proper- on the viscoelastic properties of wheat gluten based materi- ties. Depending on the polymer nature, other properties, as als, the results points to a way of controlling these viscoelastic adesivity could be also improved. During the development properties towards the achievement of tunable biodegradable of nanostructured advanced recovering materials to be used and sustainable biomaterials based on gluten. in petroleum industry distinct organically modified Brazilian [1] Kuktaite, R. et al. Biomacromolecules, 2011, 12, 1438- montmorillonite-EVA nanocomposites have been prepared by 1448. using a Brazilian clay, in the range 0.5 to 10% w/w. In this communication X-ray diffraction (XRD), thermogravimetry ACKNOWLEDGMENTS: FAPESP, CNPq analysis (TGA), hydrophobicity degree by using the contact angle, Fourier transform infrared spectroscopy, 27Al single TH093: Effects of Pretreatment on Morphology, pulse solid state NMR, 13C cross-polarization high resolu- Chemical Composition and Enzymatic Digestibility tion solid state NMR, and 1H low field NMR were used to

96 Poster Abstracts – Liquid state NMR address the structure of the EVA-organoclays. The results Center of the City University of New York were compared with those obtained by using modified car- Bacterial tyrosine kinases (BY-kinases), and their cognate ty- bon nanotubes-EVA nanocomposites, and evidenced that de- rosine phosphatases, form counteracting enzyme pairs regu- pending on the nanostructured component distinct molecular lating diverse physiological processes in both Gram-negative dynamics for EVA moiety was detected. and Gram-positive species. In Escherichia coli (K12), the Acknowledgements: Rede Nano (Petrobras), PIBIC CNPq- BY-kinase Wzc, plays a critical role in production of the ex- UFRJ, CAPES. opolysaccharide, colanic acid, and the formation of biofilms. Wzc kinase contains a cluster of tyrosine residues (YC) at its References C-terminus that is autophosphorylated and subsequently de- Aranha, I.B. (2007) Preparação, caracterização e pro- phosphorylated by Wzb phosphatase. This Wzb phosphatase priedades de argilas organofílicas. Tese de Doutorado, In- mediated phosphorylation/dephosphorylation cycle in Wzc stituto de Química, Universidade Federal do Rio de Janeiro. kinase, rather than its quantitative phosphorylation state, is critical for colanic acid production. Despite extensive ge- Bain,D.C.,Smith,B.F.L.(1994),Chemical analysis,In:Clay netic and biochemical evidence for Wzb-mediated dephos- Mineralogy, Spectroscopic and Chemical Determinative phorylation of the Wzc YC, the nature of the interactions Methods. Edited by M. J. Wilson, Chapman & Hall, London, between these two enzymes, and the regulatory mechanisms 312. that govern dephosphorylation, are unknown for this or for Menezes, R.R., Souto,P.M., Santana, L.N.L., Neves, G.A., any other BY-kinase/phosphatase pair. Here, using solution Kiminami, R.H.G.A., Ferreira, H.C.(2009) Argilas ben- NMR methodology, we provide the first insight into the reg- tonitas de Cubati, Paraíba, Brasil: Caracterização física- ulatory interactions between a BY-kinase and its opposing minerológica. Cerâmica,55,163-169. phosphatase at residue-level resolution. Wzb phosphatase docks onto the catalytic domain of Wzc (WzcCD) utilizing TU095: Molecular dynamic of modified graphene ox- a surface that is distal to the catalytic elements of the lat- ide/poly(butylene succinate) nanocomposite deter- ter. Mutations in this region reduce Wzb phosphatase medi- mined by longitudinal relaxation time NMR ated dephosphorylation of the kinase YC. WzcCD binds Wzb phosphatase in a YC-independent manner near the catalytic Fernando Luis Miranda Filho, ∗Emerson Oliveira Da site of the latter, inducing allosteric changes therein. Wzb Silva phosphatase principally recognizes the phosphate moiety of IMA/ UFRJ phosphotyrosine, and derives tyrosine-selectivity through in- teractions with a conserved tyrosine at its active site. YC- In this work, graphite was treating with oxidizing agents dephosphorylation is proximity-mediated and reliant on the (modified Hummer method) to obtain graphite oxide (GO). increased phospho-YC concentration near the Wzb phos- Besides that, the GO was treating with an excess of suc- phatase active site due to docking interactions with WzcCD. cinic acid to obtain a graphene succinate sample (SG). Nanocomposites were prepared by the following method: MO097: qNMR for Analysis of Methyldopa- Poly(butylene succinate), PBS, was solubilized in chloroform Containing Pharmaceutical Formulations: Compar- for two hours. At the same time, the nanoparticle was ison Between Free and Marketed Drugs dispersed in the same solvent with appropriate proportion, which was also in ultrasonic apparatus for two hours. Then, Wilian da Silva Nunes, ∗Glaucia Braz Alcantara both dispersions had been mixed and kept in ultrasonic ap- Mato Grosso do Sul Federal University paratus for more two hours. The sample obtained was dried to constant mass in a laboratory oven. The nanocomposites The Brazilian government has provided various free were characterized by low field NMR, to analyze the disper- medicines for the population through the national health care sion of nanoparticles at polymer matrix. The results showed system (SUS). However, it can often observe some doubts that the PBS presented three domains of mobility. The ma- from people and even health professionals about the effec- jor domain, in high relaxation time, is due the crystalline tiveness of these medicines. The effectiveness depends on phase. The other domains are the amorphous phase in and the amount of the active principle and its availability for or- out the crystal structure. The GO/PBS system presented ganism (for example, polymorphs species can decrease the only two domains shifted to shorter times. Furthermore, all bioavailability), among other factors. the domains showed wider curves. This indicates that the Considering that qNMR has been established as a success- system became more heterogeneous and with higher mobility ful analytical method, this work proposes to apply 1H NMR as a result of interaction with the GO particles. Probably, spectra for quantification of methyldopa on several pharma- the GO particles cause the decrease of the crystalline phases ceutical formulations, as a first step for comparison between of matrix which increase the mobility. The influence of SG in free and marketed drugs. We have compared the results ac- the PBS matrix was different. The SG/PBS system showed cording to the official method (titration in non-aqueous me- three mobility domains. All of them wider than the PBS sys- dia) described in Brazilian Pharmacopeia, as well as with the T tem and in lower 1 values. This occurs because the SG has permitted values by ANVISA (Brazilian agency of sanitary a better interaction with the PBS matrix what results in a vigilance). good dispersion. This morphology causes a stronger effect on 1 relaxation time of matrix. The PBS presented three domains The measurements of H NMR were performed on a Bruker 1 13 of mobility. The major domain, in high relaxation time, is DPX300 (7.05T) spectrometer, equipped with a H/ C dual due the crystallinity phase. probe, using the ZGPR pulse sequence, NS: 32; d1: 10s; AQ: 7.29s; relaxation delay > 5 T1; TD: 128k and SW: 30 ppm. The singlet of TMSP-d4 was integrated and referenced to Liquid state NMR calculate the methyldopa concentration, through its methylic singlet (δ 1.53). The quantification by NMR showed results between 89.76% TH096: Regulatory Interactions between a Bacterial and 109.96% of the methyldopa amount, according to the Tyrosine Kinase and its Cognate Phosphatase package label. The variation is according to values deter- 1,4Deniz B. Temel, 2Kaushik Dutta, 1Sébastien Alphonse, mined by ANVISA in 93.33% of cases. Methyldopa was found 3Julian Nourikyan, 3Christophe Grangeasse, 1,4∗Ranajeet in similar contents in both kinds of pharmaceutical formula- Ghose tions, corroborating that the absolute amount of this active 1The City College of New York, 2New York Structural Bi- principle in free drugs is in concordance to reference drugs. ology Center, 3Institut de Biologie et Chimie des Protéines, qNMR has proved a more reliable method for quantifica- CNRS, Université Lyon 1, Université de Lyon, 4Graduate tion of methyldopa-containing drugs than titration method,

97 Poster Abstracts – Liquid state NMR particularly due to the subjectivity in determining the color et al [2]. However, in the case we present here, neither the change of the indicator and the equivalence point in titration. proton-proton nor the carbon-carbon couplings are negligi- ble, which implies that the two zero quantum subspaces of Acknowledgment: PET/Química. the Hamiltonian that describe this four-spin system evolve in a different way during the magnetisation-to-singlet (M2S) TU098: Frequent applications of NMR in the IPT as pulse sequence. We measure the singlet relaxation time to a customer demand. be 20 s for both carbon and proton pairs. This shows that although 1H-NMR could benefit from the slower relaxing sin- 1∗Martha González-Pérez, 1Shoko Ota, 1,2Eli da Silva glet, for 13C-NMR it would be detrimental. Ferreira Júnior 1Instituto de Pesquisas Tecnológicas do Estado de São Paulo [1] Gallagher, F. A., Kettunen, M. I., Hu, D.-E., Jensen, P. S.A – IPT, 2Instituto de Química, Universidade de São R., in ’t Zandt, R., Karlsson, M., Gisselsson, A., Nelson, S. Paulo K., Witney, T. H., Bohndiek, S. E., Hansson, G., Peitersen, T., Lerche, M. H., and Brindle, K. M. (2009) Production of NMR spectroscopy is a powerful analytical tool for chemi- 13 13 hyperpolarized [1,4- C2]malate from [1,4- C2]fumarate is cal analysis and structural identification. For that reason, a marker of cell necrosis and treatment response in tumors, the Chemical Analysis Laboratory (LAQ) from Technologi- Proc. Natl. Acad. Sci. USA, 106, 19801-19806. cal Research Institute of the State of São Paulo recently has acquired two VARIAN spectrometers operating at 400 MHz. [2] Feng, Y., Davis, R. M., and Warren, W. S. (2012) Acessing One spectrometer is used for analyzing liquid samples and long-lived nuclear singlet states between chemically equiva- the other for solid state. On customers demand, the common lent spins without breaking symmetry, Nature Physics, 2425. applications in our Research Institute are the followings: MO100: RNA induced folding of the first double Qualitative Analysis: stranded RNA binding domain from A. thaliana 1- Chemical structure identification of organic compounds DCL1. used for inspection and classification of custom office, 1Paula Burdisso, 1Irina Paula Suarez, 1Guillermo Hails, 2- Monomer identification and degree of polymerization used 2Matthieu Benoit, 2Jerome Boisbouvier, 1∗Rodolfo Maxim- for evaluating the kind and quality of production processes, iliano Rasia 1 3- Determination of average ethoxylate number of polymers Instituto de Biología Molecular y Celular de Rosario, Fac- 2 for confirming physical and chemical properties. ultad de Ciencias Bioquímicas y Farmacé, Institut de Bi- ologie Structurale (IBS) Jean-Pierre Ebel, Commissariat à Quantitative analysis: L’Energie Atomique (CE 1- Monomer’s quantification of two types of polymers: MicroRNAs are essential gene regulators in multicellular or- Acrilonitrile - Butadiene – Styrene (ABS) and Styrene – Bu- ganisms. Plant miRNAs are processed in the nucleus by a tadiene (SBR), necessary for confirming raw matter quality protein complex formed by DICER-LIKE1 (DCL1), HYL1 in the automobile industry. and SERRATE. DCL1 has a central role in the recognition and processing of the heterogeneous plant precursors. Lit- 2- Chain configuration analysis of polypropylene (evaluation tle is known about the structural aspects of this protein. of randomic copolimerization through Randall method) for In a previous work, we characterized the second dsRBD of custom verification. DCL1. Here we present a structural characterization of the This work will show some of results carried out on client’s first DCL1 double stranded RNA binding domain. demands, i.e., analysis of very different samples, in order to In contrast with canonical dsRBDs, this domain binds ds- illustrate the possibilities and versatility of IPT Laboratory. DNA with a similar affinity as dsRNA, the same as DCL1- Acknowledgment: IPT dsRBD2. We found that both domains cooperate for sub- strate binding, but not for DNA binding. 13 TH099: Singlet NMR of [1,4- C2]fumarate Quite unexpectedly we found that dsRBD1 is intrinsically disordered, even in the context of flanking domains, but folds 1,2∗Irene Marco-Rius, 3Michael C. D. Tayler, 1,2Timothy upon binding substrate RNA. We have assigned the backbone J. Larkin, 1,2Kevin M. Brindle resonances corresponding to the free unfolded and bound 1 University of Cambridge, Department of Biochemistry, UK, folded protein. The free protein, though intrinsically dis- 2 3 Cancer Research UK, UK, Radboud University, Institute ordered, shows a tendency to populate folded conformations for Molecules and Materials, NL that correspond to those expected for a dsRBD. The assign- Hyperpolarised NMR, for instance using dissolution DNP, ment of the bound form allowed us to calculate the fold of is a non-invasive technique to image tissue metabolism in the protein in complex with dsRNA employing CS-Rosetta. vivo. However, the range of reactions that can be investi- This analysis shows that this domain acquires the dsRBD gated is limited by the fast T1-dependent decay of the nu- fold when bound to substrate. We further found that the clear spin order. In metabolites with coupled nuclear spin-1/2 negatively charged surface provided by SDS micelles shifts pairs, polarization may be maintained for a longer time by the protein towards folded conformations, but these are dif- exploiting the non-magnetic singlet state of the pair. This ferent from that found in the complex with RNA. Experimen- may allow preservation of hyperpolarization in vivo during tal comparison with dsRBDs from other Dicer proteins show transport to tissues of interest, such as tumours, and de- that the intrinsically disorder nature is exclusive of DCL1- tection of slow metabolic processes. One of the promising dsRBD1. Sequence analysis and in vivo function of the pro- substrates in which to combine DNP with the longer-lived tein suggests that in this domain the instability of the free singlet state to study metabolism is fumarate, the precursor form and the ability to fold upon binding the substrate have of malate, which is a marker of cell necrosis. Hyperpolarised to be essential for the function of the whole DCL1 enzyme in 13 [1,4- C2]fumarate has been used previously to monitor re- miRNA processing. sponse to cancer treatment [1], and its longitudinal relaxation times in a D2O buffer were measured to be 15 s for proton and TU101: NMR analysis of asphaltenes extracted from 30 s for carbon at a magnetic field strength of 11T. To test field deposits whether the singlet state of the carbon or proton pairs of [1,4- 1 2∗ 1 13 Camila P. Silveira, Peter R. Seidl, Ljubica Tasic, C2]fumarate was slower relaxing than their corresponding 2 2 longitudinal relaxation time, we accessed the singlet state by Fernanda B. da Silva, Maria José C. O. Guimarães breaking the symmetry of the pairs. This was done using 1University of Campinas (UNICAMP), 2Federal University the J-couplings in a similar way to that described by Feng of Rio de Janeiro (UFRJ)

98 Poster Abstracts – Liquid state NMR

Asphaltenes correspond to the heaviest and most polar frac- We describe herein the study of the aminophosphazenes or- tion of petroleum. They are defined by their solubility prop- tho lithiation process through the use of advanced multinu- erties (soluble in aromatics and insoluble in n-parafins). The clear magnetic resonance methods. The powerful combina- price attributed to a crude oil is related to its asphaltene con- tion of standard NMR techniques and 2D nOe, 7Li,31P- and tent: the higher the content, the lower its price when mar- 7Li,15N-HMQC experiments helped us to unravel the struc- keted, since asphaltenes tend to aggregate and deposit during ture of the key intermediate lithium species involved in the production and to form coke on refining. Despite the con- DoLi reactions of secondary aminophosphazenes. 1H, 7Li and siderable investment in determination of molecular proper- 31P NMR spectra as a function of temperature and concen- ties of asphaltenes, their individual structures have not been tration will be also showed in order to obtain critical coupling completely elucidated. In view of the large number of con- constants and determining the aggregation state of the exist- stituents of heavy oils that are soluble in aromatics, we are ing species. In this sense, the signal observed at d 208.9 ppm developing fractionation procedures in order to better under- in the 13C NMR spectrum measured at -100 ◦C that is as- stand the relationships of asphaltene structures with their signed to the lithiated ortho carbon defines a specific type of physical and chemical properties. structure. The multiplicity of this carbon signal, a quartet with a 13C,7Li coupling constant of 30.2 Hz, establishes the Asphaltene samples were extracted from field deposits, frac- monomeric nature of the lithiated species. tionated by two different methods and compared mainly by NMR analysis. Our objective was to verify if the traditional [1] (a) Fernández, I.; Oña-Burgos, P.; Oliva, J. M.; López- but time-consuming and relatively costly IP 143 procedure Ortiz, F. J. Am. Chem. Soc. 2010, 132, 5193. (b) García- could be substituted by an alternative method, developed in López, J.; Morán-Ramallal, A.; González, J.; Oña Burgos, P.; our labs and based in a solvent blend, without losing infor- Iglesias, M. J.; Roces, L.; García-Granda, S.; López-Ortiz, F. mation on the sample’s asphaltene composition. 1H Nuclear J. Am. Chem. Soc. 2012, 134, 19504. Magnetic Resonance (NMR), 13C NMR without NOE, 13C ACKNOWLEDGEMENT: We thank the Ministerio de NMR DEPT, elemental analysis, infrared spectra and X-ray Economía y Competitividad (MINECO) and FEDER pro- fluorescence data were collected for both types of samples. It gram for financial support (project CTQ2011-27705). M.C. was possible to verify that the new extraction method is ca- thanks MICINN for a predoctoral fellowship. pable of isolating asphaltenes that contain the same type of organic functions as samples obtained by the IP method, but in different proportions. It was also shown, mainly by NMR TU104: Experimental and Theoretical Study of Tam- analysis, that the alternative method can be applied to frac- iflu NMR Data: a Comparative Analysis tionation of heavy petroleum as well as vacuum residues. 1∗ 1 Diego Alves Rodrigues, Vinicius Sousa Ferreira, 2Felipe Terra Martins, 1Luciano Morais Lião, 1Luiz Henrique TH102: Cold-denaturation of a protein dimer moni- Keng Queiroz Júnior tored at atomic resolution. 1Universidade Federal de Goiás, Instituto de Química, Lab- 2 1Mariusz Jaremko, 1Lukasz Jaremko, 1Hai-Young Kim, oratório de RMN, Universidade Federal de Goiás, Instituto 1Min-Kyu Cho, 2Charles D. Schwieters, 1Karin Giller, de Química, Laboratório de Cristalografia 1 1,3∗ Stefan Becker, Markus Zweckstetter Oseltamivir is an antiviral drug, marketed under the trade 1Department for NMR-based Structural Biology, Max Planck name Tamiflu, used in the treatment and prophylaxis of in- Institute for Biophysical Chemistry, 2Computational Bio- fections by the influenza A and B viruses. It is one of the science, Center for Information Technology, NIH, 3Center promising therapeutic agents for the treatment of avian in- for Neurodegenerative Diseases (DZNE) fluenza H5N1 infection. The objective of the study is to cor- relate the theoretical results, with and without the solvent’s Protein folding and unfolding are crucial for a range of bio- effect, with the experimental 1H and 13C NMR data for the logical phenomena and human diseases. Defining the struc- drug Tamiflu, aiming to confirm the unequivocal assignment tural properties of the involved transient species is therefore of all the NMR signals of this compound. of prime interest. Using a combination of cold-denaturation with nuclear magnetic resonance spectroscopy we reveal de- The NMR experiments of a Tamiflu solution were obtained tailed insight into the unfolding of the homodimeric repressor in a spectrometer Bruker Avance III 11.75 T. The theoreti- protein CylR2. Seven three-dimensional structures of CylR2 cal calculations were performed using the Gaussian 03 pro- at temperatures from 25 ◦C to -16 ◦C reveal a progressive dis- gram. The lower energy structures were obtained by the opti- sociation of the dimeric protein into a native-like monomeric mization at MP2/cc-pVDZ, and the magnetic properties (δ) intermediate followed by transition into a highly dynamic, were calculated with the method GIAO, using the B3LYP/cc- partially folded state. The core of the partially folded state pVTZ model. All the calculations were done with and with- appears critical for biological function and misfolding [1]. out solvent’s effect (H2O – PCM model). 13 [1] Jaremko, M., Jaremko, L., Kim, H.-Y., Cho, M.-K., Comparing the theoretical and the experimental data of C Schwieters, C. D., Giller, K., Becker, S., Zweckstetter, M. NMR, we can observe that there was no great variation in (2013) Cold denaturation of a protein dimer monitored at coefficient of linear correlation (R = 0.99), either with or atomic resolution, Nat. Chem. Biol. Accepted without the consideration of the solvent’s effect. This fact can be justified due these 13C nuclei are located more inter- nally in the molecule. As for the 1H nuclei, the inclusion of MO103: Solution Structure of a Lithium Organyl: the solvent’s effect in the calculations provided a better cor- Dilithiated Aminophosphazenes in Action roboration between the theoretical and experimental data, María Casimiro, Ignacio Fernández, ∗Fernando López Or- what can be verify by the value of the linear correlation co- tiz efficient (R = 0.98) and because the solvent’s effect leads to lower values of mean and standard deviations. It was not University of Almería observed discrepancies between the theoretical and experi- As part of an ongoing project on the synthetic applications mental assignments. So, theoretical calculations provided to of phosphorus-bearing lithium organyls, we have developed be an excellent tool for unequivocal assignment of all the 1H efficient methodologies for the ortho functionalization of sev- and 13C NMR signal of the Tamiflu drug. eral phosphinic amides and phosphazenes via directed ortho lithiations (DoLi) and subsequent electrophilic trapping [1]. TH105: 1H NMR Spectroscopy used as an Analyti- These reactions proceed in some cases with very high di- cal Technique for Gasoline astereoselectivities and provide rapid access to multidentate hemilabile ligands which are being explored in asymmetric 1Vinícius S. Pinto, 1∗Francisco F. Gambarra-Neto, catalysis. 2Marcos Roberto Monteiro, 1Luciano Morais Lião

99 Poster Abstracts – Liquid state NMR

1Universidade Federal de Goiás, Instituto de Química, Lab- This work is supported by FAPESP (Proc. 2011/11160-3). oratório de RMN, 2Universidade Federal de São Carlos, DEMA, CCDM, Laboratório de Combustíveis TU107: 1H NMR spectroscopy and SIMCA model The development of instrumentation in Nuclear Magnetic for classification of common and premium gasolines Resonance (NMR) has increased the sensitivity of the 1∗Vinícius S. Pinto, 1Francisco F. Gambarra-Neto, technique, allowing the analyses of samples in micro and 2 1 nanogram scales. The new technologies enable the NMR Marcos Roberto Monteiro, Luciano Morais Lião technique to be applied as an analytical tool for the quantifi- 1Universidade Federal de Goiás, 2Centro de Caracterização cation of the substances in different organic matrices. A com- e Desenvolvimento de Materiais – CCDM, DEMA, Univer- mercial gasoline is composed by alkanes (paraffin), cycloalka- sidade Federal de São Carlos nes (naphthenic), alkenes (olefin), aromatic and may addi- The NMR spectroscopy has been used as a powerful analyt- tionally contain oxygenates. These information appears in 1 ical tool for fuel analysis due to its versatility and low time the H NMR spectrum, which was separated in 0.46 to 3.21, for spectrum acquisition. Several examples of fuel quality 2.49 to 5.93, and 6.25 to 8.34 ppm regions for the construc- control performed by NMR and chemometric treatment are tion of Partial Least Squares (PLS) calibration models. The found in literature. In Brazil, automotive gasoline is commer- Brazilian gasoline control quality is based on various physic- cialized as common gasoline (CG), additived gasoline (AG) ochemical parameters such as amounts of xylene, toluene, and premium gasoline (PG), with different prices. The dif- distillation temperature for 50% and 90% (T50 and T90), ference between CG and PG lies in the octane number, a saturated, olefins and aromatic compounds, ethanol (ETOH), parameter for quality control required by National Agency of methyl tert-butyl ether (MTBE), compounds of oxygen, tert- Petroleum, Natural Gas and Biofuels - ANP. The 1H NMR amyl methyl ether (TAME) and diisopropyl ether (DIPE) spectroscopy associated with Soft Independent Modeling of calculated by laboratories accredited by National Petroleum Class Analogies (SIMCA) was used for classifying samples Agency (ANP). In this work these parameters for 11 gasoline 1 1 collected in São Carlos- SP/Brazil region in CG or PG. H samples were calculated through H NMR spectroscopy com- NMR spectra of 5 CG and 6 PG samples were analyzed in bined with quantitative chemometrics, and presented an R2 triplicate. All samples were applied in the development of the value of 0.98, 0.98, 0.99, 0.99, 0.99, 0.98, 098, 0.98, 0.96, 0.97, prediction model. The SIMCA model identified the regions 0.94, 0.97, respectively, in PLS full cross-validation model. 1 of paraffinic, aromatic and unsaturated hydrogens as the re- Therefore, these high R2 values demonstrated that H NMR sponsible for distinction between the CG and PG. Individual and PLS provided quantitative and alternative tools for gaso- analysis of each region in SIMCA indicated aromatic hydro- line analyses. This is something absolutely new for the qual- gen region as the most important for CG and PG classifica- ity control of Brazilian gasoline. tion. The successful result in SIMCA model was attributed to the difference in octane number of premium and common ACKNOWLEDGEMENTS: CNPq, CAPES, UFG, FU- 1 NAPE, CCDM/DEMa/UFSCar gasolines. Finally, the results showed the potentiality of H NMR and SIMCA association for gasoline analyses presented in this work. MO106: Uses of Steady-State Free Precession pulse sequences with phase alternation and phase incre- ACKNOLEDGEMENTS: CNPq, UFG, FUNAPE and ment for fast high resolution NMR acquisition CCDM/DEMa/UFSCar. 1,2Tiago Bueno de Moraes, 2∗Luiz Alberto Colnago TH108: Structural dynamics affect the allergenic po- 1University of São Paulo, 2Embrapa Instrumentação tential of Bet v1 Steady State Free Precession (SSFP) sequences have been 1Aline Batista, 2Claudia Asam, 1Adolfo G. Moraes, 1Fabio used to enhance signal to noise ratio in high resolution 2 2 1∗ NMR spectrum but it introduces severe phase and ampli- C. L. Almeida, Fatima Ferreira, Michael Wallner, Ana tude anomalies. These distortions are essentially caused by Paula Valente the truncation of the signal and the strong interaction be- 1Centro Nacional de Ressonância Magnética Nuclear de tween the free induction decay (FID) and echo component. Macromoléculas, Instituto de Bioquímica Médica - Univer- sidade Federal do Rio de Janeiro, 2Christian Doppler Lab- Problems involving the use of SSFP in high-resolution appli- oratory for Allergy Diagnosis and Therapy, University of cations were observed by Ernest and Anderson, Freeman and Salzburg Hill and Schwenk. In the 1970s some new methods were pro- posed to overcome this limitation, like the Scrambled Steady- Bet v 1 is the major allergen of birch pollen with molecular State (SSS) and Quadriga Fourier Transform (QFT) meth- mass of 17.5 kDa. It is estimated that 100 million people ods, but none have been able to complete overcome the lim- are sensitized to Bet v 1, in Northern American and West- itations. Recently new methodologies in Steady State pulse ern Europe. Bet v 1 secondary structure arrangement is β- sequences are being proposed for fast acquisition in low reso- α2-β6-α where seven anti-parallel β-sheet surround a long lution and for rapid imaging in MRI. Similarly, we are propos- C-terminal α-helix. Also, a Y-shaped hydrophobic pocket ing SSFP sequences with phase alternation and phase incre- is present inside the protein. Its biological function is as- ment for fast acquisition in high resolution NMR. sociated with the transport of hydrophobic molecules such as brassinosteroid (plant hormone). In this work we used A theoretical treatment of the steady state was already pre- Na-deoxycholate (Doc) that is structurally similar to plant sented in the literature, but for SSFP sequences with phase steroids, in order to analyze the influence of ligand binding alternation and phase increment the magnetization behav- on dynamics, conformation and IgE-binding. Recombinant ior is not well described. To understanding the effect of 15N isotope labeled Bet v 1 was produced in E. coli and puri- the phase alternation and phase increment in SSFP signals fied. We used NMR spectroscopy to probe dynamics aspects 1 15 we have been performing numerical simulations using Bloch using R1,R2 and H- N-NOE for free and bound state. equations. To further understand the free state dynamics we performed 15 The 1H and 13C experiments have been performed in an In- N CPMG relaxation dispersion experiments at 800 and 600 ova 400 Varian spectrometer (9.4 T). Results show that it is MHz. Moreover, we performed experiments to map IgE epi- possible to enhance S/N using SSFP with phase alternation, topes in the free and Doc-bound state of Bet v 1 using purified reducing the echo component and signal suppression. These human IgE antibodies from birch pollen allergic patients. Bet techniques widens the band spacing acquired resulting in bet- v 1 is a dynamic protein with several residues in conforma- ter spectra, however still have some modulation in the signal tional exchange. The epitopes for three patients were slightly magnitude. different but several residues are in common. Doc interaction changed slightly the protein conformation and profoundly the Acknowledgments dynamics. These changes did not affected the IgE epitope

100 Poster Abstracts – Liquid state NMR but there was an increase in antibody afinity. Understanding TU110: Identification and Quantification of the structural dynamics of Bet v 1 and its interaction with Meningococcal Serogroups C and W135 Polysac- IgE with or without ligands may help to develop hypoallergic charides by NMR derivatives for immunotherapy and as well as understand the 1Ana Paula F. Leal, 1Milton Neto da Silva, 1Ivna A. F. B. correlation between dynamics of allergens and the forces that 2∗ drive the allergenicity. Silveira, Erika Martins de Carvalho 1Bio-Manguinhos/FIOCRUZ, 2Instituto de Tecnologia em Fármacos Far-Manguinhos/FIOCRUZ MO109: Calculation of Average Molecular Descrip- tions of Heavy Petroleum Hydrocarbons by Com- Bio-Manguinhos has been, in recent years, the only man- bined Analysis by Quantitative 13C and DEPT-45 ufacturer of meningococcal vaccines that are used for the NMR Experiments meningococcal disease control. To ensure the effectiveness 1∗ 2 of these vaccines are verified two determining factors in the John C. Edwards, A. Ballard Andrews quality control: the purity and O-acetyl concentration of 1Process NMR Associates, Danbury, CT, USA, polysaccharide. The meningococcal C polysaccharide (PSC) 2Schlumberger-Doll Research, Cambridge, MA, USA consists of sialic acid while meningococcal W135 polysac- charide (PSW135) consists of sialic acid and galactose. The Much debate has centered around the validity and accuracy classic method used in the control of these molecules is the of NMR measurements to accurately describe the sample UV-Vis spectroscopy, which requires a large number of ma- chemistry of heavy petroleum materials. Of particular issue nipulations in the sample preparation, negatively impacting has been the calculated size of aromatic ring systems that the final result of analysis. NMR spectroscopy is capable of in general seem to be underestimated in size by NMR meth- minimizing such problem, enabling yet, the reduction in the ods. This underestimation is principally caused by variance sample amount and analysis duration. This paper aims the in chemical shift ranges used by researchers to define the use of 1HNMR to identify and quantify PSC and PSW135 aromatic carbon types observed in the 13C NMR spectrum, using the GARP experiment (qNMR) [1,2]. in particular the bridgehead aromatic carbons that can be shown to overlap strongly with the protonated aromatic car- First, spectra were obtained using standard of sialic acid bons. The ability to discern between bridgehead aromatic (NANA) and galactose for selecting the signal to be quan- carbons and protonated carbons in the 108-129.5 ppm re- tified. The analysis of these spectra showed the signals at > gion of the spectrum is key in the derivation of molecular 2.06 and 2.15 ppm regarding to methyl groups hydrogen of - parameters that describe the "molecular average" present in NAc and -Oac, respectively in PSC and 2.09 ppm in PSW135, the sample. Utilizing methodologies developed by Pugmire are suitable for the quantitation of NANA; while the signal 13 and Solum for the solid-state C NMR analysis of coals at > 5.07 ppm is the most suitable for the quantification of and other carbonaceous solids [1], we have developed a new galactose in PSW135. The parameters have been optimized 13 liquid-state C NMR method that allows the relative quan- for the quantification as a function of the signal-to-noise ratio, tification of overlapping protonated and bridgehead aromatic and the delay was determined using the inversion-recovery carbon signals to be determined [2]. method. The qRMN results showed that both polysaccha- rides have purity above 80%, similar to that one found in The NMR experiments involve the combined analysis of both 13 the classical method. The qRMN method was able to de- quantitative C single pulse excitation which observes all termine the identity, purity and degree of O-acetylation of carbons quantitatively, and a DEPT45 polarization transfer polysaccharides in a single experiment with excellent repro- which observes only the protonated carbons in the sample. ducibility and efficacy. These preliminary results suggest that Though the DEPT45 results are not quantitative across all the methodology would be applied in the quality control of carbon types (CH, CH2, and CH3) due to polarization trans- meningococcal polysaccharides. fer differences, the technique is well enough understood that simple multiplication factors allow the relative intensities of Reference the different carbons to be determined. The average ring [1] Jones C. & Lemercinier X. J. Pharm. Biom. Anal. 2002, system sizes derived from these NMR experiments tend to 30, 1233-1247. be several ring systems larger than has been calculated in previous studies. In heavy petroleum asphaltenes the av- [2] Jones, C. J. Pharm. Biom. Anal., 2005, 38, 840-850. erage aromatic ring system is 5-7 rings in size which is in agreement with FTICR-MS and fluorescence measurements, ACKNOLEDGEMENTS: FARMANGUINHOS, BIOMAN- rather than the 3-4 rings previously reported. GUINHOS

The results obtained by liquid-state NMR are compared to TH111: Monomer-dimer equilibrium of the carbon-type NMR and molecular parameters derived canecystatin-1 studied by high-resolution NMR from the solid-state 13C analysis of the same samples. A 1Ítalo Augusto Cavini, 1Richard Charles Garratt, 2Hans chemometric approach to the derivation of the parameters 1∗ has been developed so that automated output of the analysis Robert Kalbitzer, Claudia Elisabeth Munte results can be routinely provided by technician level person- 1Physics Institute of São Carlos, University of São Paulo, nel. Brazil, 2Institute for Biophysics and Physical Biochemistry, University of Regensburg, Germany Finally, a quantitative 13C NMR method is described that Cystatin superfamily comprises tight and reversibly binding utilizes internal standards to quantify the amount of carbon inhibitors of cysteine proteases. They are present in a wide in the sample observed in the NMR experiment. This 13C variety of organisms including vertebrates, invertebrates and quantitation yields information on the degree of aggregation plants. Plant cystatins, which are named phytocystatins, in the asphaltene components. are distinguished by the absence of disulfide bonds. The Oryza [1] Carbon-13 solid-state NMR of Argonne-premium coals, best studied phytocystatin, oryzacystatin I from rice ( sativa Mark S. Solum , Ronald J. Pugmire , David M. Grant, En- ), has its three-dimensional structure solved by high- ergy Fuels, 1989, 3 (2), pp 187–193. resolution NMR, showing a monomeric tertiary fold consist- ing of a five-stranded antiparallel β-sheet wrapped around [2] "Comparison of Coal-Derived and Petroleum Asphaltenes an α-helix. The inhibition is caused by action of three struc- by 13C Nuclear Magnetic Resonance, DEPT, and XRS", A. tural motifs, including L1 loop, region that connects β-2 Ballard Andrews, John C. Edwards, Andrew E. Pomerantz, and β-3 strands. We recently reported the X-ray structure of Oliver C. Mullins, Dennis Nordlund, and Koyo Norinaga, En- canecystatin-1 from Saccharum officinarum, that presented a ergy Fuels, 2011, 25 (7), pp 3068–3076. domain-swapped dimeric form, similar to the topology found in other non-plant cystatins like human cystatin C, human

101 Poster Abstracts – Liquid state NMR stefin A and B. These domain-swapped structures exhibit the 1Grupo de Biofísica Molecular Sérgio Mascarenhas, In- hairpin L1 loop in a β-strand conformation, linking β-2 and stituto de Física de São Carlos, USP, 2Laboratório de β-3 strands and forming the contiguous β-2,3 strand. The Biofísica Molecular, Faculdade de Filosofia, USP, 3Instituto dimer is not expected to be active due to the accessibility de Química de Araraquara, Universidade Estadual Paulista restriction of L1 loop. Complementing the X-ray studies, our Human dihydroorotate dehydrogenase (HsDHODH) cat- NMR spectra showed the presence of two conformations for alyzes the fourth and only redox step of the de novo canecystatin-1: a low populated monomeric state and a high pyrimidine biosynthesis. HsDHODHs are monomeric and populated domain-swapped form, coexisting in solution in a membrane-bound proteins and their inhibition represents slow exchange process. an important strategy for antineoplasic and antiproliferative Our present work aims to explore the physiological relevance drugs. Here we report structural studies of the HsDHODH of the canecystatin-1 domain-swapped structure by studying N-terminal peptide, which is responsible for anchoring the its equilibrium with the active monomers in solution. For enzyme to the inner mitochondrial membrane and then to this, uniformly 15N-labeled canecystatin-1 samples were pro- allow access to ubiquinones, essential for the enzyme catal- duced and a set of 1H-15N-HSQC spectra varying pressure ysis. Fluorescence studies using Tyr7 residue showed that (up to 240 MPa, 303 K) and temperature (278 to 318 K, am- the peptide interacts with micelles and vesicles and that this bient pressure) were recorded on Bruker 800-MHz and 600- residue is located inside the membrane. CD studies indi- MHz spectrometers. Thermodynamic parameters associated cate that the molecule, when in TFE and model membranes, with conformations in fast exchange were obtained and a new acquired a high amount of α-helix. A complete sequence- intermediate conformation was identified. The equilibrium specific proton resonance assignment of the peptide in the characterization of this cystatin may be helpful to better un- presence of d38-DPC micelles was performed by means of two- 1 1 derstand the action mechanism of this inhibitor. dimensional H- H NOESY, TOCSY and QDF-COSY anal- yses. Based on the chemical shift index, two helical segments MO112: Use of 1H NMR to analyze Leishmania sp. (Gly1-Gln15 and Pro20-Gly34) separated by a turn were pre- under different conditions dicted. In addition to the backbone Hα-HN (i,i+1) and HN- HN (i,i+1) NOEs, a large number of medium-range NOEs 1,2∗Paulo Falco Cobra, 3Matheus Pereira Postigo, 3Otavio Hα-HN (i,i+3)/(i,i+4) between residues 2-15 and 21-33, a Henrique Thiemann, 2Luiz Alberto Colnago typical pattern of a helical structure, were observed. In both 1Instituto de Química de São Carlos, IQSC/USP, 2Empresa cases, proline residues showed only trans conformation, con- Brasileira de Pesquisa Agropecuária, EMBRAPA-CNPDIA, firmed by strong NOEs between Met11CαH-Pro12CδH and 3Instituto de Física de São Carlos, IFSC/USP Asp19 CαH-Pro20CδH. The d38-DPC induced structure of the peptide is a well defined amphipathic helix (Glu3-Gly16 Leishmaniasis is a tropical disease caused by protozoa of the and Ser22-Ser32) connected by a short loop (Leu17-Glu21) genus Leishmania, transmitted by a sand-fly of the genus with average backbone and heavy atoms r.m.s.d. of 0.71 and Phlebotomus. It affects mainly Africa and the Americas, 1.1 Å, respectively. Our findings suggest that the peptide manifesting different forms and can lead to skin sores (cuta- adopts a hairpin-like conformation laid on the membrane, neous leishmaniasis) or to liver and spleen injuries (visceral with the hydrophobic face in contact with the acyl chains leishmaniasis), when it’s generally fatal. The World Health of the membrane mimetic and the hydrophilic face pointing Organization (WHO) estimates that approximately 350 mil- towards the aqueous surface. These results can open new lion people live in areas under constant risk of contamination, insights regarding the development of antineoplasic and an- while currently near 12 million are infected and 1.6 million tiproliferative drugs that could bind to this specific region new cases are reported each year. There is still not an ef- while anchored to the membrane. fective treatment for leishmaniasis, and the use of pentava- lent antimonials, amphotericin B or pentamidines, which are Supported by FAPESP, CNPq and CAPES. toxic, and the combat to the vector of the disease are the only forms of controlling it. Besides, it is not exactly known TH114: Antimicrobial Peptide Tritrpticin (TRP3): how these drugs act in the organism of the parasite. Conformational Variability and Membrane Interac- Metabolomics and Metabonomics are tools of modern Bio- tion evaluated by NMR chemistry widely employed nowadays in the study of 1Talita LoSan, 2∗Shirley Schreier, 1Fabio C. L. Almeida, metabolism for several organisms and are often associated 1 to analytical chemistry techniques. Ana Paula Valente 1IBqM-UFRJ, 2IQ- USP In an attempt of better understanding these parasites, and in the future to propose a new treatment for leishmania- Antimicrobial peptides (AMPs) play a major role in the in- sis, this work shows the first metabolic analysis applied to nate immune system of all living organisms and they are L. donovani and L. major cultures, under different condi- generally thought to kill by disrupting lipid membranes. tions, including the use of new drug candidates. The result- Due to the increase in antibiotic resistant-organisms, AMPs ing metabolomic and metabonomic studies by high-resolution are being considered as an alternative antimicrobial agent. Nuclear Magnetic Resonance (NMR) are providing valuable Tritrpticin (TRP3) is a 13 residue antimicrobial peptide, information about the metabolism of the parasites and how from cathelicidin family, with a high proportion of trypto- it varies in the presence of a biologically active molecule. phan residues. TRP3 is highly selective to bacterial mem- brane and does not act in mammalian cells. TRP3 does Leishmania The NMR analyses of cells and extract shows the not present a unique conformation in solution and confor- main metabolites inside the protozoa cell matrix, when these mational exchange occurs in slow exchange regime that is grow in the appropriate medium. Afterwards, these data will possible to be detected by 1D-1H NMR spectra. Since NMR be employed as a comparative basis for a metabonomic eval- spectroscopy is capable of not only determination of ter- uation of possible variations in the chemical composition of tiary structure as well as monitoring movements of chain these organisms. in diverse timescales, we studied structure and dynamics of ACKNOLEDGEMENTS: FAPESP, CNPq, CAPES peptide TRP3, trying to correlate its conformational vari- ability with its mechanism of action and selectivity. TRP3 TU113: Spectroscopic studies of HsDHODH N- structural dynamics was studied in four membrane mimetic terminal domain: an evaluation of the peptide- systems, lyso-phosphocholine (LPC), dodecylphosphocholine membrane interaction (DPC), n-octyl-β-D-glucopyranoside (NOG) and N-dodecyl- N,N-(dimethylammonio)butyrate (DDMAB). The stabilized 1Edson Crusca, 3Eduardo Festozo Vicente, 2Antonio José structure in LPC, NOG and DDMAB was analyzed through Costa-Filho, 1Claudia Elisabeth Munte, 3∗Eduardo Maffut 2D-1H NMR spectra and compared with stabilized structure Cilli in DPC (Schibli and co-workers, 2006). Although TRP3 pre-

102 Poster Abstracts – NMR of Membrane Proteins sented different interaction pathway in each system, the fi- MetaboAnalyst (metaboanalyst.ca) for principal component nal structure in all mimetic systems was very similar. Our analysis (PCA). Since NMR allows the direct evaluation of data show that TRP3 interact through conformation selec- liquid, solid and semi-solid samples without sample destruc- tion with the four kinds of micelles and the final structure is tion or extensive preparation or extraction steps, it can be achieved through peptide accommodation. applied as a strong and practical tool for complex mixtures studies. In our experiments, this technology displayed robust ACKNOLEDGEMENTS: FAPERJ, CAPES, and CNPq sensibility for the identification and quantification of skin metabolites and formulation compounds over time. There- MO115: Quality control of Beer by NMR concerted with, here we describe that the combination of a skin perme- with Chemometric Analysis ation approach designed for the biological activity mainte- nance, with a robust detection technique, can be a powerful 1Camilla do Nascimento Bernardo, 1Raquel Pantoja 2 1∗ tool for evaluation of compound absorption associated with Barreiros Rodrigues, Paula Fernandes de Aguiar, Jochen skin health and drug metabolism assessment. We also believe Junker that the evolution of similar approaches, employing human 1Fundação Oswaldo Cruz - CDTS, 2IQ, UFRJ skin samples and combined detection systems, can improve Beer is fermented beverage produced from malted grains, efficacy and safety studies for cosmetic and pharmaceutical hops, yeast, and water. Over 800 compounds constitute development. beer, and numerous of then give taste. The major groups [1] Xia, J., Mandal, R., Sinelnikov, I., Broadhurst, D., and of compounds are carbohydrates, prenylflavonoids, amino Wishart, D.S. (2012). Nucl. Acids Res. first published online acids, amines among other inorganic and organic com- May 2, 2012. pounds. Beer is the main dietary source of xanthohu- mol and prenylflavonoids. Xanthohumol and other preny- [2] Xia, J., Psychogios, N., Young, N. and Wishart, D.S. lated flavonoids from hops bring positive effects to health: (2009. Nucl. Acids Res. 37, W652-660.) flavonoids are natural antioxidants; weak estrogenic activity 51 was observed in hops and xanthohumol is a potent cancer TH117: V NMR in solution of substituted Keggin 6− chemo preventive agent. ion [a-SiV2W10O40] ∗ Beer composition can be correlated to origin and quality. Rodrigo de Paiva Floro Bonfim, Luiza Cristina de Compounds related to flavor and aroma usually are identified Moura, Jean Guillaume Eon by sensorial analysis. Identification and quantitative analysis Universidade Federal do Rio de Janeiro of substances in beers is done by various techniques, such as HPLC/Electrospray mass spectrometry, NMR spectroscopy, Heteropolianions are oxides of transition metals which have MALDI-TOF mass spectrometry and others. a high versatility in composition and properties. The het- eropolianions substituted Keggin has the general formula x− High resolution NMR spectroscopy technique has been de- [XM’yM12−yO40] where X = P and Si, M = Mo and W, M veloped to aim at a rapid identification of the nature and ’= transition metal and y = 1, 2 and 3. These ions are synthe- n− n− origin of the beer sample, outside recognition aromatic com- sized from species lacunar [XM11O39] , [XM10O36] and 1 n− 4− pounds, carbohydrates, amino acids and others. H NMR [XM9O34] or [α-SiW12O40] . The obtaining of mixed spectroscopy is an efficient method used to discriminate or- Keggin ions is hampered by the complexity and stability of ange and grapefruits juices and to prevent of frauds. compounds can be formed. The pH is a determining factor for the formation of these compounds in aqueous solution. In this 6− Quality control of beers by NMR methods concerted with work the synthesis of Keggin ions [α-SiV2W10O40] were chemometric analysis has been studied. The combination of accompanied by 51V NMR in solution. We used the Keggin 7− 10− spectroscopy and Principal Components Analysis provide in- lacunar [α-SiW11O39] and [α-SiW9O34] as precursors formation about compositional properties like authenticity, and pH between 5.5 and 8.0. All syntheses were performed quality and origin. Furthermore, spectroscopic quantifica- following the methodology in which the pH of the reactional tion of peaks on spectra gives quantitative information of medium is kept constant. compounds of sample. The 51V NMR spectra in solution of the Keggin ion [α- 6− SiV2W10O40] were obtained from aliquots of the reac- TU116: A Metabolomic Assessment of the In Vitro tion medium and the products crystallized. The spectra of Skin Permeation Method 7− the products obtained from lacunar [α-SiW11O39] showed 2Ana Paula Azambuja, 2Julia Menegola, 1Rafael Renatino three peaks at -519.9 ppm, -545.8 ppm and -549.6 ppm. The Canevarolo, 1∗Ana Carolina de Mattos Zeri peak at -545.8 ppm is more intense at pH 6.5. The peak at 1 2 -549.6 ion can be attributed to the mono-substituted Keggin Brazilian Biosciences National Laboratory, Natura 5− [α-SiVW11O40] . The spectra of the solutions of the crys- 7− The in vitro skin permeation approach is considered a clas- tals obtained in pH 6.5 from lacunar [α-SiW11O39] and 10− sical tool in the development and improvement of topically [α-SiW9O34] showed an intense peak at -544.8 ppm indi- 6− applied pharmaceutics and cosmetics. Besides its wide em- cating the formation of the Keggin ion [α-SiV2W10O40] . ployment as predictive of in vivo absorption for safety and The diffractograms and infrared show the formation of a sin- formulation quality control purposes, skin permeation tests gle product and confirms the results of NMR 51V. are mainly applied as a barrier test, were no biological activ- ity is assessed. Aiming at the establishment of a model to ACKNOLEDGEMENTS: CAPES, CNPq evaluate the metabolic profile and probe skin conditions dur- ing the permeation studies, we optimized the classical method for in vitro skin absorption. Here we depict that, either under NMR of Membrane Proteins control conditions or after treatment with a cosmetic formu- lation, pig ear skin samples mounted on Franz diffusion cells, MO118: Solid state NMR studies of membrane pro- with Dulbecco’s Modified Eagle Medium (DMEM) as recep- teins tor fluid, can sustain metabolic activity as described by the identification of biomarkers from the respiratory, amino acid, 1∗Vladimir Ladizhansky, 1Shenlin Wang, 1Sanaz Emami, and nucleic acid metabolism. Metabolomics studies were per- 1Rachel Munro, 2Lichi Shi, 1Ying Fan, 3Izuru Kawamura, formed assessing receptor fluid samples from 0, 2, 4, 10 and 1Leonid Brown 24 hours of in vitro permeation employing Nuclear Magnetic 1University of Guelph, 2University of Toronto, 3Yokohama Resonance spectroscopy (NMR) at 600MHz, and evaluating National University the data using Chenomx NMR Suite (7.5 version; Chenomx Inc., Edmonton, Canada) for targeted profiling analysis, and Magic angle spinning (MAS) solid-state NMR shows great

103 Poster Abstracts – NMR of Nucleic Acids potential for studying structure and dynamics of membrane [1] Denisov et al. (2004), J. Am. Chem. Soc., 126, p. 3477. proteins, which are not readily amenable to X-ray crystallog- [2] Hagn et al. (2013), J. Am. Chem. Soc., 135, p. 1919. raphy and solution NMR. In this presentation we will describe the latest developments in our group, including advances in sample preparation procedures for obtaining MAS spec- tra with optimal spectral sensitivity, experimental strategies NMR of Nucleic Acids for obtaining spectroscopic assignments, methods for mea- suring long-range structural restraints for the determination MO121: Investigation of RNA-small molecule inter- of high resolution structure and supramolecular organization actions by NMR spectroscopy of membrane proteins in the lipid environment. We will present high-resolution structure of a seven-helical photore- 1∗Romana Spitzer, 2Roland Huber, 1Christoph Wunder- ceptor Anabaena Sensory Rhodopsin, and discuss progress lich, 2Klaus Liedl, 1Christoph Kreutz towards structural characterization of a eukaryotic membrane 1University of Innsbruck, Institute of Organic Chemistry, protein human Aquaporin 1. 2University of Innsbruck, Institute of Inorganic and Theo- retical Chemistry TU119: Structural determinants and functional as- The starting point of the presented research was a recently pects of specific lipid binding to potassium channels published X-ray crystal structure of the cyclic di guanosine as seen by coarse grained MD and solid state NMR monophosphate (c-di-GMP) class I riboswitch aptamer in 1∗Markus Weingarth, 1,2Alexander Prokofyev, 1Elwin A. complex with its cognate ligand c-di-GMP. [1,2] W. van der Cruijsen, 1Deepak Nand, 1Alexandre M. J. J. 2 1 Riboswitches are RNA sequences that are capable to respond Bonvin, Olaf Pongs, Marc Baldus to the concentration level of a metabolite and subsequently 1Bijvoet Center for Biomolecular Research, Utrecht Univer- regulate gene expression by binding of the metabolite.[3-5] sity, 2Saarland University, Faculty of Medicine, Department During this gene regulation process two very interesting prop- of Physiology erties of RNA sequences are highlighted, namely the ability to CGMD simulations overcome the typical length- and recognize binding partners (small molecules up to large pro- timescale limits of atomistic simulations, which greatly en- teins) with high affinity and selectivity and secondly, RNAs hances the potential to compare computational observations have the intrinsic propensity to undergo large scale structural to powerful experimental techniques like confocal microscopy rearrangements triggered by a specific event (e.g. binding of or solid state NMR. a ligand or addition of a complementary sequence partition).

Here we have investigated specific lipid binding to the pore Based on the molecular details of ligand recognition we domain of potassium channels KcsA and KcsA-Kv1.3 on rationally designed a ligand, cyclic iso-cytidine-guanosine the structural and functional level using coarse-grained and monophosphate (c-iCG), which binds with even higher affin- atomistic MD simulations, solid state NMR and single chan- ity than the native one and under preservation of specificity. nel conductance measurements. We show that while KcsA ac- In the first step we confirmed the binding properties of c- tivity is critically modulated by the specific and cooperative iCG by an in silico approch using molecular dynamics (MD) binding of anionic non-annular lipids close to the channel’s simulations. We then set out for the experimental valida- selectivity filter, the influence of non-annular lipid binding tion by NMR spectroscopy of the results obtained from the on KcsA-Kv1.3 is much reduced. These finding provided the computational methods. basis to deduce a molecular mechanism of lipid modulated [1] N Kulshina, NJ Baird, AR Ferré-D’Amaré, Nat. Struct. activity in KcsA [1]. Mol. Biol. 2009, 16 (12), 1212-7. [1] Markus Weingarth, Alexander Prokofyev, Elwin A. W. [2] KD Smith, SV Lipchock, TD Ames, J Wang, RR Breaker, van der Cruijsen, Deepak Nand, Alexandre M. J. J. Bonvin, Nat. Struct. Mol. Biol. 2009, 16, 1218 - 1223 Olaf Pongs, and Marc Baldus, J. Am. Chem. Soc., 2013, 135 [3] RK Montagne, RT Batey, Annu. Rev. Biophy. 2008, 37, (10), pp 3983–3988. 117-33.

TH120: NMR Structural Characterization of Mem- [4] M Mandal, RR Breaker, Nat. Rev. Mol. Cell Biol. 2004, brane Proteins in a Lipid Environment 5, 451-63. [5] KF Blount, RR Breaker, Nat. Biotechnol. 2006, 24 (12), 1,2 , 1Franz Hagn, 1Thomas Raschle, Rasmus Linser 1558-64. 1∗Gerhard Wagner 1 Harvard University, Biol. Chem. and Mol. Pharm., TU122: New Tagging Strategies for DNA-Strands of 2 University of New South Wales, Sydney Modified Nucleobases and Cys-Ph-TAHA tag Membrane proteins are naturally constituted into a lipid bi- Sebastian Täubert, Florian Siepel, Andrei Leonov, layer environment. This setting is decisive for the function ∗Christian Griesinger of the majority of such proteins. For most structural bi- ology techniques, however, it poses major hurdles. Besides Max Planck Institute approaches using, e.g., cubic lipid phases in crystallogra- Paramagnetic tagging of proteins introduces paramagnetic phy, techniques potentially amenable to membrane-integral parameters such as residual dipolar couplings, pseudocon- biopolymers are solid-state and solution-state NMR. A mile- tact shifts and paramagnetic relaxation into otherwise dia- stone for a native setting of membrane proteins is the nan- magnetic proteins. From those information on structure and odisc concept,[1] which we could use successfully for NMR dynamics of domains and within domains can be derived. structural characterization recently.[2] We employ this tech- For proteins several tags are known in literature with a wide nique to gain a functional understanding of different mem- range of different properties. However, there are neither con- brane proteins. One target presented is a membrane anchor venient tags nor accessible binding sites for oligonucleotides domain of a transcriptional activator precursor protein that which we address in this contribution. is immanent for regular cell physiology and has been found to be associated to cancer, a second one is an alpha-helical Here we introduce two modified nucleobases to provide an pore protein. exposed thiol moiety for tagging reactions via a disulfide for- mation. These nucleobases are synthesized within six steps We will show NMR data for the two proteins in both settings and can be introduced into oligonucleotides easily by chemi- and discuss differences of nanodisc embedding with respect cal synthesis. The yields during the DNA synthesis are nearly to detergent micelle reconstitution. the same as for unmodified nucleobases. The first version

104 Poster Abstracts – Molecular Dynamics by NMR of the modified nucleobase contains a cyanoethyl protected formed helical binding interface via conformational selection sulfur-atom. The protection group is removed after the DNA and subsequently completes folding upon binding. synthesis. The second version provides a terminal acetylene moiety where the sulfur-atom is introduced via a copper cat- TU125: Study of Brazilian Asphaltene Agregation alyzed click-reaction after the DNA synthesis. We used the by Nuclear Magnetic Resonance Spectroscopy Cys-Ph-TAHA tag developed in our department to test the new approach. The preloaded tag was incubated with the 1∗Emanuele Catarina da Silva Oliveira, 1Alvaro Cunha DNA and subsequent HPLC purification leads to the tagged Neto, 1Valdemar Lacerda Júnior, 1Eustáquio Vinícius DNA quantitatively. Ribeiro de Castro, 2Sonia Maria Cabral de Menezes 1 2 Furthermore, we synthesized a new paramagnetic tag based Universidade Federal do Espírito Santo, Centro de on the Ph-TAHA construct which will be attached to phos- Pesquisas e Desenvolvimento Leopoldo Américo Miguez de phorothioate DNA. Mello (Cenpes – Petrobras/RJ) Heavy crude oils have attracted a growing interest from oil After the tagging protocol was successfully established with industries around the world. In this scenario, asphaltenes, the diamagnetic species NMR spectra will be shown for a which are molecules known for their low reactivity and high self-complementary DNA strand of 24 nucleobases and Cys- molecular weight, have received special attention in recent Ph-TAHA tag with various paramagnetic lanthanides. The decades, due to the problems they cause during oil process- prospect of characterizing DNA and RNA dynamics with this ing. In an effort to minimize these problems, researchers have approach will be discussed. increasingly looked for a better understanding of aspects such as molecular structure, stability and physicochemical proper- TH123: Conformational capture mechanism of the ties of asphaltenes, as well as for the development of methods neomycin B riboswitch to prevent their unwanted precipitation. In recent decades, nuclear magnetic resonance (NMR) has been widely used as a Christoph Wunderlich, ∗Christoph Kreutz tool for the study of petroleum and its derivatives. The anal- University Innsbruck ysis of NMR spectra allows the direct determination of a se- The goal of the presented project is the characterization of ries of structural parameters such as the fractions of aromatic a low populated state of a gene-regulative RNA sequence, carbons. Diffusion-ordered NMR spectroscopy (DOSY) is a namely an artificial riboswitch responsive to neomycin B. For new developed NMR technique highly useful for the elucida- that purpose we apply a multi-disciplinary approach using tion of complex mixtures. . This work sought to identify the chemical synthesis of 13C-modified building blocks and the differences in average parameters of asphaltenes from na- state-of-the-art solution NMR spectroscopy. In a favourable tional crudes and to correlate them with the physicochemical case we will be able to determine the population of the ex- properties of the asphaltene and of the original crude. The cited state (i.e. thermodynamic), the exchange rates from aim was also to investigate and to compare the asphaltenes the ground to the excited state and for the backward reaction among themselves, using data such as diffusion coefficient, (i.e. kinetics), and also information on the structure of the concentration of these compounds in the oil and state of ag- excited state (i.e. chemical shift) using CPMG (Carr-Purcell- gregation. Extraction and quantification of the amount of Meiboom-Gill) relaxation dispersion NMR spectroscopy. The asphaltenes were performed using ASTM 6560-00 standard excited state of the artificially designed neomycin B ri- and the characterization was performed using the following 1 13 boswitch will be compared to the ligand bound state. Us- analytical techniques: elemental analysis, H and C NMR ing the proposed approach we will be able to differentiate and DOSY. Significant changes were observed in almost all between various ligand-host recognition modes. This will studied parameters. To study the asphaltene aggregation be- deepen the understanding of RNA and its way to interact havior using DOSY, the asphaltenes were diluted in various with ligands allowing the design of high-affinity binder with concentrations of deuterated toluene and the DOSY spectra applications in the field of drug development. were correlated with their respective states of aggregation. The diffusion properties were highly dependent on the con- centration and type of oil from which the asphaltene was extracted. Molecular Dynamics by NMR

TH126: Novel intramolecular dynamics within the MO124: Conformational Dynamics of the Partially N-Cap-SH3-SH2 regulatory unit of the c-Abl tyro- Disordered Yeast Transcription Factor GCN4 from sine kinase reveal targeting to the cellular membrane Molecular Dynamics Simulations and NMR 1Guilherme A. P. de Oliveira, 1Elen G. Pereira, 1Giulia ∗Paul Robustelli, Nikola Trbovic, Arthur G. Palmer III D. S. Ferretti, 1Ana Paula Valente, 2Yraima Cordeiro, 1∗ Columbia University Jerson L. Silva 11 Programa de Biologia Estrutural, Instituto de Bioquímica We have employed molecular dynamics (MD) simulations to Médica, Instituto Nacional de Biologia, 22 Faculdade de study the conformational dynamics of the partially disor- Farmácia, Universidade Federal do Rio de Janeiro, Rio de dered yeast transcription factor GCN4. We demonstrate that Janeiro, Brazil. a detailed analysis of back-calculated NMR chemical shifts and spin-relaxation data provide complementary probes of c-Abl is a key regulator of cell signaling and is under strict the structure and dynamics of partially disordered protein control via intramolecular interactions. In this study, we ad- states and enable comparisons of the accuracy of multiple dress changes in the intramolecular dynamics coupling within MD trajectories. Back-calculated chemical shifts provide a the c-Abl regulatory unit by presenting its N-terminal seg- sensitive probe of the populations of residual secondary struc- ment (N-Cap) with an alternative function in the cell as c- ture elements while spin-relaxation calculations are sensitive Abl becomes activated. Using small angle X-ray scattering, to a combination of structural factors including the stability nuclear spin relaxation experiments and confocal microscopy, of local secondary structure elements, the presence of ter- we demonstrate that the N-Cap and the Src homology (SH) tiary contacts, and the presence of residual structure in more 3 domains acquire µs-ms motions upon N-Cap association distal regions of the protein. We demonstrate that an analy- with the SH2-L domain, revealing a stabilizing synergy be- sis of back calculated chemical shift and spin-relaxation data tween these segments. The N-Cap-myristoyl tether likely can be used to evaluate the populations of specific interac- triggers the protein to anchor to the membrane because of tions in disordered states and identify regions of the phase this flip-flop dynamic, which occurs in the µs-ms time range. space that are inconsistent with experimental measurements. Chemical shift perturbation (δδ) analyses of 1H-15N by nu- Our analyses suggest that GCN4 recognizes DNA with a pre- clear magnetic resonance (NMR) HSQC spectra were carried

105 Poster Abstracts – Molecular Dynamics by NMR out to map critical moieties that are affected by intramolec- sessment of accuracy. Moreover, novice CPMG experimen- ular interactions within the regulatory unit. The N-Cap seg- talists face an additional barrier because current software op- ment not only presents the myristate during c-Abl inhibition tions lack an intuitive user interface and extensive documen- but may also trigger protein localization inside the cell in a tation. Hence, we present the open-source software package functional and stability-dependent mechanism that is lost in GUARDD (Graphical User-friendly Analysis of Relaxation Bcr-Abl+ cells, which underlie chronic myeloid leukemia. Dispersion Data), which is designed to organize, automate, and enhance the analytical procedures which operate on MO127: Analysis of the Pirouetting Motion in [2]Ro- CPMG RD data (http://code.google.com/p/guardd/). This taxanes by Dynamic 1H NMR MATLAB-based program includes a graphical user inter- face, permits global fitting to multi-field, multi-temperature, 1Clarissa Piccinin Frizzo, 1∗Lilian Buriol, 1Letícia V. multi-coherence data, and implements χ2-mapping proce- Rodrigues, 1Marcos A. P. Martins, 2José Berná, 2Mateo dures, via grid-search and Monte Carlo methods, to enhance Alajarín and assess fitting accuracy. The presentation features allow 1Federal University of Santa Maria, 2Universidad de Murcia users to seamlessly traverse the large amount of results, and the RD simulator feature can help design future experiments Due to their characteristic of controlling motion, as well as serve as a teaching tool for those unfamiliar with mechanically-interlocked molecules like [2]rotaxanes have RD phenomena. Based on these innovative features, we ex- received much scientific interest over the last decade, espe- pect that GUARDD will fill a well-defined gap in service of cially in the development of biological machines [1,2]. The the RD NMR community. [2]rotaxanes interlinked by benzylic amide macrocycles have shown chair-chair co-conformational changes, where methy- lene hydrogens present in the pseudoaxial position change to TH129: Inter-domain motions of calmodulin from the pseudoequatorial position and vice versa. The molecu- NMR chemical shifts and MD simulations lar dynamics of this rotation may be accomplished by the ∗ coalescence method using 1H NMR experiments in solution Predrag Kukic, Carlo Camilloni, Michele Vendruscolo [3]. Given this context, our aim in this work is to demon- Cambridge University, UK i strate the effect that different stoppers, such as -butyl (1) Around 80% of all proteins in eukaryotes consist of multiple and butyl (2) substituents attached at succinamide template domains that are connected by flexible linkers. Although the in the pirouetting motion of the benzylic amide macrocycle 1 multi-domain proteins are involved in a range of important bi- around a thread, by using a dynamic H NMR study in ological processes, detailed knowledge of their structure and 1 d solution. Thus, through H NMR experiments in DCM- 2 dynamics requires extensive experimental characterization. as part of the coalescence method we measured the energy Here, we study the possibility of using the informational con- barriers for the rotation of the macrocycle around the two tent encoded in the most readily and accurately measured different threads. The calculated energy barriers for the NMR observables, such as chemical shifts, to characterize macrocycle pirouetting process were: 12.90 kcal.mol−1 at −1 inter-domain motions in proteins. For this purpose, we use 293 K for [2]rotaxane (1), and 13.39 kcal.mol at 303 K for chemical shifts measured for the flexible linker of calmodulin [2]rotaxane (2). The different energy barrier values between in its calcium-bound state (Ca2+-CaM) and chemical shifts the two [2]Rotaxanes are attributed to structural changes in measured for its two domains. Using cross-validation analysis the stoppers of the thread. The pirouetting submolecular of the reference ensemble, we firstly demonstrate the ability motion of these interlocked molecules can be correlated with of chemical shifts implemented in replica-exchanged molecu- ••• π CH interactions between the thread stoppers and lar dynamics simulations to reproduce the inter-domain mo- the benzylic amide macrocycles and other hydrogen-bonded tion of Ca2+-CaM by accurately reproducing the dynamics molecules observed in [2]rotaxane (1). of its flexible linker. We then use the same procedure with 2+ References: experimentally measured chemical shifts of Ca -CaM and determine an ensemble of conformations that represents the [1] Stoddart, J. F. Chem. Soc. Rev. 2009, 38, 1802. free-energy surface of Ca2+-CaM in the solution. The ensem- ble is validated against the previously obtained ensemble of [2] Kay, E. R.; Leigh, D. A.; Zerbetto F. Angew. Chem. Int. Ca2+-CaM determined from RDC and SAXS measurements. Ed. 2007, 46, 72. The results confirm the dominance of Ca2+-CaM conforma- [3] Berná, J.; Alajarín, M.; Martínez-Espín, J. S.; Buriol, L.; tions with both hydrophobic patches oriented towards oppo- Martins, M. A. P.; Orenes, R.-A. Chem. Commun. 2012, 48, site sides with respect to the central helix. In these confor- 5677. mations, hydrophobic patches of N- and C-terminal domains of Ca2+-CaM are highly exposed to the solution and fully Acknowledgements: accessible to their target proteins. This work was supported by the MICINN (Project CTQ2009- 12216/BQU) and the Fundación Séneca-CARM (Project MO130: Refinement of protein conformational en- 08661/PI/08). José Berná thanks the MICINN for the sembles using methyl chemical shifts Ramón y Cajal contract (RYC-2008-02647) which was co- financed by the European Social Fund. Thanks for financial Arvind Kannan, Carlo Camilloni, Aleksandr Sahakyan, support is also acknowledged by Lilian Buriol and Letícia V. Andrea Cavalli, Juuso Lehtivarjo, ∗Michele Vendruscolo Rodrigues (to CAPES), by Marcos A. P. Martins (to CNPq), Cambridge University and by C.P. Frizzo (to FAPERGS). The last decade has sparked considerable interest in the de- velopment of methods for protein structure determination us- TU128: GUARDD: User-friendly MATLAB soft- ing only the information encoded in NMR chemical shifts. ware for rigorous analysis of CPMG RD NMR data Chemical shifts are the NMR observables that can be mea- Ian R. Kleckner, ∗Mark P. Foster sured under the widest range of conditions and with the greatest accuracy. These parameters are often the only struc- Ohio State University tural data that can be obtained for large protein complexes, For the past fifteen years, the Carr-Purcell Meiboom-Gill re- making them an invaluable experimental tool for probing the laxation dispersion (CPMG RD) NMR experiment has af- structure and dynamics of these systems. Recent work has forded advanced NMR labs access to kinetic, thermodynamic, resulted in methods for predicting the dihedral angles and and structural details of protein and RNA dynamics in the secondary structure populations of amino acids from their crucial µs-ms time window. However, analysis of RD data chemical shifts. Moreover, it is now known that the accuracy is challenging because datasets are often large and require of structure-based chemical shift predictions improves dra- many non-linear fitting parameters, thereby confounding as- matically when the calculations are averaged over ensembles

106 Poster Abstracts – New Methods in solution NMR of protein conformations, suggesting that chemical shift data can be used to generate accurate structural ensembles that New Methods in solution NMR reflect the true dynamics of proteins in solution. We have recently established the feasibility of this approach by imple- menting chemical shifts for backbone atoms as structural re- TH132: Padé via Moments: Squeezing the FID and straints in replica-averaged molecular dynamics simulations, going beyond the limits of the FFT in NMR in analogy to previous work with other NMR observables. In ∗ this study we demonstrate that the same methodology can Carlos Cobas, Santiago L. Ponte also be applied to methyl proton shifts, leading to further re- MESTRELAB RESEARCH S.L. finement of the resulting structural ensembles and improved Since the Fourier Transform (FT) was first applied in NMR agreement with the experimental data. In order to validate by R. Ernst and W. Anderson almost 50 years ago,[1] it has this approach, we have run a series of simulations of four become a de facto mathematical procedure for converting the model proteins - the autophagy related protein Atg8, a BMP encoded time signal (FID) into its spectral representation in receptor, the DNA-bending HU dimer, and the GPCR reg- the frequency domain. There are a number of reasons that ulator RGS10 - involving (1) no restraints, (2) restraints us- justify why it has been so widely used for NMR data pro- ing only the backbone shifts, (3) restraints using only the cessing, even to date, ranging from the fact that it is compu- methyl shifts, and (4) both sets of restraints. Our results tationally very efficient and robust to its steady convergence indicate that methyl chemical shifts can provide complemen- with increasing acquisition time. tary structural information to that obtained from backbone data, and that this complementarity induces global improve- However, the FT presents several limitations. In one hand, ments in the accuracy of free energy landscapes computed it can be considered as a low-resolution estimator as its res- from ensembles 3 and 4. olution is pre-determined exclusively by the total acquisition time. Attempts to increase resolution by acquiring the signal for a longer period of time will lead to worsened SNR. More- TU131: NMR Analyses of the Keto-Enol Equilib- over, the FT yields an envelope spectrum with no quantifica- rium of Avobenzone Type UVA Photoabsorbers tion on its own, and some additional processing or analysis procedures such as peak picking, integration or line fitting 1Patrícia Coelho Nascimento, 2,3Vinícius Soares da Paixão (deconvolution) must be applied should any quantitative in- Correia, 2,3Luiz Antônio Soares Romeiro, 1∗Edilberto formation is required. Rocha Silveira 1Universidade Federal do Ceará, 2Universidade Católica de Considering the quantum-mechanical origin of the interest- Brasília, 3Universidade de Brasília ing, genuine signals, an alternative approach is proposed in this work that circumvents the fundamental limits of the FT. Avobezone [1-(4-methoxyphenyl)-3-(4’-tert-butylphenyl)- It is based on a new mathematical procedure aimed at build- propane-1,3-dione] is a photoabsorber of UVA type radia- ing stable and accurate rational Padé approximants[2] from tion (320-400 nm). It is present in almost all commercially which all the spectral parameters, including all elementary available sunblockers, where it also works to prevent skin frequencies, the corresponding amplitudes and the true num- age development. However, avobenzone has low photosta- ber of physical resonances from the time domain signal can be bility. This has motivated the syntheses of avobenzone derived. We will show how this new method can resolve and analogs as a lead to more stable UVA absorbers. The quantify tightly overlapped signals and discriminate between keto-enol tautomerization of dibenzoylmethanones seems genuine and spurious signals. to be associated with their activity, thus NMR raised as a suitable method to analyse this behavior. Either a 300 [1] R.R. Ernst and W.A. Anderson, Rev. Sci. Instrum. 37, MHz DPX or a 500 MHz DRX Bruker spectrometer was 93 (1966) used in this task. The uni and bidimensional NMR spectra of compound 1 [1-(2-hydroxyphenyl)-3-phenyl-propane-1,3- [2] Dž. Belkic, Quantum Mechanical Signal Processing and dione] and of its derivatives 2 [1-(2-hydroxyphenyl)-3-(4’- Spectral Analysis (Taylor & Francis Publishers, London, methylphenyl)-propane-1,3-dione], 3 [1-(2-hydroxyphenyl)- 2005) 3-(4’-methoxylphenyl)-propane-1,3-dione] and 4 [1-(2- hydroxyphenyl)-3-(4’-fluorophenyl)-propane-1,3-dione], in MO133: Quantitative study chemical exchange of di- CDCl3 solution, showed the presence of only one enol moiety lute sample by inversion transfer preserving the carbonyl "ortho" to the hydroxyl substituent of ring A and enolysing just the carbonyl "para" to the Qing Luo, Weiping Jiang, Haidong Li, Qianni Guo, Ji substituent of ring B. The HMBC spectra allowed the as- Zhang, Zhao Li, Shizhen Chen, Chaohui Ye, ∗Xin Zhou signment of the phenol hydrogen at about 12.0 ppm, while Wuhan Institute of Physics and Mathematics the hydrogen of the enol hydroxyl appeared about 15.0 ppm, as expected. Compounds 5 [1-(2-hydroxyphenyl)-3-(4’- The remarkable advantage of NMR over other spectroscopy nitrophenyl)-propane-1,3-dione] and 6 [1-(2-hydroxyphenyl)- methods, e.g. IR or MS, is the comprehensive ability for the 3-(4’-cyanophenyl)-propane-1,3-dione], in Pyr-d5, showed an kinetic studies. In addition to tracing the time dependence of almost 1:1 keto-enol equilibration what has been charac- chemical reaction, NMR is able to provide the detailed kinetic terized through the AB quartet of the β-diketone methylene information, such as chemical exchange, under conditions of around 3.4 ppm and HSQC correlation with the carbon about the dynamic equilibrium. Exchange-correlated spectroscopy 50 ppm and the vinyl hydrogen of the enol form at 7.5 ppm (EXSY) [1] is a regular method for the chemical exchange correlating with the carbon at 97.0 ppm. The compounds studies, which can provide both qualitative and quantitative 1 and 7 [1-(2-hydroxyphenyl)-3-(4’-dimethylaminephenyl)- information. However, the usage of such method is unable to propane-1,3-dione], in Pyr-d5, showed the same behavior of study the dilute samples due to the low detection sensitivity, only one enol form predominance. As a conclusion it seems the intrinsic weakness of NMR. Chemical exchange saturation that "meta" orientating deactivating substituents in ring B transfer (CEST) [2, 3, 4] is an indirect detection method with favor the 1:1 equilibrium while "ortho"/"para" orientating a remarkable sensitivity for the species of low concentrations substituents in ring B favor the enolization of the carbonyl or short lifetimes, and it is typically applied for MR molec- conjugated to ring B, preserving the carbonyl of ring A ular imaging of proteins/peptides which participate in the hydrogen bonded to the "peri" hydroxyl. proton chemical exchange with surrounding water. Some en- vironmental properties, such as pH and temperature, which strongly associate with the chemical exchange, can also be ACKNOLEDGEMENTS: CNPq, PRONEX, CAPES, FUN- imaged. With these advantages, the CEST becomes a pow- CAP erful tool for MR molecular imaging.

107 Poster Abstracts – New Methods in solution NMR

Theoretically, the CEST effect contains the information of mentation in Origin® unfeasible a friendly interface con- chemical exchange rates. However, the mechanism of magne- struction, rendering complex software for users not experts. tization during the CEST experiment can be described by a The main goal of this project is to implement the FDM al- series of Bloch equations are difficult to be analytically solved. ® Zhou et al. solved these equations with several simplified as- gorithm on the Matlab platform, inserting facilities to the sumptions and approximations, which were not very close to users and make the code simpler to new applications. Un- the experimental conditions. Sherry et al. gave the numeri- til now we have finished the implementation of the KBDM cal solutions of these equations, with several fixed parameters method; two regularization method: SVD and Tikhonov; and including the concentrations of the chemical exchange sites. many simple functions at the interface. The FDM software But the concentrations are practically difficult to measure for implemented in Matlab has been tested with several known the dilute samples, especially for the in vivo experiments. spectra and compared with Fourier Transform.

Recently, we developed a novel method, called Chemical ex- TH135: Thermodynamic, Volumetric and Hy- change Inversion Transfer (CEIT), based on the similar sig- drophobic Characterization of Micelles by NMR to nal enhancement strategy to the CEST, but with an ex- Understand Detergent Stabilization of Membrane tremely improved spin control manner. By removing the Proteins radio frequency (RF) during the chemical exchange dura- ∗ tion, the Bloch equations of the magnetization processes of Rohan Alvares, Shaan Gupta, Peter M. Macdonald, Scott the new method are greatly simplified. Thus, the analytical Prosser solutions are readily achieved. We applied this method to Department of Chemistry, University of Toronto study the Xe-Xe chemical exchange, i.e., exchange between Xe dissolved in water and Xe trapped inside the function- Membrane proteins are involved in functions such as catal- ally modified Cryptophane-A molecular cage, a 129Xe based ysis, transport and ion conductance. Furthermore, thirty MRI biosensor [5, 6]. The chemical exchange rate calculated percent of genes code for membrane proteins and seventy by CEIT method, without any pre-information of concentra- percent of drugs target them. Consequently, determination tions, is in agreement with that measured by 1D EXSY exper- of structure and dynamics of membrane proteins is of con- iment. Furthermore, such novel method achieved the same siderable importance. One of the main techniques used to sensitivity of the CEST with a much low duty cycle, which study membrane proteins, NMR, requires solubilization of hints a great potential with a higher sensitivity in biomedical these hydrophobic proteins in detergents. The choice of de- applications. tergent, which is imperative to maintaining proper protein structure, is still an empirical process. We have developed a References: high-throughput NMR methodology to determine detergent thermodynamic and volumetric properties upon micellization [1] J. TAMMY et al, Chem. Rev. 90:935 (1990) in hopes of elucidating the characteristics of detergents that dictate their ability to solubilize membrane proteins. Ther- [2] K. M. Ward et al, JMR 143, 79 – 87 (2000) modynamic and volumetric properties were indirectly ob- [3] J. Y. Zhou et al, MRM 51:945 (2004) tained by observing subtle differences in chemical shifts be- tween micelle and monomeric states of the detergent which [4] D.E. Woessner et al, MRM 53:790–799 (2005) yielded the critical micelle concentration (CMCs) and subse- quently the Gibbs free energy of micellization. Temperature [5] Schroder L, et al„ Science, 314:446, (2006) dependencies of the free energy yielded enthalpic, entropic [6] Meldrum T, et al, JMR, 213:14 (2011). and heat capacity changes upon micellization, whereas, high pressure studies provide changes in the isothermal compress- ibility and partial molar volume. We compare differences in TU134: Implementation of the Filter Diagonaliza- these properties between four common detergents: anionic tion Method (FDM) in Matlab® for signal analysis (sodium dodecyl sulfate, SDS), zwitterionic (dodecylphos- in nuclear magnetic resonance. phocholine, DPC and cyclofos, CyF) and neutral (dodecyl 1,3Joao Felipe Alves da Cruz, 2,3∗Tiago Bueno de maltoside, DDM) detergents to understand why some are Moraes, 2Cláudio José Magon, 3Luiz Alberto Colnago better mimetics of the membrane than others. Furthermore, these measurements are complemented by using a paramag- 1Universidade Federal de São Carlos - UFSCar, 2Instituto netic oxygen molecule to probe the hydrophobicity of the mi- de Física de São Carlos - IFSC-USP, 3Empresa Brasileira celle interior. This technique can, in theory, provide methy- de Pesquisa Agropecuária - Embrapa Instrumentação lene specific estimates of hydrophobicity along the detergent In 1990, Neuhauser introduced a numerical method for ex- chain. The results indicate increasing hydrophobicity across traction of eingenvalues and eingenvectors in a given energy the series SDS < DPC < DDM which is generally consistent range of a large quantum system, named "Filter Diagonal- with tendencies of these detergents to stabilize membrane ization". It apply a Fourier filter for remove the correla- proteins. tion between distant eigenstates, and then, diagonalizing the Hamiltoniana matrix in the subspace to calculate the eigen- MO136: Real-time mechanistic monitoring of an ac- states of a short range of energy. In 1997 Mandelstam using etal hydrolysis by ultrafast 2D NMR a rectangular filter, achieved convergence, velocity and relia- bility and the name "Filter Diagonalization Method" (FDM) 1,2∗Luiz Henrique Keng Queiroz Júnior, 3Patrick Gi- was introduced. The FDM was explored in several areas of raudeau, 1Fabiane Aparecida Batalha dos Santos, 1Kleber Physics where the harmonic inversion is necessary. Partic- Thiago de Oliveira, 1Antonio Gilberto Ferreira ularly, one of the most interesting applications of the FDM 1Universidade Federal de São Carlos, Departamento de is Nuclear Magnetic Resonance (NMR). We recently showed Química, Laboratório de RMN, 2Universidade Federal that with the use of FDM it is possible to quickly obtain spec- de Goiás, Instituto de Química, Laboratório de RMN, tra with excellent signal/noise ratio. With these techniques 3cUniversité de Nantes, CNRS, Chimie et Interdisciplinar- we can suppress the echo component SSFP signal allowing ité: Synthèse, Analyse, Modélisation (CEISAM) the suppression of signals anomalies in the frequency domain spectrum or to suppress intense solvent signals in biological Ultrafast (UF) 2D NMR makes it possible to obtain a 2D samples or broad signal in heterogeneous materials. NMR spectrum in less than a second.[1] UF-NMR is emerg- ing as a potential technique to study mechanisms in organic Our current software developed runs on Origin®, with rou- reactions at natural abundance. Acetals are a very impor- tines in C/C++ and Fortran. Initially was developed for tant class of organic compounds and are normally hydrolyzed signal analysis in EPR (Electronic Magnetic Resonance) and using water and an acid catalyst. However, identifying the subsequently modified to resolve NMR signals. This imple- intermediates of these hydrolysis reactions is problematical

108 Poster Abstracts – New Methods in solid-state NMR since the lifetime of these species are very short. This work Department of Electronic Science, Xiamen University, Xia- presents the real-time monitoring of an acid catalyzed acetal men, Fujian 361005, China hydrolysis reaction by UF-HSQC experiments, with no need 13 High-resolution NMR spectroscopy has been a powerful tool for C-labeling.[2] It was monitored the hydrolysis of the for the study of molecular structures and chemical dynamics 2-(4-nitrophenyl)-1,3-dioxolane acetal reacting with trifluo- of a vast range of compounds, materials and reaction pro- roacetic acid (TFA) in the presence of residual water from the cesses, from the metabolites of an organism to the composi- solvent (deuterated chloroform). This reaction was carried tions of promising new materials. However, there are many out in a 5 mm NMR tube placed in a 400 MHz spectrometer circumstances where the magnetic field homogeneity is de- equipped with a 5 mm BBI probe. UF-HSQC experiments graded or the testing object is subject to intrinsic variation were used for the real-time mechanistic study of this acetal 13 in magnetic susceptibility so that the spectral lines are broad- hydrolysis at C natural abundance, and it was possible to ened and the spectrum becomes devoid of any chemical infor- characterize the presence of the hemiacetal, an intermediate mation. This precludes NMR spectral analyses based upon with a well-known short lifetime. This monitoring result in the interpretation of the familiar NMR parameters, such as the characterization of a hemiacetal intermediate, which we chemical shifts and scalar coupling constants. In this study, could not observe by 1D NMR, in spite of numerous attempts, we put forward an approach to remove the influence of mag- probably due to the very short lifetime of this intermediate. netic field inhomogeneity on NMR spectroscopy via inter- These assignments were confirmed and rationalized by quan- 1 13 molecular multiple-quantum coherence technique [1]. The tum calculations of H and C NMR chemical shifts and 2D NMR acquisition is accelerated by spatially encoded tech- NBO (Natural Bonding Orbital) analysis. While this work nique proposed by Frydman and co-workers [2] and improved showed the potentialities of UF-NMR for real-time mechanis- by Pelupessy [3]. To increase the signal-to-noise ratio, only tic studies, it also allowed the characterization of the short the solvent spin is excited to evolve during the spatially en- lifetime hemiacetal intermediate. coded period. The area of the decoding gradients is small [1] Frydman, L.; Scherf, T.; Lupulescu, A.; Proc. Natl. Acad. and the solutes do not experience the diffusion decay from Sci. USA 2002, 99(25), 15858-15862. the spatial encoding gradients. The principles and advan- tages of this new approach are presented, and its application [2] Queiroz Jr., L. H. K.; Giraudeau, P.; Santos, F. A. B.; is exemplified with a sample packed with pig brain tissue. Oliveira, K. T.; Ferreira, A. G.; Magn. Res. Chem. 2012, 50(7), 496-501. This work was supported by the NNSF of China under Grant 11204256. Acknowledgements: CNPq, CAPES and FAPESP. [1] Huang, Y. Q.; Cai, S. H.; Chen, X.; Chen, Z. J. Magn. TU137: Ultrafast High-Resolution J-resolved Spec- Reson., 2010, 203, 100- 107. troscopy in Inhomogeneous Magnetic Fields [2] Frydman, L.; Scherf, T.; Lupulescu, A. Proc. Natl. Acad. ∗Shuhui Cai, Hao Chen, Zhiyong Zhang, Zhong Chen Sci. U. S. A., 2002, 99, 15858- 15862. Department of Electronic Science, Xiamen University, Xia- [3] Pelupessy, P. J. Am. Chem. Soc., 2003, 125, 12345- 12350. men 361005, China Ultrafast 2D NMR accelerates the acquisition of 2D NMR spectrum by orders of magnitude. However, like conventional New Methods in solid-state NMR methods, ultrafast methods suffer from line broadening and loss of spectral features in inhomogeneous fields. To circum- 13 13 vent the influence of field inhomogeneity, various approaches MO139: Fast and ultra-fast MAS C- C correla- based on intramolecular or intermolecular multiple-quantum tions applied to biological systems coherences have been proposed. The protocol based on co- 4 4 1 1 herence transfer can obtain high-resolution spectrum in arbi- Guangjin Hou, Si Yan, Julien Trébosc, Olivier Lafon, 2Bingwen Hu, 3Bernd Reif, 2Qun Chen, 3,4Sam Asami, trary inhomogeneous field, but it has difficulty in spectral as- 4 1∗ signment [1]. The spatially encoded technique combined with Tatyana Polenova, Jean Paul Amoureux homonuclear intermolecular zero-quantum coherence (iZQC) 1Lille University, France, 2East China Normal Univer- can obtain a high-resolution 2D J-resolved spectrum with two sity, Shanghai, Chine, 3Munchen University, Germany, scans [2], but it undergoes difficulty in implementing when 4University of Delaware, Newark, USA the chemical shifts of solvent and solutes are close to each Proteins benefit from high-field spectrometers for the resolu- other. Here we propose a new scheme to ultrafast achieve 2D tion. However, sidebands may obscure the 13C-13C spectra J-resolved spectroscopy in inhomogeneous fields via heteronu- and spinning speeds faster than 30 kHz are thus required. clear iZQC. To evaluate the efficiency of the new scheme, ex- periments were performed. The results show that accurate The assignment of the resonances must first be done with chemical shifts and J-coupling information can be retrieved 1st-order methods. DQ-SQ experiments present the advan- in the 2D J-resolved spectrum. As the solvent and solutes are tages of: (i) allowing the observation of only one-bond corre- excited through different channels, selective pulses required in lations, (ii) short recoupling times, and (iii) no uninformative homonuclear iMQC methods are no longer necessary. More- diagonal peaks, which simplify the assignment of resonances over, the loss of signal-to-noise ratio caused by solvent sup- with close frequencies. In a second step, SQ-SQ experiments pression can be got rid of. All these greatly improve the are useful to observe two-bond cross-peaks. We have devel- spectral quality. oped two such methods, H-BR2 (DQ-SQ)[1] and RFDR-xy8- 41 (SQ-SQ),[2] which (i) require low rf-power, (ii) are robust [1] Pelupessy, P.; Rennella, E.; Bodenhausen, G. Science, to CSA and offset, (iii) are very simple to use with only the 2009, 60, 429-448. recoupling time to optimize, (iv) work at ultra-fast MAS, and 1 [2] Lin, Y. L.; Zhang, Z. Y.; Cai, S. H.; Chen, Z. J. Am. (v) do not require H decoupling. Chem. Soc., 2011, 20, 7632-7635. Once the assignment achieved, long range correlations pro- ACKNOLEDGEMENTS: This work was supported by the vide constraints to get the protein structure and folding. This NNSF of China under Grant 11275161. requires sequences less sensitive to dipolar truncation. The most used sequence, DARR, does not work at fast MAS, TH138: An Approach for High-Resolution Spatially and PARISxy is currently the most broadband sequence.[3] Encoded Intermolecular Multiple-Quantum Coher- We present several new sequences, SHANGHAI,[4] SHANG- ence NMR Spectroscopy in Inhomogeneous Fields HAI+,[5] and CORD,[6] which are more robust and efficient than previous methods. They even allow observing contacts Yulan Lin, ∗Zhong Chen, Shuhui Cai, Zhiyong Zhang up to 1000 pm.[5]

109 Poster Abstracts – New Methods in solid-state NMR

To decrease the experimental time, covariance can be used cases (long T2’). We also demonstrate the gain in quad-quad along the indirect dimension. However, this treatment in- 17O-27Al SS-J-HMQC correlations. troduces uninformative diagonal peaks that can be avoided with new covariance treatments.[7] Another way is using TH141: Probing the proximities and connectivities non-uniform sampling with maximum-entropy interpolation between close Larmor frequencies nuclei (NUS-MINT).[8] 1Julien Trebosc, 1∗Frédérique Pourpoint, 1Régis Gau- All the sequences and data treatments have been demon- vin, 2Qiang Wang, 1Olivier Lafon, 2Feng Deng, 1Jean Paul strated on YajG, SH3 and LC8 proteins, and also compared Amoureux with PAR and DREAM.[1,6] Moreover, the proteins were ei- 1 ther fully or only 15% protonated. UCCS – ENSCL - University Lille North of France, Vil- leneuve d’Ascq 59652 France, 2State Key Laboratory of Mag- [1] Lafon, Trébosc, Hu, DePaëpe, Amoureux, Chem. Comm. netic Resonance and Atomic and Molecular Physics Wuahn 47 (2011) 6930. Center for Magneti [2] Shen, Hu, Lafon, Trébosc, Chen, Amoureux, JMR. 223 The usual NMR probe design prevents the observation of (2012) 107. connectivities and proximities between numerous pairs of iso- topes (1H-19F; 31P-7Li; 13C-27Al; 13C-51V . . . ) exhibiting [3] Weingarth, Demco, Bodenhausen, Tekely, CPL. 469 close Larmor frequencies. The absence of such information is (2009) 342; Weingarth, Bodenhausen, Tekely, JACS. 131 a limitation for numerous important systems, including poly- (2009) 13937. Weingarth, Bodenhausen, Tekely, CPL. 488 mers, biomolecules, energy materials or catalysts. (2010) 10 Through the case study of 13C-27Al isotopes, we introduce [4] Hu, Lafon, Trébosc, Chen, Amoureux, JMR. 212 (2011) efficient methods to probe proximities and connectivities 320. between nuclei exhibiting close Larmor frequencies. Over- coupled resonators allow the tuning and matching of a single [5] Hu, Trébosc, Lafon, Chen, Masuda, Takegoshi, 27 13 Amoureux, ChemPhysChem. 13 (2012) 3585 channel at the close Al and C Larmor frequencies. This commercial device permits the successive irradiation of 27Al [6] Hou, Yan, Trébosc, Amoureux, Polenova, submitted. and 13C isotopes in a pulse sequence and accurate Al-C dis- tances are measured using the S-RESPDOR sequence.[1] We [7] Lafon, Hu, Amoureux, Lesot, Chem. Eur. J. 17 (2011) show that in 13C natural abundance, this approach allows 6716. measuring distances of 216 pm between covalently bonded Al- [8] Paramasivam, Suiter, Hou, Sun, Palmer, Hoch, Rovnyak, C atoms in lithium tetra-alkyl-aluminate, a commonly used Polenova, JPC-B. 116 (2012) 7416. co-catalyst in olefin polymerization processes, and distances ranging from 274 to 381 pm for non-bonded Al-C atoms in aluminum lactate. TU140: Satellite Saturation HMQC for quadrupolar to spin 1/2 nuclei correlations: transfer faster than We also report the first two-dimensional heteronuclear cor- the interaction relation experiments between 27Al and 13C nuclei. HMQC scheme using J coupling allows detecting the 13C-27Al co- 1Julien Trebosc, 2∗Qiang Wang, 3Yixuan Li, 1Olivier La- valent bonds, while HMQC using dipolar coupling reveals fon, 2Jun Xu, 1Jean Paul Amoureux, 3Qun Chen, 3Bingwen multiple proximities between the different 13C and 27Al sites Hu, 2Feng Deng proximities. 1Unit of Catalysis and Chemistry of Solids, CNRS-8181, Lille North of France University, 59652 Villen, 2State Key These methods are expected to be useful for the characteriza- Laboratory of Magnetic Resonance and Atomic and Molec- tion of supported heterogeneous catalysts, which lack long- ular Physics, Wuhan Center for Magnet, 3Physics Depart- range order or to gain insight into the structure of metal ment & Shanghai Key Laboratory of Magnetic Resonance, organic frameworks. It will help to bring further understand- East China Normal University, Sh ing in the nature of the Al-C bond in terms of distances and proximities and hence, of reactivity. When quadrupolar nuclei are involved in HMQC correlation experiments, one usually applies CT selective pulses to ma- Other pairs of nuclei available for distance measurement and nipulate only the central transition. If the quadrupolar nu- correlation with our over-coupled resonator include 13C-51V. cleus is detected in the indirect dimension using CT selective We demonstrate such methods on oxovanadium complexes. pulses then approximation of fictitious spin-1/2 applies and There are potential application for metallo-proteins like van- the buildup curve of J-HMQC transfert follows the case of abins. 1/2 spin with an optimum mixing time equal to 1/2J. How- We also managed much closer frequency nuclei pair by get- ever, the HMQC mixing mecanism can also populate energy ting correlations in between 23Na, 27Al and 31P in a 3D levels other than +1/2 or -1/2. Actually, the energy level n/2 experiment of a sodium alumino phosphate glass. Indeed we is populated with an optimum mixing time of 1/2nJ. At this managed to turn a triple resonance probe into quadruple res- point, SS-HMQC saturates the n/2 energy levels by irradi- onance one. That can be useful to discrimate such phases in ating the satellite transition in a manner similar to Double complex mixture of recrystallized glasses. Frequency Sweep (DFS) or other techniques used to enhance signal of the central transition. [1] F. Pourpoint et al. Chem. Phys. Chem, 2012 (13), 3605- 3615 We show that Satellite Saturation (SS) HMQC can reduce by a factor S+1/2 the required mixing time in a HMQC se- quence. Significant gain is thus achieved in sensitivity due to MO142: Structural Elucidation from Prednisone ap- lower attenuation resulting of transverse relaxation. plying Residual Dipolar Coupling 1,2∗ 1 Consequences of such results are of first importance as it José Adonias Alves de França, Rubens Rodrigues 1 opens the technique to a much broader set of materials for Teles, Fernando Hallwass which transverse (T2’) relaxation and weakness of J coupling 1Universidade Federal de Pernambuco, 2Instituto Federal de make the experiment very insensitive or impossible. We show Alagoas that this method extends to dipolar based D-HMQC. Structural elucidation of new organic compounds and natu- We study the robustness of the method to quadrupolar inter- ral products is a challenge for chemists. In some cases, even action by simulation and show experimental results of 31P- very elaborated NMR experiments are not sufficient to find 27Al J-HMQC, D-HMQC using the satellite saturation tech- the correct molecular structure. Because of this, different ap- nique with gains over 50% in sensitivity even in favorable proaches have been carried out in order to provide additional

110 Poster Abstracts – Exotica information to overcome this limitation as, for instance, the School of Molecular Bioscience, University of Sydney, Aus- realization of experiments in oriented media, where molec- tralia ular orientation is restrained in the NMR tube, recovering 1H NMR spectra of high concentrations (several M) of urea the anisotropic parameter, such as Residual Dipolar Coupling in high-concentration gelatin (denatured collagen) gels have (RDC) and Residual Chemical Shift Anisotropy (RCSA). two well-separated resonances from the amide 1H-atoms and In this work we have investigated the molecular structure residual water (HDO); this implies slow exchange of urea pro- of the steroid Prednisone (17,21-dihydroxypregna-1,4-diene- tons with aqueous solvent, on the NMR timescale. Holding 3,11,20-trione). It is a synthetic corticosteroid drug that is the gel stretched [1-3] generates residual dipolar splittings particularly effective as an immunosuppressant drug, used to in urea resonances (and other incorporated guest molecules) treat certain inflammatory diseases and some types of cancer. and residual quadrupolar splitting in HDO. Association of urea with collagen reduces, but does not completely quench, An examination of the structure of Prednisone makes it pos- the anisotropic behavior of stretched and compressed gelatin sible to propose 64 different diastereomers. All 64 structures gels, potentially allowing the structure determination of pep- were optimized by molecular mechanic calculations at the tides under denaturing conditions. DFT level of theory, using Gaussian03. The B3LYP func- 1 tional along with the 6-311++G(d,p) basis set was used. We We describe the anisotropic H NMR splitting pattern of carried out NMR experiments, such as 1H, 13C, gHSQC, urea under these anisotropic conditions with varying degrees 15 gHMBC, NOESY, gCOSY, using a commercial Prednisone of deuteration as well as with N-urea labelling. 1 13 sample in DMSO-d6 solution. In order to obtain the H, C We have also refined the method of estimating the values of RDC values we performed coupled gHSQC experiments with relaxation time constants of spin states of all ranks and or- PAN Gel, as the alignment media. ders of selected quadrupolar ions in stretched gels [4]. This 7 + 23 + We have compared the experimental RDC values with back- has been done for Li and Na [5], and work is in progress calculated ones (obtained by NMRDev software) for the 64 on others, using a Markov chain Monte Carlo algorithm. proposed diastereomers. The smallest Q factor was observed References: for the molecular structure of the correct stereoisomer. The same result would be not possible to obtain if only isotropic [1] Kuchel. P. W., Chapman, B. E., Mueller, N., Bubb, W. parameters had been used. Therefore, RDCs can be seen as a A., Philp, D. J., and Torres, A. M. (2006) J. Magn. Reson. powerful tool to solve assignment ambiguity and to elucidate 180, 256-265. chemical structures. [2] Naumann, C., Bubb, W. A., Chapman, B. E., and Kuchel, In conclusion, we have shown that measurement by RDCs P. W. (2007) J. Am. Chem. Soc. 129, 5340-5341 is fundamental to define the correct molecular structure of Prednisone. [3] Naumann, C., and Kuchel, P. W. (2008) J. Phys. Chem. A. 112, 8659-8664. ACKNOLEDGEMENTS: PRONEX/FACEPE/CNPq e UFPE [4] Philp, D. J., Naumann, C., and Kuchel, P. W. (2012) Concepts Magn. Reson. A 40, 90–99. TU143: Simple and accurate determination of X-H [5] Puckeridge, M., Chapman, and Kuchel, P. W. (2012) distances under ultra-fast MAS NMR Magn. Reson. Chem. 50, S17-S21. 1∗Piotr Paluch, 1Tomasz Pawlak, 2Jean Paul Amoureux, 1Marek J. Potrzebowski MO145: New Model of Electron Spin Polarization 1Polish Academy of Sciences, Centre of Molecular and in GaAs and AlGaAs/GaAs Arising from Optical Macromolecular Studies„ 2Unit of Catalysis and Chemistry Pumping and Detection by Optically-Pumped NMR of Solids (UCCS), CNRS-8181, University Lille North of 1Dustin Wheeler, 1Erika Sesti, 2Dipta Saha, 2Christopher J. France Stanton, 1∗Sophia E. Hayes We demonstrate that a very simple experiment, Cross- 1Washington University in St. Louis, 2University of Florida, Polarization with Variable Contact-time (CP- Gainesville VC), is very efficient at ultra-fast MAS (νR ≥ 60 kHz) to Optically-pumped nuclear magnetic resonance (OPNMR) is measure accurately the C-H and N-H distances, and to ana- a radio-frequency detection method to study the coupling lyze the dynamics of bio-molecules. This experiment can be of nuclei with both bulk conduction electrons and two- 13 15 performed with samples that are either C or N labeled dimensionally confined electrons in quantum wells. OP- or without any labeling. The method is very robust exper- NMR takes advantage of the hyperpolarization of the nu- imentally with respect to imperfect Hartman-Hahn setting, clei through the Fermi-contact interaction with electrons that and presents a large scaling factor allowing a better dipolar have been excited by laser light. Our experiments focus on determination, especially for long C-H or N-H distances. At both bulk GaAs and multiple-quantum well samples of GaAs, ultra-fast MAS, it can be used quantitatively in a 2D way, and we study the effects of photon energy on 69Ga OPNMR because its scaling factor is then little dependent on the off- signal intensities. sets. This robustness with respect to offset is related to the ultra-fast spinning speed, and hence to the related small rotor An ongoing line of inquiry involves the fundamental physical diameter. Indeed, these two specifications lead to efficient n interactions between optical selection rules, steady-state elec- = ±1 zero-quantum Hartman-Hahn CP-transfers with large tron polarization, and transfer of that polarization to nuclei rf-fields on proton and carbon or nitrogen channels, and large in the impacted sample area. We have made progress on a dipolar scaling factor. model for predicting OPNMR signal intensity as a function of input photon energy, including insights into electron spin polarization. Exotica Electron polarization is calculated at a range of photon en- ergies. This photon-energy dependent and variable quantity is informative because it allows us to more accurately model TH144: New findings with NMR of stretched hydro- the dependence of the OPNMR signal on optical pumping gels: peculiar urea resonance splittings and fitting wavelength. z-spectra

∗Philip W. Kuchel, Christoph Naumann, Max Puckeridge, TU146: Measuring Bipartite Quantum Correlations Bogdan E. Chapman in NMR Systems

111 Poster Abstracts – Instrumentation

1Isabela Almeida Silva, 3Davide Girolami, 2Ruben Auc- the quantum state tomography procedure. The experiments caise, 2Roberto Silva Sarthour Júnior, 2Ivan dos Santos were performed on a Varian 500 MHz (11.7 T) spectrometer Oliveira, 1Tito José Bonagamba, 1∗Eduardo Ribeiro de at 25 ◦C. We observed the dynamics transition of the sys- Azevêdo, 3Gerardo Adesso, 1Diogo O. Soares Pinto tem between a linear to a nonlinear regime. This evolution 1Universidade de São Paulo, Instituto de Física de São Car- is interpreted using the Josephson Junction model, which is los, São Carlos-SP, Brazil, 2Centro Brasileiro de Pesquisas commonly applied in superconductivity. We achieved that Físicas, Rio de Janeiro-RJ, Brazil, 3University of Notting- behavior when we varied one of the quadrupolar Hamilto- ham, School of Mathematical Sciences, Nottingham, United nian parameters. The change in the system dynamics lead Kindon to a supercritical pitchfork bifurcation that we were able to evaluate and to show the quantum control in the NMR spin How quantum states differ from classical states? For a long scenario. time, entanglement was considered the only responsible for that difference. When it was demonstrated that it is not AKNOWLEDGEMENTS: IFSC/USP, FAPESP, FAPERJ, possible to produce entanglement in conventional room tem- CNPq, and CAPES. perature liquid state NMR, the use of the technique as a test bench for quantum information processing (QIP) was severely criticized. This last until it was shown that separable Instrumentation states, as found in most of the NMR implementations, may present other types of non-classical correlations, for instance, the quantum discord proposed by Ollivier and Zurek. Besides MO148: Multi-Band Automatically Tunable High- a time costly quantum state tomography process, the experi- Sensitive Nuclear Quadrupole Resonance Spectrom- mental evaluation of the quantum discord involves large num- eter ber of mathematical manipulations. Because of that, other alternative discord measures have been proposed. For a two 1,2∗Lucas Matías Cerioni, 1,2,3Daniel José Pusiol qubit system, Girolami and Adesso proposed the quantity 1SPINLOCK SRL, Córdoba, Argentina, 2Argentinian Na- Q(ρ), which allow the calculation of the so called geomet- tional Research Council, 3Instituto de Física Enrique Gavi- ric quantum discord DG, and Nakano et al. defined another ola, CONICET, Ciudad Universitaria, Córdoba, Argentina discord measurement, named the negativity of quantumness A QN . We demonstrate that in NMR systems both quantifiers Nuclear quadrupole resonance (NQR) is a nondestructive, are calculated from spin correlations, which are measured di- highly specific, noninvasive spectroscopic technique. NQR rectly from the magnetization of one nucleus (qubit) after can be used to detect signals from solids and its parameters applying a specific pulse sequence. The equivalency between are highly sensitive to local environment changes. These fea- A direct and tomography based measurements of DG and QN tures make NQR a powerful technique for identifying and is then demonstrated using a two-qubit NMR system imple- study the properties of different polymorphs as well different mented by J-coupled 1H and 13C nuclei. Thereafter, we apply hydrates forms in pharmaceutical agents, providing effective these quantities to determine the relative amount of quantum assistance at the main steps of drug development, manufac- correlations present in two-qubit pseudo-pure states typically turing process and quality control. used in NMR QIP. At last, NMR is used to experimentally observe that, for some selected states and finite time period, In this work we present the development and implementation the amount of discord is preserved even under the presence of a high sensitive pulse NQR spectrometer, capable of mea- of relaxation (freezing of quantum correlations). We further suring in different frequency bands, allowing the scan of new observe the phenomenon of sudden change in the relaxation NQR signals in a fast, automatic and unattended way. behavior of quantum correlations as measured by DG and A The spectrometer was developed for measuring in two dif- QN . ferent ranges: 1.9-4.1 MHz (14N) and 34-36 MHz (35Cl). In ACKNOWLEDGMENTS: FAPESP, FAPERJ, CAPES order to make the spectrometer capable of measuring in both bands, a two capacitively coupled high-Q coils probe-head TH147: Observation of classical bifurcation in a was built. To cover the whole bandwidth, the high-Q tuning 133Cs quadrupolar NMR system and matching capacitors are mechanically adjustable. The spectrometer also includes a full bandwidth Q-damper sys- 1Arthur Gustavo de Araújo Ferreira, 2Ruben Auccaise tem; a switchable transmitter filters; and a sample temper- Estrada, 3Roberto Silva Sarthour Júnior, 3Ivan dos Santos ature control. The performance of the two-coil circuit was Oliveira Júnior, 1∗Tito José Bonagamba, 3Itzhak Roditi evaluated and compared with the standard single coil config- 1Instituto de Física de São Carlos - Universidade de uration. São Paulo, 2EMBRAPA Solos - Rio de Janeiro, 3Centro The tuning and matching capacitors are adjusted by a fast Brasileiro de Pesquisas Físicas auto-tuning algorithm, feedbacked by reflected power. Q- The investigation of classical bifurcation on quantum sys- damper frequency band and power amplifier filters are also tems has been the focus of many theoretical and experi- fast and automatically adjusted. This allows making a full mental studies. This behavior is typically addressed in the frequency sweep, performing measurements on the whole context of Bose-Einstein condensates (BEC) systems. Bi- range of interest in a complete automatic way. furcation is a signature of phase transition between different regimes, thus the interest in such phenomena. In this work, The spectrometer sensibility was tested using the stan- 14 we use a quadrupolar NMR system based on 133Cs nuclei dard samples hexamethylenetetramine (hmt) for N, and 35 with spin I=7/2 to study the bifurcation behavior in analogy p-diclorobenzene (pdb) for Cl. The spectrometer allows to what happens in BEC systems. A two-mode BEC can detecting 19 mg of hmt in 30 minutes and 7 mg of pdb in 1 be described via the Schwinger pseudo-spin representation minute. and entirely mapped onto a quadrupolar NMR Hamiltonian, enabling us to study this type of dynamics using NMR quan- The spectrometer was tested reconstructing the previously tum information processing tools. For observing the classical reported spectra of pharmaceutical samples including carba- bifurcation we used a lyotropic liquid crystal sample com- mazepine, furosemide and hydrochloro-thyazide. Unreported posed of cesium-pentadecafluorooctanoate. The 133Cs NMR lines were found in diclofenac sodium, aripiprazole and clo- experiments were performed using specially designed radio- pidigrel bisulfate. No NQR signal of these last two samples frequency pulses in order to implement spin coherent states had been reported before. as initial states. Next, appropriate evolutions were imple- ACKNOLEDGEMENTS: CONICET, FONTAR, SPIN- mented to achieve the desired linear or non-linear regime. LOCK Finally, the read out of the quantum state was done using

112 Poster Abstracts – Medicinal Chemistry

TU149: A Cryogen-Free 300 MHz Desk-Top NMR effect on nanodiamonds for the sake of improving NVCs con- Magnet for Industrial Applications centration in nanodiamonds. Nanodiamonds generally con- ∗ tain sub-ppm nitrogen atoms as impurity and annealing with Jonathan Cole, Steven J. Green, Eugeny Kryukov, Jeremy vacancies results in NVCs. However, because only few va- A. Good cancies exist in commercially available nanodiamonds, it is Cryogenic Ltd. necessary to create more vacancies by ion irradiation pro- cess to increase NVC concentration in nanodiamonds. We Since the 1990s, Cryogenic Ltd. have been developing ver- explored various irradiation conditions with H+, He+, Li+ satile experimental platforms based on cryogen-free magnets and N+ ions and tested the effect on the NVC concentration and variable temperature inserts for measurements at high in nanodiamonds. magnetic field (< 20 Tesla) and low temperature (> 30 mK) for research in condensed matter physics. MO151: Development of an Ultra-High-Power More recently, this proprietary technology is being adapted H/C/N NMR Probe for Membrane Proteins to magnetic resonance. In the mid-2000s, Cryogenic built several cryogen-free EPR magnets at 9 to 14 tesla, thereby ∗F David Doty, John Staab, George Entzminger, JB helping to launch the field of mm-wave EPR spectroscopy [1, Spitzmesser, Daniel Arcos, Laura Holte, Paul Ellis 2, 3]. Then, the first cryogen-free 600 MHz wide bore mag- Doty Scientific Inc net was developed for NMR studies involving quadrupolar- broadened lines up to tens of MHz wide [4, 5]. Last year, the Available methods for molecular structure determination company installed the most compact cryogen-free 1.5 tesla have had limited success on the insoluble proteins that are pre-clinical MRI magnet of its type in the world [6]. critical to biological function, though various recent de- velopments have enhanced the effectiveness of solids NMR Here, we describe a cryogen-free 300 MHz narrow bore NMR methods incorporating Magic Angle sample Spinning (MAS). system that may be operated on an office desk or laboratory However, static (non-MAS) high-power methods, such as bench. The magnet is suspended in vacuum inside a radia- PISEMA, have been equally fruitful thus far in yielding struc- tion shield held at 40 K and is kept at 3.5 K by a pulse tube tures of large, complex, helical membrane proteins. Prelim- cryocooler rated to deliver 0.5 W of cooling at low tempera- inary work from several leading research groups has demon- ture. The cooler consumes no helium and works on a closed strated the value of advanced 3D methods that could not cycle driven by a 3.5 kW compressor connected to 3-phase be carried out at high fields using commercially available mains electricity. Little cryogenic experience is required to probes because large samples are needed with very high rf operate the magnet which may be safely sited in limited space field strengths at 1H, 13C, and 15N simultaneously. A novel without quench ducting. Following on-site installation, the 1H resonator and novel circuits were developed and demon- magnet is left to cool overnight; it may also be left switched strated in a 900 MHz narrow-bore probe that provided order- off when experiments are not being run. The cryocooler cold- of-magnitude reduction in RF sample heating compared to heads have proven to be very reliable, requiring infrequent single-coil circuits, along with substantial improvements in maintenance at low cost. each of the remaining three most important and technically Our compact cryogen-free desk-top system offers industrial demanding specifications simultaneously: RF field strength, researchers a practical solution for high field NMR spec- spectral resolution, and S/N. The probe also included a gradi- troscopy with low overall cost of ownership. ent coil to permit novel methods in static NMR experiments, including solvent and background suppression, coherence se- [1] T.I. Smirnova et al., in ESR Spectroscopy in Membrane lection, and diffusion experiments. The gradient coil was Phys., 2005 tested to 157 G/cm in the 21.1 T magnet and demonstrated [2] A.I. Smirnov et al., Rev. Sci. Instrum., 2006 excellent linearity, efficiency, and recovery time. The tests in the 900 MHz magnet at the NHMFL in Tallahassee in- [3] E.J. Reijerse et al., JACS, 2010 cluded CP experiments for 1H/13C, 1H/15N, double CP, and [4] J.W. Harter et al., Phys. Rev. B, 2007 gradient-based coherence selection. The maximum power lev- els available were 320 W 1H, 700 W for 13C, and 1100 W for [5] L.K. Alexander et al., Phys. Rev. B, 2010 15N. The probe demonstrated superior efficiency and had no difficulty handling these powers under CP conditions, as the [6] J.A. Good et al., ENC 2012 1H is on an outer resonator while the 13C and 15N are on a doubly balanced inner solenoid. The probe is expected to be TH150: Selective Imaging of Fluorescent Nanodia- able to handle at least 40% higher power on all channels. monds and Study of Ion Irradiation Effect on Nan- odiamonds Acknowledgements: 5R44 GM079888-03 1Shingo Sotoma, 1Ruji Igarashi, 2Yosuke Yoshinari, 2Hiroaki Yokota, 1Hidehito Tochio, 2Yoshie Harada, 1,2∗Masahiro Shirakawa Medicinal Chemistry 1Graduate School of Engineering, Kyoto University, 2Instisute for Integrated Cell-Material Science (WPI- iCeMS) TU152: NMR-Based screening exposes new frag- ment detection possibilities at the pVHL:HIF-1α Fluorescent imaging is a powerful tool for visualizing intra- protein-protein interface cellular event. Recent development of this technique has enabled us to investigate the localization and dynamics of 1David M. Dias, 1Inge Van Molle, 1Matthias Baud, 1Carles bio-molecule in real time at a single molecule level. How- Galdeano, 2Carlos F. G. C. Geraldes, 1∗Alessio Ciulli ever, such sensitive detection is often hampered by intrinsic 1Chemistry Department, University of Cambridge, Cam- fluorescence arising from endogenous molecules. Techniques bridge, United Kingdom, 2Department of Life Sciences, Fac- which reduce intrinsic fluorescence have been investigated but ulty of Science and Technology, Centre for Neurosciences there are no ways to shut that out perfectly. Here, we de- and Cell Bi scribe a method for the selective imaging of nitrogen-vacancy center (NVCs) in nanodiamonds. This method relies on the The growing interest in Protein-Protein interactions and In- property of NVCs that the fluorescence intensity sensitively terfaces (PPIs) has been hampered by a lack of lucid methods depends on the ground state spin configuration which can capable of exploring its ligandability. Modulation of large be regulated by inducing electron spin magnetic resonance. multiprotein complexes has remained incredibly challenging Here, we present a technique for selectively imaging NVCs of and the relative low hit rates have encouraged other ap- diamonds in real-time. We also studied the ion irradiation proaches e.g. peptidomimetics to assist lead discovery against

113 Poster Abstracts – Natural Products and Small Molecules

PPIs. However, is the classical fragment-based biophysical anticancer activity. This information is important to under- screening set-up able to prime and command this challenge? stand the form of interaction of this compound with DNA and with enzymes of microorganisms. Here, we investigate the ability of NMR-based screening tech- niques to guide a hit-to-lead optimization campaign at von The molecule 9,10-phenantrenequinone guanylhydrazone has Hippel-Lindau protein/hypoxia inducible factor 1a interface a C(NH2)2 terminal group allowing the rotation of the (pVHL:HIF-1a). The recently disclosed first lead-like small amines. The structure was studied by NMR under differ- molecules disrupting this interaction were de-fragmented to ent temperatures and the results confirmed that the amines assess the minimal pharmacophore for the interaction since rotation angle is achieved when the compound is submitted pivotal components of the inhibitor’s binding affinity had to a temperature above 33 ◦C. This result was confirmed by been mostly elusive to binding detection by standard screen- molecular modeling. ing procedures. The interaction of this compound with oligonucleotides was Isothermal Titration Calorimetry (ITC) experiments further studied by NMR using relaxation time (T1 and T2), diffu- defined the range of detection for the fragments. Fascinat- sion coefficients (D) and chemical shifts, indicating that it is ingly Ligand-based NMR techniques (STD, CPMG and Wa- a DNA ligand. terLOGSY) accommodate the entire range of affinity con- trasting with ineffective X-ray screening efforts. AKNOWLEDGEMENTS: CAPES, INBEB and Military In- stitute of Engineering. We postulate that NMR is an invaluable tool to detect binders with such affinity and ligand efficiencies as found be- tween protein surfaces and small molecules, contributing to Natural Products and Small Molecules higher hit rates at PPIs.

TH153: Interaction study of Bovine Serum Albumin TU155: The Keto-Enol-Equilibrium of Tetra- (BSA) with guanylhydrazones by Nuclear Magnetic Acetones Resonance 3Queli Aparecida Rodrigues de Almeida, 2Raphaela Teresa ∗ Denise Cristian Ferreira Neto, José Daniel Figueroa- Sant’Anna, 1Raquel Pantoja Barreiros Rodrigues, 1Marcela Villar, Sirlene O. F. Azeredo, Elaine da C. Petronilho Christina Oliveira Nogueira, 2Sérgio de Paula Machado, Military Institute of Engineering, Brazil. 2Roberto de Barros Faria, 1∗Jochen Junker 1 2 3 The interaction between biomolecules and ligands has Fundação Oswaldo Cruz - CDTS, UFRJ - IQ, IFRJ - IQ aroused interest, being fundamental for design and evaluation The dimerization of di-acetones to tetra-acetones is a key of new potential drugs. In the last decades many researches step for the synthesis of highly substituted pyrrols. The have been involved on developed of procedures, including on tetra-acetones like 3,4-diacetyl-hexane-2,5-dione are not only NMR, to study how these interactions occurs. Albumin is very symmetrical, they also feature two highly acidic protons, a important protein, and its main function is transport and which could form six membered rings if in the enol-form of the binding different substances, including drugs, in the blood. molecule. Further reactivity suggests that that tetra-acetone Substances that interact strongly with albumin present diffi- is mainly in enol-form, but this contradicts the literature. culty in their pharmacological function. However, molecules 1 that do not interact with the albumin are likely to be de- Different from older literature, we could observe from H stroyed by other proteins present in blood. Therefore, to NMR that in most solvents the enol-form is actually favored display appropriate action, the drug must provide a medium over the keto-form. This also has been confirmed by DFT interaction with albumin, a process that have been used by calculations considering different solvents. NMR with many molecules. Our research group developed inedited guanylhydrazones as new potent antibiotics, which TH156: NMR Crystallography: Chemical Shift are effective against different bacteria. These compounds dis- Driven Crystal Structure Determination of the An- play good potential for treatment of intoxication with antibi- tibiotic Amoxicillin Trihydrate otic resistant Gram negative bacteria, especially against E. coli ST131. In this work we study the interaction of bovine ∗Sérgio M. Santos, João Rocha, Luís Mafra serum albumin (BSA) with guanylhydrazones, via nuclear Department of Chemistry, CICECO, University of Aveiro, magnetic resonance (NMR), using as experimental measure- Portugal ments methods the effect on relaxation times (T1 and T2), saturation transfer difference (STD), nuclear Overhauser ef- The recent developments in the field of solid-state NMR spec- fect (NOE), and diffusion coefficients by diffusion ordered troscopy (ssNMR) has lead to the emergence of NMR crys- spectroscopy (DOSY). Preliminary results by NMR using the tallography in which ssNMR data is used to derive the 3- dimensional crystal packing of powdered samples. In this techniques described, showed appropriate changes on T1 and communication, we report a new strategy for NMR crystal- T2 of guanylhydrazones interacting with albumin. These lography of a molecular solid, in which ssNMR data, in the results are confirmed by diffusion coefficient variations and 1 STD, indicating the interaction ligand-BSA. The preliminary form of H chemical shifts, enter directly in a structure gen- results indicate that tested guanylhydrazones are potentially eration step, and are further used in a refinement step as stable and transported drugs bacterial infections and cancer. pseudo-forces acting on the models, driving them towards the final structure. This procedure was successfully applied Acknowledgments: CAPES, IMBEB and Brazilian Army. to powdered amoxicillin trihydrate, a widely used β-lactamic antibiotic. MO154: Conformational Analysis of 9,10- The methodology takes, as input, unit cell parameters, space Phenanthrenequinone Guanylhydrazone by Nuclear 1 group information and solid-state H chemical shifts, and Magnetic Ressonance (NMR) and Molecular Mod- yields a final refined structure compatible with the input elling. data. Processing of the data proceeds in 3 steps: (i) gen- Clara Simões, ∗José Daniel Figueroa-Villar, Sirlene O. F. eration of an ensemble of structures; (ii) refinement of the Azeredo generated structures; (iii) energy minimization of the refined structures. Step (i) employs a genetic algorithm where the fit- Military Institute of Engineering, Brazil. ness of each structure is given by Etotal = Elattice+Eδ, where In this work we studied the conformational changes as a Elattice is given by the General Amber Force Field (GAFF) temperature function in 9,10-phenantrenequinone guanylhy- force field and Eδ is a term accounting for the deviation of the drazone, a novel compound with potential antibiotic and on-the-fly-calculated from the experimental chemical shifts.

114 Poster Abstracts – Natural Products and Small Molecules

The genetic algorithm yields, in step (i), a set of generated Smith, S. Yang, E. Holmes, H.R. Tang, G. Zhao, J. Nichol- structures, which moderately obey the input data. In step son, L. Li, L. Zhao, "Human symbiotic gut microbes specifi- (ii), these are refined by means of MD simulated annealing, cally modulate metabolic phenotypes"PNAS, 105:2117-2122, governed by forces Fi = -∂Etotal/∂ri. Finally, in (iii) the 2008. structures are energy minimized using tight-binding density [8] C. N. Xiao, H. Dai, H. B. Liu, Y.L. Wang, H. R. Tang, functional theory. In the whole process, Elattice is respon- "Revealing the metabonomic variation of rosemary extracts sible for the physical and chemical validity of the generated using 1H NMR spectroscopy and multivariate data analysis. structures, whereas Eδ is responsible for imposing the exper- Agric. Food Chem., 56:10142-10153, 2008. imental ssNMR restraints. [9] E. Holmes, R. Loo, O. Cloarec, M. Coen, H.R. Tang, Acknowledgements E. Maibaum, S. Bruce, Q. Chan, P. Elliottm, J. Stamler, We thank FCT for financial support to project I. Wilson, J. Lindon, J. Nicholson, "Detection of urinary PTDC/QUIQUI/100998/2008 and for the post-doc grant drug metabolite (Xenometabolome) signatures in molecular SFRH/BPD/64752/2009. This work was financed by epidemiology studies via statistical total correlation (NMR) FEDER, via ”Programa Operacional Factores de Compet- spectroscopy", Anal. Chem., 78:2629-2640, 2007. itividade – COMPETE”, and by FCT (Portugal), PEst- [10] E. Holmes, H.R. Tang, Y.L. Wang, C. Seger, "The assess- C/CTM/LA0011/2011. ment of plant metabolite profiles by NMR- based methodolo- gies", Plant. Med. 72:771-785, 2006. MO157: Integrated NMR-MS Approaches for Dis- covering New Metabolites and Metabotyping TU158: Studying the gem-diol generation in imida- zole and pyridine aldehydes derivatives using NMR ∗Huiru Tang, Yulan Wang and X-ray crystallography Chinese Academy of Sciences, Wuhan 1,2Juan Manuel Lázaro Martínez, 3Daniel Vega, Metabolism represents all biochemical changes in biological 1,4Gustavo Alberto Monti, 1,4Ana Karina Chattah, processes and is the basic feature of living systems. Anal- 2∗Graciela Yolanda Buldain ysis of the metabolite composition (metabonome) is there- 1 2 fore an essential aspect of molecular phenotyping. The IFEG-CONICET, Universidad de Buenos Aires, 3 4 integrated NMR-MS techniques have shown great poten- Comisión Nacional de Energía Atómica, Universidad Na- tial for new metabolite discovery and metabolic phenotyp- cional de Córdoba ing (metabotyping). In this presentation, we will report The adduct formed upon addition of water to an aldehyde some recent progresses in developments of the combined or ketone is called a hydrate or germinal diol (gem-diol). NMR/LC-MS metabonomic analytical methods for under- These compounds are rarely stable (and only in aqueous standing the metabotypic alterations induced by various ex- solution),i.e., the equilibrium is greatly dependent on the posomic stresses. We will report some discoveries of various structure of the hydrate. Thus, formaldehyde in water at new plant secondary metabolites with novel skeletons and 20◦C exists 99.99% in the hydrated form, while for ac- the combined NMR-MS approaches in conjunction with DFT etaldehyde this figure is 58%, and for acetone the hydrate calculations will be discussed for de novo absolute structure concentration is negligible. For that reason, the few stable determination for these newly discovered natural products. crystalline hydrates known (such as polychlorinated aldehy- We will further discuss current bottleneck of metabonomic des) are those that have a strongly electronegative group analysis techniques and possible future developments in the associated with the carbonyl group since, in general, the NMR-based metabotyping methods with the effectiveness of hydrates can seldom be isolated because they readily revert integrated metabonomic analysis particularly reflected. to the parent aldehyde. References: The aim of the present work was to study the existence and stability of the aldehyde-hydrate form of some pyridine or [1] Y. Tian, L.M. Zhang, Y.L. Wang, H.R. Tang, "Age-related imidazole carboxaldehyde derivatives commonly employed as topographical metabolic signatures for the rat gastrointesti- reagents in the synthesis of active compounds (formylimi- nal contents", J. Proteome Res. 11:1397-1411, 2012. dazoles and formylpyridines), using solution- and solid-state 13 1 13 [2] H.B. Liu, A.M. Zheng, H.L. Liu, H.Y. Yu, X.Y. Wu, C.N. NMR experiments ( C CP-MAS and H- C HETCOR). In Xiao, H. Dai, F.H. Hao, L.M. Zhang, Y.L. Wang, H.R. Tang, addition, some of the derivatives presented here were studied "Identification of three novel polyphenolic compounds, orig- through single-crystal X-ray crystallography. anine A-C, with unique skeleton from Origanum vulgare L. In particular, the 2-formylimidazole hydrate is a stable crys- using the hyphenated LC-DAD-SPE-NMR/MS methods", J. talline substance because, in order to revert to the aldehyde- Agric. Food Chem., 60:129–135, 2012. form, a water molecule must be left out, and this is dif- [3] H. Dai, C.N. Xiao, H.B. Liu, H.R. Tang, "Combined ficult by the electron-withdrawing character of the imida- NMR and LC-MS analysis reveals the metabonomic changes zolium cation. That was probed since the 2-formylimidazole in Salvia Miltiorrhiza Bunge induced by water depletion", J. in D2O-TFA solution evolved completed to its hydrated Proteome Res., 9:1460–1475, 2010. form (100%), however, the 4-formylimidazole and 4-methyl- 5-formylimidazole existed 40% and 10% in the hydrated form, [4] H. Dai, C. Xiao, H. Liu, F. Hao, H.R. Tang, "Com- respectively. bined NMR and LC-DAD-MS analysis reveals comprehen- sive metabonomic variations for three phenotypic cultivars Interestingly, single crystal for the hemiacetal derivative was N of Salvia Miltiorrhiza", J. Proteome Res. 9:1565–78, 2010. obtained from -methyl-2-formylimidazole in TFA solution and confirmed by X-ray diffraction techniques. In addition, [5] H.R. Tang, C.N. Xiao, Y.L. Wang, "Important roles the hydrate and hemiacetal form coexisted in the solid, after of the hyphenated HPLC-DAD-MS-SPE-NMR technique in the completely removal of the TFA, according with the 13C metabonomics", Magn Reson Chem, 47:S157-S162, 2009. CP-MAS results. [6] C. Xiao, H. Liu, H. Dai, L. Tseng, M. Spraul, Y. TH159: Complete 1H and 13C NMR Spectral Data Wang, H.R. Tang, "Rapid and efficient identification of some Assignment of a new Nemorosinic Acid from Clusia rosemary metabolites using HPLC-DAD-SPE-CryoNMR-MS Nemorosa (Clusiaceae) method", Chin. J. Magn. Reson., 26:1-16, 2009. 1∗Mário Geraldo de Carvalho, 1Rafaela Oliveira Fer- [7] M. Li, B. Wang, M. Zhang, M. Rantalainen, S. Wang, H. 2 Zhou, Y. Zhang, J. Shen, X. Pang, M. Zhang, H. Wei, Y. reira, Tania Maria S. da Silva Chen, H. Lu, J. Zou, M. Su, Y. Qiu, W. Jia, C. Xiao, L. 1Universidade Federal Rural do Rio de Janeiro,

115 Poster Abstracts – Natural Products and Small Molecules

2Universidade Fedeal Rural de Pernambuco (CC) yielded eight groups of the fractions after TLC analy- Clusia nemorosa Mey. is a tree widespread in the northeast sis. The A7 group was subjected to CC with flash silica gel region of Brazil, where it is popularly known as ’pororoca’. and the fractions were analyzed by TLC and reunited in 4 groups. The fraction A73 furnished a white solid that was The previous studies of this species described some polyiso- 1 13 prenylated benzophenones and alkylarylketones, known as analyzed by H and C NMR spectral data to define the nemorosinic acids.[1] The phytochemical investigation of the structure which molecular formula was confirmed by ESI- dichloromethane extract from fruits ofC. nemorosa allowed MS. The dammarane triterpene skeleton was proposed by analysis of 1D and 2D NMR experiments, including 1H and the isolation of a yellow gum identified as a new polyisopreny- 13 lated ketone, named as nemorosinic acid C (1). The complete C NMR (DEPTQ and DEPT-135 and DEPT-90), COSY, proton and carbon NMR assignment of 1 was accomplished HSQC and HMBC pulse sequences, besides comparison with by 1D and 2D NMR spectra analysis, including COSY, literature data of different part of the structure.[1,2,3] 30 mg HSQC and HMBC sequences. 30 mg of 1 were dissolved in of the saponin were dissolved in pyridine-d5, placed in a 5 mm NMR tube to obtain the spectra with a Bruker Avance MeOD4, placed in a 5mm NMR tube, and the spectra were 1 13 obtained with a Bruker Avance III 500 spectrometer [500.13 III 500 spectrometer (500.13 MHz for H and 125.77 for C). MHz for 1H and 125.77 for 13C]. NMR spectra analysis of 1 In the analysis of these spectra, besides the number of CH3, and comparison of the data with those of nemorosinic acid B1 CH2, CH and C carbon, the chemical shift values detected in 1 13 1J suggests a similar structure with a hydroxyl-isopropyl-furan the H and C NMR spectra, the corresponding HC in the 2,3J group. The molecular formula was deduced as C31H44O7 HSQC, and the connection by HC of methyl group with δ from its [M-H] at m/z 527 in the MS spectrum. Character- CH-3, -5, -9, -13, -17 ( CH 88.7, 56.2, 50.3, 42.3, 50.8, re- δ istic 13C NMR resonances for a six-member ring consisting spectively) and C-10, -8, -14, -20 and -25 ( c 36.8, 40.4, 50.5, of two carbonyl groups [δ 205.5 (C-6) and 191.8 (C-2)], and 73.8, 131.9, respectively) allowed to define the dammarane δ β three sp2 carbons [δ 109.6 (C-3), 170.3 (C-4) and 103.4 (C-5)] skeleton. Besides the CH2 75.06, three ,D-glucopyranoside besides one quaternary carbon [δ 60.8 (C-1)]. The 1H NMR were in agreement with other oxygenated carbons chemical 2,3J δ spectrum exhibits signals at δ 4.79 (H-23) of double bond, shift values. The HC of H-1’, with CH-3 ( CH 88.7), H- δ δ two vinylic methyl groups [δ 1.55 (H-25) and 1.48 (H-26)] and 1" with CH2-6’ ( CH2 69.7), and H-1’" with CH2-26 ( CH2 Na+ two allylic protons [δ 2.52 (H-22)], indicating the presence 75.06). All of these data, and the m/z 969.5384 (M + ) of one isopent-2-enyl group. Additionally, NMR analysis and 945.5463 (M - H), are in agreement with the molecular shows signals for one carboxyl function [δ 172.2 (C-21)], four formula C48H82O18, of this new dammarane saponin, identi- β vinyl carbons [δ 145.7 (C-14), 114.1 (C-15), 142.5 (C-18) and fied as 20-hydroxydammar-24-ene-3-O- -D-gentiobiosyl-26- β 127.5 (C-19)], two vinylic methyl group [δ 17.3 (C-16) and O- -D-glucopyranosyde, and allowed to make the complete 12.3 (C-20)], two allylic methylene carbons [δ 40.4 (C-12) proton and carbon-13 chemical shift assignment. and 33.2 (C-17)] and one methine carbon [δ 44.2 (C-13)], References: [1] Hu L., Chen Z., and Xie Y. J. Nat. Prod. which correspond to the presence of an oxidized lavandulyl 1996, 59, 1143-1145. [2] Orihara Y. and Furuya T. Phyto- chain. The cross peaks, observed in the HMBC spectrum, chemistry, 1993, 34|4| 1045-1048. [3] Ky P. T., at all Phyto- between methylene protons at C-22 (δ 2.52) and at C-12 (δ chemis try 2010, 71, 994-1001. 2.24 + 2.11) with C-1 (δ 60.8) indicated that the isopentenyl and oxidized lavandulyl chain are attached to quaternary Acknowledgment: FAPERJ / CNPq / CAPES / CPP carbon C-1. The COSY, HMQC and HMBC data indicated and INAU-MT. the involvement of H3C-10,11 with C-9 (d 71,8) and CH-8(d 92,7) in a 2-(2-hydroxypropyl)-dihydrofuran ring. The cross TU161: How useful is Inclusion of 13C NMR Data peaks between methylene protons at C-7 (δ 2.91) and C-3 into an Automated Structure Verification Algo- (δ 109.5) and C-4 (δ 170.3) indicated that the dihydrofuran rithm? ring is formed with C-3 and C-4. The additional signals at 196.5 (C), 40.7(CH), 26.6(CH2) and 11.9, 17.2 of two CH3 1∗Sergey Golotvin, 1Rostislav Pol, 2Patrick Wheeler justify the 1-hydroxy-1-iso isopenthylene group (C-27–C-31). 1ACD Moscow, Moscow, Russia, 2ACD/Labs, Toronto, ON Thus, this analysis allowed to make the complete 1H and 13 Automated Structure Verification (ASV) using the combina- C NMR chemical shift assignment of 1, identified as a new 1 1 13 compound named nemorosinic acid C. tion of 1D H and 2D H- C single-bond correlation spec- trum such as HSQC or HMQC is continuing to gain interest REFERENCE as a routine application for qualitative evaluation of large compound libraries produced by synthetic chemistry [1]. [1] Monache, F. D.; Monache, G. D.; Gács-Baitz, E. Phyto- chemistry, 1991, 30 (2), 703-705. However this method is not always well suited for hetero- cycles or condensed aromatic structures; they offer a very CNPq/CAPES/FAPERJ. limited number of correlations in HSQC spectra, and much spectral information is concentrated in unprotonated carbon 1 13 MO160: Complete H and C NMR Spectral Data positions. Ongoing progress in cryoprobe development and Assignment of New Dammarane Saponin Isolated deployment has considerably shortened the time necessary from Siolmatra brasiliensis (Cogn.) Baill (CUCUR- to acquire directly detected 1D 13C spectra. The progress BITACEAE) makes it possible to acquire 13C data in semi-routine fashion and thus expand the NMR dataset used for ASV. 1∗Mário Geraldo de Carvalho, 1Carlos Henrique Corrêa dos Santos, 2Virginia Cláudia da Silva, 2Paulo Teixeira de This study highlights the advantages and disadvantages of Sousa Júnior addition of 1D 13C data to the combination of 1D 1H and 1Universidade Federal Rural do Rio de Janeiro, 2D HSQC spectra for ASV activity. The overall performance 13 1 2Universidade Federal de Minas Gerais of expanded NMR datasets ( C, H and 2D HSQC) is eval- uated against "standard" (1D 1H and 2D HSQC) combined Siolmatra brasiliensis (Cogn.) Baill is popularly known as datasets using commercially available compounds. For com- "taiuia" or "cipó taua" and often occurs in the central region parison of accuracy of verification, false structures of spec- of Brazil. The plant material was collected in Jangada, Mato troscopically similar isomers are generated by human and by Grosso State, Brazil. A voucher specimen is deposited at the software [2]. Of special interest are such performance met- herbarium of the Federal University of Mato Grosso do Sul, rics as the False Negative rate, in which correct structures MS, Brazil, under the number CGMS: 31.643. The fraction- are improperly rejected, and the False Positive rate, in which ation by solvent partition of the hidroalcoholic extract from improper structures are passed through spectroscopic filters. the stem of this plant yielded the chloroform (SBCMC), ethyl acetate (SBCMA), and methanol (SBCMM) frac- [1] "Automated structure verification based on a combina- tions. The fractionation of SBCMA on a silica gel column tion of 1D 1H NMR and 2D 1H13C HSQC spectra", MAG-

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NETIC RESONANCE IN CHEMISTRY; Volume 45, Issue and increasingly in metabolomics studies, to characterise the 10, October 2007, Pages: 803–813, Sergey S. Golotvin, Eu- structural features of known and unknown metabolites in gene Vodopianov, Rostislav Pol, Brent A. Lefebvre, Antony samples. Here we present Seneca 2.0, an enhanced version of J. Williams, Randy D. Rutkowske and Timothy D. Spitzer Seneca 1.0 [1], a fully open-source java based cross-platform desktop application to perform stochastic CASE. The CASE [2] "Concurrent combined verification: reducing false posi- engine uses the Chemistry Development Kit (CDK) [2], an tives in automated NMR structure verification through the open source java library, for the chemical structure repre- evaluation of multiple challenge control structures", MAG- sentation and manipulation. We have implemented a new NETIC RESONANCE IN CHEMISTRY; Volume 50, Issue evolutionary algorithm scheme within Seneca 2.0, that per- 6, June 2012, Pages: 429–435, Sergey S. Golotvin, Rostislav forms successful CASE in a fully automated fashion. We will Pol, Ryan R. Sasaki, Asya Nikitina and Philip Keyes also present the evaluation of applying Natural product like- ness [3] - a score for a given molecular structure based on TH162: Progressive Structuring of a Branched An- a statistical model relying on the differences in occurrence timicrobial Peptide on the Path to the Inner Mem- of circular fingerprint of atoms in compound collections - in brane Target performing CASE. 1,2Bai Yang, 2Roger W. Beuerman, 1∗Konstantin Per- References vushin [1] Steinbeck C, SENECA: A platform-independent, dis- 1School of Biological Sciences, Nanyang Technological Uni- tributed, and parallel system for computer-assisted structure versity, 2Singapore Eye Research Institute elucidation in organic chemistry. J Chem Inf Comput Sci. In recent years, interest has grown in the antimicrobial prop- 2001 erties of certain natural and non-natural peptides. The strat- Nov-Dec;41(6):1500-7 egy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host bio- [2] Steinbeck, C., Han, Y. Q., Kuhn, S., Horlacher, O., compatibility. However, little is known regarding possible Luttmann, E., & Willighagen, E. structural transitions as the peptide moves on the access (2003). The Chemistry Development Kit (CDK): An open- path to the presumed target, the inner membrane. Estab- source Java library for lishing the nature of the interactions with the complex bac- terial outer and inner membranes is important for effective chemo- and bioinformatics. Journal of Chemical Information peptide design. Structure-activity relationships of an am- & Computer Sciences, phiphilic, branched antimicrobial peptide (B2088) are exam- 43(2), 493–500. ined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high reso- [3] Jayaseelan, K.V., Moreno, P., Truszkowski, A., Ertl, P., lution NMR in solution and in condensed states. The peptide & Steinbeck, C.(2011). Natural product-likeness score re- is reconstituted in bacterial outer membrane lipopolysaccha- visited: an open-source, open-data implementation. BMC ride extract as well as in a variety of lipid media mimicking Bioinformatics. 13:106. the inner membrane of Gram-negative pathogens. Progres- sive structure accretion is observed for the peptide in wa- TU164: Quantitative 1H NMR Applied to ter, LPS, and lipid environments. Despite inducing rapid Bioethanol Production aggregation of bacteria-derived lipopolysaccharides, the pep- 1Thiago I. B. Lopes, 2Antônio M. Júnior, 2Maria I. Berto, tide remains highly mobile in the aggregated lattice. At the 2 3 1∗ inner membranes, the peptide undergoes further structural Glauciane R.D. Silva, Daniel I.P. Atala, Anita J. Mar- compaction mediated by interactions with negatively charged saioli lipids, probably causing redistribution of membrane lipids, 1State University of Campinas – UNICAMP, 2Institute which in turn results in increased membrane permeability of Food Technology. Laboratory for Applied Microwave- and bacterial lysis. These findings suggest that peptides pos- LMA/GEPC, 3British Petroleum sessing both enhanced mobility in the bacterial outer mem- Greenhouse gas emissions and fossil fuel reservoir deple- brane and spatial structure facilitating its interactions with tion increase the biofuel production appeal. Fermentation the membrane-water interface may provide excellent struc- processes using yeasts to convert sugars to ethanol yield a tural motifs to develop new antimicrobials that can overcome mixture of ethanol, saccharides, yeast cells and unwanted antibiotic-resistant Gram-negative pathogens. byproducts (as lactate and acetate). Typically these pro- 1 References: cesses are monitored by HPLC, here quantitative H NMR was applied as an alternative method. The comparison be- 1. Bai, Y.; Liu, S.; Jiang, P.; Zhou, L.; Li, J.; Tang, tween quantitative NMR and the reference method (HPLC) C.; Verma, C.; Mu, Y.; Beuerman, R. W.; Pervushin, K., was performed using two sets of fermentation samples rep- Structure-dependent charge density as a determinant of an- resenting 8 hours of process monitoring. All samples were timicrobial activity of peptide analogues of defensin. Bio- analyzed in triplicate. The test showed that 1H NMR data chemistry 2009, 48 (30), 7229-39. are in good agreement with HPLC results and the comparison by linear regression and ethanol showed the best correlation 2. Bai, Y.; Liu, S.; Li, J.; Lakshminarayanan, R.; Sarawathi, (R = 0.9929; LOQ = 4.248 g·L−1), followed by glucose (R = P.; Tang, C.; Ho, D.; Verma, C.; Beuerman, R. W.; Per- 0.9831; LOQ = 7.055 g·L−1) and sucrose (R = 0.9791; LOQ vushin, K., Progressive structuring of a branched antimicro- = 3.207 g·L−1). Additionally lactate, acetate and pyru- bial peptide on the path to the inner membrane target. J vate were detected only by NMR method and these have Biol Chem 2012, 287 (32), 26606-17. quantification limits equal to 0.766; 2.316 and 0.291 g·L−1, respectively. The results demonstrated that 1H NMR can be MO163: Seneca 2.0: an open source, open data tool used to monitor: i) amount of produced ethanol, ii) amount for Computer Assisted Structure Elucidation of small of remaining sugars (sucrose and glucose) in the fermentation molecules. broth and iii) concentration of byproducts and intermediates (lactate, acetate and pyruvate). Present method was fully , Luis de Figueiredo, ∗Christoph Kalai Vanii Jayaseelan validated (regarding accuracy, precision, selectivity, linearity Steinbeck and robustness) and presented many advantages compared Chemoinformatics and Metabolism, European Bioinformat- to reference method: faster and larger number of monitored ics Institute (EBI), Cambridge, UK compounds. Nuclear Magnetic Resonance (NMR) spectroscopy is the ACKNOLEDGEMENTS: FAPESP, CAPES, e CNPq most informative analytical tool in natural product chemistry

117 Poster Abstracts – Natural Products and Small Molecules

TH165: Condensed Symmetry Structure: an ap- α-Santonin has 4 chiral centers, making it possible to draw proach for automatic assignment of NMR spectra sixteen diastereomers. The structures of these 16 dias- 1 2 3 4∗ teromers were optimized by molecular mechanic calculations Andrés Bernal, Andrés M. Castillo, Luc Patiny, Julien at the DFT level of theory, using Gaussian03. The B3LYP Wist functional along with the 6-311++G(d,p) basis set was 1Grupo de Química Teórica, Universidad Nacional de used. One-dimensional and two-dimensional NMR experi- Colombia, Colombia, 2Facultad de Ingeniería, Universidad ments were performed (1H, 13C, gCOSY, gHSQC, gHMBC, Nacional de Colombia, Colombia, 3Institute of Chemical J-Resolved and NOESY). Moreover, coupled gHSQC exper- Sciences and Engineering, Ecole Polytechnique Fédérale de iments in isotropic and anisotropic media were carried out. Lausanne, 4Chemistry Department, Universidad del Valle, PH gel was used as the alignment media. Colombia Analyzing J-coupling and NOE data made it possible to se- Full-search NMR assignment methods aim to score all pos- lect four suitable structures among the 16 diastereomers. Af- sible assignments of a structure to an observed spectrum in terwards, the RDC values in the assay were included. Com- order to choose the best. The main challenge of this meth- paring the Q values of these four structures made it possible ods consists in dealing with combinatorial explosion on the to select the correct structure. size of the solution space. We noticed that the suitability of an assignment on the light of any given property should In conclusion, RDC values are fundamental to achieve the remain invariant under permutations of nuclei/peaks with correct tridimensional structure of α-Santonin. the same property value. As a consequence, assignments dif- fering only by permutations of homotopic and enantiotopic ACKNOLEDGEMENTS: PRONEM/FACEPE/CNPq , nuclei are always equally adequate, since such nuclei have PRONEX/FACEPE/CNPq, UFPE identical magnetic properties. Thus, rather than assigning the full chemical structure of the candidate molecule, we can TU167: Candidate Structures Generated from assign a Condensed Symmetry Structure representation of Molecular Fragments that Match NMR Information the molecule (CSS) in which each vertex corresponds to a ∗ complete family of magnetically equivalent nuclei. This CSS A.K. Velez-Jurado, I. Tischer, Julien Wist contains the most relevant structural information that corre- Universidad del Valle sponds to the NMR spectra of the candidate molecule (some At present, the process of structural elucidation using nu- information related to multiplicity and chemical shift is lost). clear magnetic resonance spectra (NMR) is a tedious task. A second consequence is that the less discriminant a prop- There is a variety of work done in this field based on different erty is, the faster it rejects suboptimal assignments, thereby approaches, e.g MS-Molgen, NMRShiftDB and SPECINFO motivating a strategy where lowly-discriminant properties make use of databases of NMR spectra, LSD performs a are used first for quickly pruning the solution tree, whereas heuristic algorithm using logical rules and CASA validate highly-discriminant properties are introduced latter to nar- the structure suggested by LSD. GENIUS predicts all pos- row it down to a few solutions. As a proof of concept, we sible molecular structures given a chemical formula, while have been evaluating a procedure that uses proton integrals StrucEluc accelerates the process using CASE "Computer- for the early filter and 2D connectivities. We find that it Aided Structure Elucidation" technique. This technique con- is possible to efficiently discard most suboptimal solutions sists in taking as initial data any information available from to the assignment problem, even in presence of peak overlap the spectra obtained by NMR, IR and MS as multiplicity and artifacts, without any input other than the candidate and chemical shifts to generate a set of chemical fragments. structure, 1H integrals, and COSY, HSQC and HMBC con- Structures that satisfy these restrictions are generated and nectivities. Remarkably, in several cases a unique solution ranked by the system exhaustively, simulating the spectra to could be obtained without considering chemical shifts. compare with the initial spectrum. Here, we developed an approach for the automatic struc- MO166: Residual Dipolar Coupling as an addi- tural elucidation using artificial intelligence methods, that tional NMR tool for defining the configuration of α- build candidate structures compatible with NMR experimen- Santonin tal data. Therefore, we created a database of 2032 molecular 1∗Rubens Rodrigues Teles, 1Fernando Hallwass, 1,2José fragments obtained by taking apart 300 known molecules, Adonias Alves de França to ensures that only "chemically" appropriate candidate are formed. As a first step, the structural information is ex- 1Universidade Federal de Pernambuco, 2Instituto Federal de tracted from 1H, 13C, TOCSY and HSQC spectra. This in- Alagoas formation is processed and randomized by a Las Vegas algo- Recently, there have been developed a complementary rithm that searches the database for fragments compatible methodology to the traditional NMR experiments applied with these experimental data, connectivities and chemical to determine tridimensional structure or to solve assignment shifts. These are represented as a covariance matrix that questions of small molecules, namely RDC (Residual Dipo- match the molecular formula. Candidates are then submit- lar Coupling). This methodology is based on measuring ted to an algorithm of optimization randomly generates new anisotropic parameters, produced by partial molecular align- feasible solutions from the initial candidates with selected ment. Consequently, all geometrical dependent parameters of fragments and a weight function that is updated with the the NMR Hamiltonian are partially recovered, providing use- simulation of the spectra. Finally, the proton spectra are ful additional structural information, when the experiments simulated for the more promising candidates to rank them in isotropic media are not sufficient. according on their degree of similarity with the experimental The aim of this work is to show how 1H,13C RDC mea- data. surement, associated to the traditional NMR analysis, is fundamental to determine the correct stereochemistry TH168: Structure elucidation by NMR of a fu- of α-Santonin -(3S,3aS,5aS,9bS)-3,5a,9-trimethyl-3a,5,5a,9b- ranocoumarin from the extract of Simaba maiana tetrahydronaphtho[1,2-b]furan-2,8(3H,4H)-dione. The natu- Casar. (Simaroubaceae) ral compound α-Santonin belongs to the class of sesquiter- 1,2∗ 1 1 pene lactone, with anti-helminthic and antipyretic activity. Edson de Souza Bento, Isis Torres Souza, Carla Karine Barbosa Pereira, 1Erika Verena Figueiredo Cam- The literature reports that the β-isomer has pronounced an- 1 2 tipyretic activity, compared to the α-isomer. Moreover, this bui, Antônio Euzébio Goulart Sant’Ana, Geoffrey Ernest class of compounds has wide application as a precursor agent Hawkes in photochemical synthesis for the production of new prod- 1Universidade Federal de Alagoas, 2Queen Mary University ucts with biological activity. of London

118 Poster Abstracts – Natural Products and Small Molecules

Simaba maiana belongs to the family Simaroubaceae and is Brasil, 3Departamento de Ciências Naturais, Universidade commonly used to treat stomach ulcer, cancer, malaria and Federal do Estado do Rio de Janeiro, 4Departamento de gastrointestinals disorders. Previous studies with an ethano- Química, Instituto Militar de Engenharia, Brasil. Simaba maiana lic extract of have indicated a molluscicidal Keto-enol tautomerism is important in many fields of chem- Biomphalaria glabrata activity against . The compounds of istry and biochemistry to better understand the structure the chloroform sub-fraction were isolated by a semi prepara- of a compound, and it has been investigated for several re- tive HPLC system consisting of a C18 column (10 x 25 cm), search groups for a long time. Different studies applied gradient 60-100%B (30 min), 100-100%B (40 min) (A – ultra- to different systems have been shown that intra and in- pure water, B – methanol), with a injection volume of 2 mL −1 termolecular interactions, solvents, temperature, concentra- and a flow of 20 mL*min . One of these compounds, here tion and solvent dielectric constant have a strong influ- called SMC2, showed to be active in a molluscicidal test, ence on this equilibrium. In this work we studied the in- and, analysis of 1D and 2D NMR spectroscopic data (1H, 13 fluence of different solvents (DMSO-d6, CDCl3, CD3OD C, COSY, HSQC, HMBC and NOESY) and comparison and acetone-d6) and temperatures (ranging from 20-80 ◦C) with literature data, gave evidence for the presence of a fura- on the NMR spectra of the 3-(2-hydroxy-4,4-dimethyl-6- nocumarin with structure, corresponding to 4-[(3-Methylbut- oxocyclohex-1-enyl)isobenzofuran-1(3H)-one. This synthetic 2-en-1-yl)oxy]-7H-furo[3,2-g]chromen-7-one, and with molec- compound can inhibit both cancer cell proliferation (lym- ular formulae C16H1404. The NMR spectra were measured phoma and myeloid leukemia) and the photosynthesis pro- in CDCl3, at 300K, on a Bruker Avance NMR spectrometer, cess. The influence of an intramolecular hydrogen bond for- operating at frequency of 400,13 MHz, for hydrogen, using mation and the dynamic characteristics of this equilibrium Bruker’s standard pulse programs, in a direct observe probe- (proton and carbon-13) were analyzed under different condi- head. The acquired NMR data were processed with the top- tions. spin Bruker program and the chemical shifts were reported in d (ppm) downfield to TMS. The structure here proposed ACKNOWLEDGEMENTS: CAPES/Embrapa (001/2011), was also confirmed by X-ray diffraction data. CTEx, FAPEMIG, CNPq.

CAPES, CNPq TH171: Discussing structural proposals by theoreti- cal and experimental NMR MO169: NMR Analysis of intermolecular interac- 1 2 tions of catechins in green tea (Camellia sinensis) Quézia da Silva Sant’Anna, Marcela Christina Oliveira Nogueira, 1∗Jochen Junker 1,2 1,2∗ Gloria Castañeda-Valencia, Luzineide Wanderley 1Fundação Oswaldo Cruz - CDTS, 2UFRRJ - IQ Tinoco 1Universidade Federal do Rio de Janeiro, 2Núcleo de The structure determination of natural products by NMR re- Pesquisas de Produtos Naturais mains one of the biggest challenges in chemistry. Although NMR correlation data is relatively accessible, the interpre- Catechins are phenolic compounds extracted from plants and tation can still be very hard. But, the use of NMR in this found in foods and beverages such as green tea. Several bi- process is not only limited by the experimental part. Fre- ological activities have been described for catechins as anti- quently molecules are found that are very similar in their cancer, antioxidants, etc. Although the aggregation of phe- constitution, and actually could not have been distinguished nolic compounds has already been described, there is a lack by NMR. In these cases complimentary methods are needed, of detailed information about the processes of catechins ag- that might be chosen based on the structural proposals. gregation in samples of green tea. Intermolecular interactions could be identified as changes in chemical shifts, in the times The identification of such cases is a challenge on its own, and of relaxation and translational diffusion coefficients. Our goal can only be achieved using computer software to interpret the was to use NMR to follow the catechin aggregation process experimental (or better theoretical) data. Over the past years in green tea (Camellia sinensis). Lyophilized green tea infu- we have found several of these molecules in the literature that sion was prepared at concentrations of 0.5, 1, 2 and 5 mg/ml deserve more attention. Some of the most challenging will be 1 in D2O. H NMR spectra and the relaxation times T1 and shown, together with suggested complimentary methods, as T2 measurements were carried out on Agilent VNMRS-500 far as possible. at 25 ◦C. Representative signals for catechins components: epigallocatechin gallate (EGCG), epicatechin (EC), epigallo- MO172: Antinociceptive and anti-inflammatory ac- catechin (EGC) and catechin (CATE) were identified in the tivity of the Oleanane-type triterpenoid 1 H NMR of green tea. It was observed a chemical shifts de- 1∗ 3 crease with the concentration increase, mainly for H-3, H-2 Cláudia Quintino da Rochaa, Fabiana Cardoso Vilela, 2Fabiana Cardoso Vilela, 2Gustavo Prione Caval- and H-2” of EGCG; H-2, H-6 and H-2’ of EGC, H-2’ of EC 1,3 2 α cante, Vinícius Ferreira Guimarães, Marcelo Henrique and H-8, H-4 and H-2’of CATE. In the most concentrated 2 1 sample (5 mg / mL) the broadening line and signals overlap- dos Santos, Alexandre Giusti-Paiva, Wagner Vilegas ping prevent the recognition of some hydrogens, which were 1Department of Organic Chemistry, Chemical Institute, clearly identified in the samples of 0.1 and 1 mg/mL. The Araraquara, Brazil., 2Department of Pharmacy, Federal 3 relaxation times T1 and T2 for the representative hydrogens University of Alfenas-MG, Brazil, Department of Biomedi- of EC, EGC and EGCG decrease with the concentration in- cal Science, Federal University of Alfenas–MG, Brazil crease. The hydrogens of CATE showed an irregular behav- Abe-01 was evaluated the analgesic and anti-inflammatory ior of the relaxation time according to concentration. These effects using two animal models. Carrageenan-induced paw data indicate that catechins, even in the presence of other edema and peritonitis were used to investigate the anti- components present in green tea, undergo a process of aggre- inflammatory activity of Abe-01 . The acetic acid-induced gation, which should be taken into consideration in assessing writhing, formalin and hot-plate tests were used to investi- the biological activity assays with green tea. gate its antinociceptive activity. At test doses of 5, 10 and 15 mg/kg p.o., Abe-01 had an anti-inflammatory effect as TU170: NMR Investigation of Keto-Enol Tau- demonstrated by the reduction of paw edema induced by car- tomerism of an Isobenzofuran-1(3H)-one Derivative rageenan and the inhibition of leukocyte recruitment. Abe- 1∗ 2 01 inhibited nociception induced by an intraperitoneal injec- Diego Arantes Teixeira Pires, Wagner Luiz Pereira, tion of acetic acid, observed by the decrease in the number 1,3Claudia Jorge do Nascimento, 4José Daniel Figueroa- 2 of writhing episodes. Additionally, Abe-01 decreased licking Villar, Róbson Ricardo Teixeira time caused by a subplantar injection of formalin. Moreover, 1Instituto de Química, Universidade de Brasília, Brasil, the hot plate test produced a increase in latency reaction, 2Departamento de Química, Universidade Federal de Viçosa, demonstrating an antinociceptive effect. The structure of

119 Poster Abstracts – Natural Products and Small Molecules

Abe-01 was identified by NMR 13C, 1H, HMQC, HMBC, Experimental and calculated NMR chemical shifts as well as COSY, DEPT, IV and Ms experimental NOE and theoretical distance calculations were used for the stereochemical characterization of the compound ACKNOLEDGEMENTS: FAPESP, CNPq, UNESP E UNI- as Z s-E. FAL ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq TU173: Structure Elucidation of epimeric Acylphloroglucinols by NMR MO175: Characterization by NMR of Triterpenes from Leaves Inga Marginata 1∗Renata Mendonça Araújo, 2Edilberto Rocha Silveira, 3Raimundo Braz Filho ∗Nerilson Marques Lima, Dulce Helena Siqueira Silva, Ni- 1Instituto de Química, Universidade Federal do Rio Grande valdo Boralle do Norte., 2Centro Nordestino de Aplicação e Uso da RMN, Universidade Estadual Paulista "Júlio de Mesquita Filho" Departamento de Química Orgânica e Inorgânica, Univer- The investigation of Inga marginata leaves evidenced the sidade Federal do Ceará., 3Laboratório de Ciências Quími- presence of triterpenoids in its EtOH extract by 1H and 13C cas,UENF - Departamento de Química, UFRRJ NMR data analysis. Phytochemical study of Harpalyce brasiliana revealed that this species is a promising source of geranyl phoroglucynol NMR spectra were obtained in Varian-INOVA 500 instru- ◦ derivatives. Flash chromatography and HPLC analysis of ment and the experiments were performed at 28 C, using the CHCl3 fraction from the ethanol extract of H. brasiliana, CDCl3 as solvent and internal standard. Pulse sequence permitted the isolation of two new epimers compounds 1 and s2pul, 45.0 degrees, relaxation delay of 1.000 seconds, ac- (MF:C22H32O6), named as rel-(2’R,3’S,6’S)-1-hexanoyl- quisition time of 2.049 s, line broadening of 0.2 Hz and 16 2 1 2’,4,6-tri-hydroxy-3’-methyl-3’-[3-hydroxy-4-methy-pent-4- repetitions were used for H NMR data acquisition, where as enyl]-benzo-dihydropyran and rel-(2’R,3’S,6’R)-1-hexanoyl- pulse sequence s2pul, 45.0 degrees, relaxation delay of 1.000 2’,4,6-tri-hydroxy-3’-methyl-3’-[3-hydroxy-4-methy-pent-4- seconds, acquisition time of 1.300 seconds, line broadening 13 enyl]-benzo-dihydropyran. Structural elucidation was estab- of 0.5 Hz and 1088 repetitions were used for C NMR data lished through spectroscopic techniques, including 1D and acquisition. 2D NMR, acquired on a Bruker Avance DRX500 spectrom- The crude EtOH extract from I. marginata leaves was sub- eter, equipped with an inverse detection probe head with 1 mitted to liquid-liquid partition and the Hexane fraction was Z-gradient accessory working at 500.13 ( H) and at 125.77 13 analysed by NMR, which disclosed the presence of lupeol, MHz ( C). α-amyrin and olean-18-ene esters, in addition to fridelin. The 1H and 13C NMR spectra of displayed typical sig- Charachteristic signals were observed at 0.8 to 1.2 ppm in 1 1 nals [106.2, 157.7, 101.1, 164.5, 96.1 (CH/δH 6.52), 166.7, the H NMR spectrum for methyl groups in a triterpene 206.4, 27.2 (CH2/δH 3.47 and 3.14), 66.8 (CH/δH 4.33), skeleton. Additional signals at 4.68 ppm (bd, J = 2.1 Hz) 82.0 (C-O)] of a 4,6-dihydroxy-benzo-dihydropyran system, and at 4.56 ppm (dd, J = 2.1 and 1.5 Hz) for the geminal with a carbonyl at C-1. The carbonyl exhibited correlations protons of a terminal olefin evidenced the presence of lupeol. α 13 with both hydrogens at δH 3.33 (CH, dd, J = 16.4, 7.5 Hz) The presence of -amyrin was determined from C NMR and 3.25 (CH, dd, J = 16.4, 8.0 Hz), in the HMBC spec- data analysis, which showed signals at 124.3 and 139.4 ppm, trum. In addition, were observed successive correlations in associated to sp2 carbons C12 and C13, respectively. Fur- 13 the gs-COSY spectrum between this hydrogen at δH 3.33 thermore, fridelin was confirmed from the C NMR data with three methylenes at δH 1.84 (qt, 7.4 Hz), 1.45 (m) and analysis which showed a signal at 210.0 ppm for a carbonyl 1.36 (qt, 7.5 Hz) and one methyl at δH 0.90 (t, 7.5 Hz), char- group and a signal at 6.80 ppm for a methyl group , typical acterizing the hexanoyl moiety. The gs-COSY spectrum also of the triterpene fridelin. Additionally, signals at 142.4 ppm showed correlations of the oxi-methine hydrogen at δH 4,44 (C-18), 129.4 ppm (C-19), as well as the signals of methyl (t, 6.0 Hz) with the methyl at δH 1,94 (sl), and the vinyli- groups indicated the presence of an ester derivative of olean- I. marginata dene hydrogens at δH 5,26 and 4,97, and with the methylene 18-ene in the hexane fraction of leaves extract, at δH 2,20 (m), identifying the 3-hydroxy-4-methylpent-4- and established its major constituents as triterpenes. enyl moiety, supported by the HMBC data. The compounds were distinguished by small differences in the chemical shifts TU176: DOSY NMR of Flavonoids and gs-NOESY analysis, due to the important NOE for the ∗ pentenyl moiety. A peracetyl derivative 3 was synthesized Gabriel R. Martins, Mauro B. de Amorim, Antonio Jorge and its NMR was compatible with the proposed structure, Ribeiro da Silva also confirming the presence of four hydroxyl groups in both Núcleo de Pesquisas de Produtos Naturais, UFRJ compounds. Pulse Gradient Spin Echo experiments has been introduced as versatile tools for the study of compound mixtures without TH174: Configurational and conformational prefer- the recourse to chromatographic methods. Politi et al[1] ap- ences of an enaminone studied by NMR and theoret- plied Diffusion-edited 1H NMR (1D DOSY) for metabolic fin- ical calculation gerprint analysis of crude commercial herbal tinctures. The 2D DOSY version allows the study of random translational ∗Gil V. J. da Silva, Viviani Nardini motion of molecules in solution, arising from the thermal en- Departamento de Química, FFCLRP, Universidade de São ergy under conditions of thermodynamic equilibrium. The Paulo application of 2D DOSY sequences provides spatial label- β-Enaminones are important organic intermediates that ing of spins and displacement monitoring generating infor- present as characteristic structural feature a group N-C=C- mation on self-diffusion of molecules in solution. Because of C=O. Due to the tautomeric forms of β-enaminones as enol- the relationship between diffusion rates and molecular radii imines, keto-imines and keto-enamines, these compounds the diffusion dimension reveals the distribution of molecu- may appear as two geometric isomers and their conformers lar sizes and allows identification of different molecules in a s-E and s-Z. mixture.[2] The term "NMR chromatography" is applied to regular application of 2D DOSY in the analysis of mixtures In the development of a synthetic work, we prepared (5Z)- where the spectra displays chemical shifts in one dimension 6-amino-1-(1,3)-dioxan-2-yl)-3-hydroxy-3-methyloct-5-en-4- and diffusion rates in the other dimension.[3] Rodrigues et one. This β-enaminone has additional polar groups that al took advantage of 2D DOSY to get preliminary identifica- may play a role in the configurational and conformational tion of the components of the ethyl acetate fraction of ethanol preferences of this compound. extract from the aerial parts of Bidens sulphurea.[4]

120 Poster Abstracts – Chemometrics and metabolomics

As part of our continuing work on the analysis of polyphenols, of the collected data using appropriate multivariate statis- we undertook a systematic study of DOSY of flavonoids. The tical techniques. 1H NMR spectroscopy facilitates the de- chosen flavonoids were rutin, isoquercitrin, quercetin, morin tection of a wide range of low molecular weight metabolites and chrysin. In the present study we intended to verify the commonly found in tissues and biofluids including urine and effect of rotational diffusion of the B ring in the DOSY of blood plasma. It typically generates thousands of resonance the flavonoids. Initial molecular modeling calculations indi- signals that can be related to the response of biological sys- cated very low barriers to the rotation of ring B for chrysin, tems to perturbation of the system via pathological events. while for rutin, the volume of C-3 substituent would increase Due to the high density of the spectroscopic data, it is de- the barrier to rotation. We observed that the aspect of the sirable to characterize these modulations by application of signals relative to protons at C2’, C3’, C5’ and C6’ were af- multivariate statistical data analysis so as to reduce the com- fected by the rotation of ring B and that the rotation itself plexity of these data and to facilitate visualization of inher- is dependent on temperature and steric effects. ent patterns in the data. Various multivariate statistical data analyze, including projection methods such as principal com- Acknowledgements: CAPES, CNPq and FAPERJ ponents analysis (PCA) and projection to latent structures References: (PLS) based methods[3]. Since the birth of metabonomics, it has been proven to be an extremely powerful analytical [1] Politti, M.; Zioh, M.; Pintado, M.E.; Castro, P.M.L.; tool and hence found successful applications in many research Heinrich M. and Prieto, J.M. Phytochemical Analysis 20:328- areas including molecular toxicology[4,5], pathology, physiol- 334 (2009) ogy, functional genomics and environmental sciences. In this [2] Parella, T, Magnetic Resonance in Chemistry 36:467-495 presentation, examples of metabonomics application in tox- (1998) icity will be given and some potentials of this technique in the area of nano-sciences will be outlined. [3] Pemberton, C.; Hoffman, R.E., Aserin, A. and Garti, N.Langmuir 27:4497-4504 (2011) [1] Tang, H. R., et al., Metabonomics: a revolution in progress. Prog Biochem Biophys 2006, 33 (5), 401-417. [4] Rodrigues, E.D.; da Silva, D.B.; Oliveira, D.C.R and da Silva, G.V.J, Magnetic Resonance in Chemistry 47:1095-1100 [2] Nicholson, J. K., et al., ’Metabonomics’: understanding (2009). the metabolic responses of living systems to pathophysiolog- ical stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 1999, 29 (11), 1181- Chemometrics and metabolomics 1189. [3] Trygg, J., Wold, S., Orthogonal projections to latent structures (O-PLS). J. Chemometr. 2002, 16, 119-128. TH177: NMR studies of infection: bacterial, sub- clinical in open heart surgeries and liver diseases [4] Zhao, X. J., et al., Dynamic Metabolic Response of Mice to Acute Mequindox Exposure. J Proteome Res 2011, 10 ∗ C.L. Khetrapal (11), 5183-5190. Centre of Biomedical Magnetic Resonance [5] Zhang, L. M., et al., Systems Responses of Rats to Afla- Continuous and rapid growth of NMR methodology and tech- toxin B1 Exposure Revealed with Metabonomic Changes in nology has been responsible for the exponential growth in the Multiple Biological Matrices. J Proteome Res 2011, 10 (2), applications of the technique as a noninvasive tool for study- 614-623. ing anatomy, structure and in vivo metabolism. However, ACKNOLEDGEMENTS: developments of innovative techniques for understanding hu- man diseases at molecular level are still in infancy and hence We acknowledge financial supports from National Basic Re- promise a bright future for research. Variations in metabolic search Program of China (2012CB934004, 2011CB933401, profile resulting from disorders and clinical intervention at 2009CB118804) and the National Natural Science Founda- molecular level are more sensitive in identifying diseases in tion of China (20825520, 21221064, 31070854). early stages and assessing the efficacy of the interventions. NMR results on bacterial infections and those arising from TU179: Structure-dependence for the Molecular Dy- open heart surgeries and liver diseases will be presented. For namics of Metabolites such studies results obtained from bio-fluids such as urine, Jing Huang, Pingping Ren, Limin Zhang, ∗Huiru Tang serum, bile acids, and pericardial fluid which have very com- plex metabolic profiles with numerous structurally similar Chinese Academy of Sciences, Wuhan metabolites will be depicted. Specific metabolic signatures Molecular dynamics of metabolites are important for for different diseases from such investigations will be illus- their interactions and biological functions in both biol- trated. ogy and for their potential applications as natural ma- terials. To understand the relationship between metabo- MO178: Potentials of metabonomics in Nano-drug lite structure and dynamics, we comprehensively inves- screening tigated the molecular motions of three sets of struc- turally related metabolites (sarcosine, N,N-dimethylglycine, 1∗ 1 2 1 Yulan Wang, Limin Zhang, Chunying Chen, Huiru betaine, β-alanine, γ-aminobutyrate, 5-aminovalerate, 6- Tang aminocaproate, a-aminobutyrate, methylamine, dimethy- 1Key Laboratory of Magnetic Resonance in Biological Sys- lamine, trimethylamine and trimethylamine oxide) by mea- tems and State Key Laboratory of Magnetic Ressonance, suring their 13C CPMAS NMR spectra, the 13C and 1H spin- 2CAS Key Laboratory for Biomedical Effects of Nanoma- lattice relaxation times (T1, T1ρ), CSA and DIPSHIFTS terials and Nanosafety, National Center for Nanosafety properties as a function of temperature.

Metabonomics is the science that studies dynamic alterations We found that three-fold reorientations of CH3 and NH3 of metabolites in a cell, organ or entire organism[1]. The groups were dominant molecular motions in most of these definition of the metabonomics was first given in 1999 as molecules. The re-orientation of whole trimethylamine "the quantitative measurement of the time-related multi- groups was detectable in betaine and its hydrochloride salt. parametric metabolic response of living systems to patho- Whilst similar rotational properties were observable for two physiological stimuli or genetic modification"[2].Technically, methyl groups in N,N-dimethylglycine and for three methyl metabonomics investigations usually consist of the collec- groups in betaine anhydrous, three methyl groups in betaine tion of metabolic profiles using nuclear magnetic resonance hydrochloride had completely different motional properties. spectroscopy or mass spectrometry techniques and analysis The proton T1 measurements detected a polymorphic phase

121 Poster Abstracts – Chemometrics and metabolomics transition for DMG at 348.5 K. The DIPSHIFT experiments a 9.4T horizontal magnet, 20 cm bore. All mice livers were showed that CH3 and CH2 moieties in the N-methylated analyzed before starting the high fat diet; then, 3 weeks, 6 glycines had dipolar dephasing properties similar to these weeks and 12 weeks after starting the high fat diet. moieties in alanine and glycine, respectively. The activa- The measurements consisted of: a Turbo-RARE sequence tion energies for CH3 rotations increased with the increase (TE= 19.5 ms, TR= 743.9 ms) to image the whole liver for of the number of substituted methyl groups. The amino estimating the liver size and a STEAM sequence (TE= 3 ms, groups and backbone reorientations were major motions for TR= 1500 ms) for the spectroscopic investigation of the liver ω-amino acids except for β-alanine. The activation energies fat deposition. for amino group were positively correlated with the strength of hydrogen-bonds involving these groups in the crystals and The preliminary results show a progressive increase in the the carbon-chain lengths whereas such energies for the back- liver lipid levels for all mice and a liver augmentation due to bone motions were inversely correlated with the carbon-chain fat deposition. Further results are under analysis. lengths. Molecular dynamics is associated with two poly- References morphic transitions in a-amino-n-butyric acid (ABA). Both 13 proton and C relaxations showed that, apart from two mo- 1. Wahrburg, U. (2004) What are the health effects of fat? tions corresponding to the reorientations of methyl and amino Eur. J. Nutr. 43(I), pp. 6 groups, ethyl motion (Ea, 16-21.5 kJ/mol) was also present in ABA. DIPSHIFT results further revealed the molecular 2. Key, T.J., Schatzkin, A., Willett, W.C., Allen, N.E., dynamics of different polymorphs and associations between Spencer, E.A., Travis, R.C. (2004) Diet, nutrition and the motions and polymorphic transitions. These findings pro- prevention of cancer. Public Health Nutr. 7(1A), pp. 187 vided essential information for molecular dynamics of these 3. Lunati, E., Farace, P., Nicolato, E., Righetti, C., Marzola, metabolites and for understanding the structural dependence P., Sbarbati, A., Osculati, F. (2001) Polyunsaturated fatty of molecular dynamics. acids mapping by 1H MR-Chemical Shift Imaging. Mag. Res. in Med. 46, pp.879 References: 4. Schultz, L.D., Ishikawa, F., Greiner, D.L. (2007) Human- [1] J. Huang, L. Jiang, P. Ren, L. Zhang, H.R. Tang, J Phys ized mice in translational biomedical research. Nat. Rev. Chem B, 116:136–146, 2012. Immunol. 7, pp. 118 [2] J. Huang, L.M. Zhang, H.R. Tang, J Phys Chem B, 5. Gailus-Durner, V., Fuchs, H. et al (2005) Introducing the 116:2096-2103, 2012. German Mouse Clinic: open access platform for standardized [3] P. Ren, D. Reichert, Q. He, L. Zhang, H. Tang, J Phys phenotyping. Nat. Meth. 2(6), pp. 403 Chem B, 115:2814-2823, 2011. MO181: 13C-biomass characterization by data min- [4] D. Reichert, M. Koverman, N. Hunter, D. Hughes, O. ing of solid/solution NMR Pascui, P. Belton, PCCP, 10:542, 2008. 1,2,3,4∗ 1,4 [5] H.R. Tang, Y.L. Wang, P. Belton, Phys Chem Chem Phys, Jun Kikuchi, Eisuke Chikayama 3694-3701, 2004. 1RIKEN Plant Science Center, 2Biomass Engineering Pro- gram, RIKEN, 3Nagoya University, 4Yokohama City Uni- [6] Y. L. Wang, H.R. Tang, P. S. Belton, J Phys Chem B, versity 106:12834-12840, 2002. Plant biomass is now highlighted by view points both ba- [7] H. R. Tang, P. S. Belton, Solid State NMR., 21:117-133, sic and applied science such as biorefinery materials [1,2]. 2002. It consists polysaccharides and lignin, both are so difficult [8] Tang, H. R.; Belton, P. S. Solid State NMR. 1998, 12:21 biomacromolecules in terms of tackling by NMR. Major dif- ficulties are attributed to their low solubility, mixture com- [9] M. Hong, J. D. Gross and R. G. Griffin. J Phys Chem B, plexity and low spectral dispersion. In order to "escape" the 101:5869-5874, 1997; C. A. McDowell, P. Raghunathan, D. problem for mixture complexity, firstly we tried NMR exper- S. Williams, J Chem Phys, 66:3240-3245, 1977. iments using 13C labeled cellulose. Several solid-state NMR measurements (VPCP, 2D-INADEQUATE and DARR), as TH180: Application of MRI and MRS in obesity re- well as solution-NMR (2D-HSQC and HSQC-NOESY) were search with mice employed for ionic-liquids solubilized and regenerated sam- ples, then statistical multivariate analysis were useful ap- 1,2∗ 1,2 1,2 Luciana Caminha Afonso, S Neschen, Helmut proach to monitor structural variations [3-5]. Next target 1,2 1,2,3 Fuchs, Valérie Gailus-Durner, Martin Hrabě de Ange- should be tackling to the mixture complexity, therefore we lis applied 13C-labeling techniques developed for plant systems 1Helmholtz Zentrum München, 2German Center for Dia- [6-10]. High level of 13C-incorporation enabled both 2D and betes Research, 3Technical University Munich 3D-NMR experiments, therefore we have been succeeded sev- eral new signal assignments that have not been reported pre- Characteristics of a diet can directly affect health, playing viously (Komatsu et al. to be submitted). Our current limi- an important role in preventing or inducing of the develop- tations both 13C-labeling as well as solid/solution NMR mea- ment of diseases. Chronic consumption of high amounts of surements will be discussed in the conference. fat results in obesity. This excess of body fat increases the risk of cardiovascular diseases, type 2 diabetes and some can- References cers.[1,2,3] 1. Kikuchi et al. (2011) J. Ecosys. Ecogr. S2, e001. Since decades research on mice is helping to understand sev- 2. Ogata et al. (2012) PLoS ONE 7, e30262. eral human biological processes, such as hematopoiesis, im- munity, infectious diseases, cancers, etc.[4] 3. Okushita et al., (2012) Biomacromolcules 13, 1323-1330. This work is was carried out in the facilities of the German 4. Mori et al. (2012) Carbohydr. Polymers 90, 1197-1203. Mouse Clinic.[5] Two groups of mice were fed with different 5. Okushita et al. (2012) Polymer J. 44, 895-900. diets. One diet has been enriched with safflower oil (com- posed mainly by polyunsaturated fatty acids), whereas the 6. Kikuchi et al., (2004) Plant Cell Physiol., 45, 1099-1104. other has been enriched with lard (composed mainly by mo- 7. Kikuchi et al., (2007) Method Mol. Biol. 358, 273-286. nounsaturated and saturated fatty acids). 8. Tian et al., (2007) J. Biol. Chem. 282, 18532-18541 The mice were analyzed using a multipurpose research sys- tem, Bruker Biospec 94/20 (Bruker Biospin), equipped with 9. Sekiyama et al. (2010) Anal. Chem. 82, 1643-1652.

122 Poster Abstracts – Chemometrics and metabolomics

10. Sekiyama et al. (2011) Anal. Chem. 83, 719-726. initiative (MSI, [1]) was formed. The MetaboLights ([2], https://www.ebi.ac.uk/metabolights/) team is coordinating TU182: Deciphering Cancer Metabolism by NMR this consortium of 14 European partners, with Metabo- Lights playing a central role for the proposed work. A key ∗ Ulrich L. Günther, John Carrigan, Christian Ludwig, aspect of this effort aims to develop efficient policies ensur- Farhat Khanim, Chris Bunce ing that metabolomics data is encoded in open standards, University of Birmingham tagged with a community-agreed and complete set of meta- data, supported by a communally developed set of open Metabolism and cancer have been associated for a long time, source data management and capturing tools, disseminated starting with Otto Warburg who discovered that cancer cells in open-access databases adhering to these standards, sup- metabolise glucose in a manner distinct from normal tissues, ported by vendors and publishers, who require deposition forming lactate, even in the presence of sufficient oxygen. upon publication, and properly interfaced with data in other While Warburg saw this as a cause of cancer, developments biomedical and life science e-infrastructures. Our aim is to in cancer biology painted a different picture, linking cancer deliver the exchange formats and terminological artifacts to altered signaling and cell cycle control. During this de- needed to describe, exchange and query metabolomics ex- velopment the aspects of metabolism were almost completely periments, using the ISA-Tab [3] as core for the description disregarded, if not rejected. Recent research supports a pro- of experiments and building additional ’layers’ for the data nounced role of altered metabolism in cancers, often induced matrices. We wish to ensure that the proposed standards by hypoxic conditions, which can considerably alter oxidative are widely accepted by involving major global players in the phosphorylation and other mechanisms. development process. We will also develop and maintain We have analyzed acute myeloid leukemia cell (AML) lines exchange formats for raw data and processed information using NMR metabolomics and tracer based metabolic flux (identification, quantification), building on experience from analysis. In our analysis we looked at the effect of a drug standards development within the Proteomics Standards combination (Bezalip and medroxyprogesterone acetate) [1] Initiative ([4], PSI). Additionally we are planning to collabo- which drive AML cells either into re-differentiation or into rate on developing the missing open standard NMR Markup apoptosis, showing that this treatment acts by generating Language (nmrML) for capturing and disseminating Nuclear large levels of reactive oxygen species (ROS). Using 13C- Magnetic Resonance spectroscopy data in metabolomics. We labeled glucose and glutamate as metabolic tracers we can aim to explore semantic web standards that facilitate linked decipher the effect of ROS, which leads to the formation of open data (LOD) throughout the biomedical and life science new metabolites, specifically malonate, which is in itself po- realms, and demonstrate their use for metabolomics data. tent inhibitor of succinate dehydrogenase. Database URL: http://www.ebi.ac.uk/metabolights This analysis also shows how NMR can be used to decipher [1] Goodacre, R., et al., Proposed minimum reporting stan- metabolism, by observing site specific label incorporation, dards for data analysis in metabolomics. Metabolomics : Of- using either signal intensities or 13C-13C-couplings. ficial journal of the Metabolomic Society, 2007. 3(3): p. 231- [1] S Tiziani, A Lodi, M Viant, C Bunce, U Günther. 241 Metabolomic profiling of drug responses in acute myeloid leu- caemia cell lines. PloS ONE, 4(1), 4251 (2008). [2] Haug, K., et al., MetaboLights–an open-access general- purpose repository for metabolomics studies and associated meta-data. Nucleic Acids Research, 2012. TH183: MetaboLights: Towards a new COSMOS of metabolomics data management and dissemination [3] Rocca-Serra, P., et al., ISA software suite: support- ing standards-compliant experimental annotation and en- 1,2∗Reza Salek, 1Kenneth Haug, 1Pablo Conesa, 1Mark 1 3 abling curation at the community level. Bioinformatics, 2010. Williams, Tejasvi Mahendraker, Eamonn Maguire, 26(18): p. 2354-6. 3Alejandra González-Beltrán, 3Philippe Rocca-Serra, 3Susanna-Assunta Sansone, 2Jules Griffin, 1Christoph Stein- [4] Taylor, C.F., et al., The work of the Human Proteome beck Organisation’s Proteomics Standards Initiative (HUPO PSI). 1European Bioinformatics Institute, Wellcome Trust Omics : a journal of integrative biology, 2006. 10(2): p. 145- Genome Campus, Hinxton, Cambridgeshire, 2University 51 of Cambridge, Department of Biochemistry and Cambridge Systems Biology Centre, Cambridge, 3University of Oxford MO184: 1HR-MAS NMR metabolomics investiga- e-Research Centre, Oxford, UK tion of human breast cancer; enhancing the results using data fusion approaches with GC and LC mass MetaboLights is a general-purpose, open-access curated spectrometry data repository for metabolomic experiments. Metabolomics is investigation of the small molecular or metabolites re- 1,7∗Reza Salek, 2Michael Eiden, 1Cecilia Castro, 3Carsten sponse in a cell, tissue or biofluid to an environmental Denkert, 4Sibylle Loibl, 5Matej Oresic, 6Oliver Fiehn, stress, the result of cellular processes or response to dis- 1,2Jules Griffin ease. Metabolomics studies are growing rapidly and there 1Department of Biochemistry and Cambridge Systems Bi- is a great need to share and disseminate data, making data ology Centre, University of Cambridge, UK, 2Elsie Wid- accessible to public as is increasingly being required by dowson Laboratory, Cambridge, UK, 3Institute of Pathol- funding organisations and journals. MetaboLights would be ogy, Charité – Universitätsmedizin Berlin, Germany, 4GBG the medium to capture the NMR and Mass spectrometry Forschungs GmbH - Neu-Isenburg, Germany, 5VTT Techni- metabolomics experiments including raw experimental data, cal Research Centre of Finland, Finland, 6Genome Center, processed and transformed data and associated metadata. University of California Davis, CA, USA, 7European Bioin- There are wide varieties of proprietary file formats by dif- formatics Institute, Wellcome Trust Genome Campus, Hinx- ferent instrument vendors in use, hence there is a need for ton, Cambridgeshire open source file formats and standardization of data. Hav- ing a standardized way of exchanging data is of immense Breast cancer is the most common malignant tumor in women importance to making vendor neutral metabolomics portals worldwide, while the disease is curable in the early stages, to automatically visualize and analyse metabolomics-data about 50% of the patients with stage II or III tumours require in web applications (i.e. as planned for MetaboLights) . potential systemic therapy and are in need of better biomark- Therefore an EU coordination action initiative for develop- ers for diagnosis. METAcancer project (www.metacancer- ing metabolomics standards in the EU and worldwide, called fp7.eu), an EU Framework 7 funded project, was setup to COordination of Standards in MetabOlomicS - COSMOS determine new metabolic biomarkers of breast cancer to pre- (http://cosmos-fp7.eu), based on Metabolomics standard dict grade and hormonal status of breast tumours using 1HR-

123 Poster Abstracts – Chemometrics and metabolomics

MAS NMR spectroscopy, gas chromatography (GC)-MS and Hydroponic production permits good control of plant growth liquid chromatography (LC)-MS, in addition to histological and development even where no suitable soil exists and/or characterisation of the tumours. Our work package task there is limitation of water resources. Classical soilless cul- was to acquire data from tumour tissue using HRMAS 1H ture causes environmental problems that can be reduced by NMR spectroscopy using a 11.7 Tesla Superconducting mag- partial recycling of the drainage solutions. However, this can net. Tissue samples were cut, if required, and packed in- produce nutrient imbalances affecting crop yield. This prob- side 4 mm Zirconium oxide rotors with Teflon caps and kel- lem is minimized by using the New Growing System NGS,® f CRAMP inserts (total sample volume of 50uL) and the a pure hydroponic production system based on the recircu- remaining volume was filled with phosphate buffered D2O. lation of nutrient solution through multilayer polyethylene The rotor was spun at 5 kHz at 27◦C. The dataset was troughs on a close system. Compared to traditional intensive acquired using a Bruker Avance III spectrometer, equipped farming, it has many advantages in relation to costs, prof- with a 1H/13C high-resolution magic angle-spinning (HR- itability, quality of production and environment. The better MAS) probe.Two different NMR pulse programs, a one- guarantee for product quality and sustainable production re- dimensional NOESY presaturation pulse sequence and T2 quires characterization of biochemical composition. This as- filter Carr-Purcell-Meiboom-Gill (CPMG) experiments (to pect of NGS® utilization has been, however, scarcely stud- minimize spectral broadening due to lipids) with solvent pre- ied. saturation, were carried out for each sample. Both exper- iments were acquired with 128 scans into 64K data-points This work is aimed at examining the effect of the hydro- across spectral width of 16.00 ppm, using a relaxation de- ponic system NGS® on the metabolic content of tomato lay 1.5s and acquisition time of 4.09 s at a proton frequency fruit using HRMAS NMR methods. Two commercial fla- of 500.3 MHz. For the acquisition of CPMG spectra a to- vor varieties (marmande type) "Tigre" and "Raf", the refer- tal spin echo delay of 400 ms was used Multivariate pattern ence cv. for the investigations in the line of tomato taste in recognition techniques were used for NMR data analysis us- Almería (Spain), were chosen. Tomatoes were harvest under ing SIMCA-P+ 12.0 (Umetrics AB, Umeå, Sweden) with the commercial culture conditions in green-house and the fruits data mean centred and Pareto scaled prior to analysis. Data were hand-picked at the stage commonly marketed during analysis results proved to be highly discriminatory between the months of January, February, March and May. "Tigre" tumours, healthy breast tissue and healthy duct tissue. In variety was grown in both sand-covered soil traditionally in addition we were able to discriminate between different tu- the area (TS) and in hydroponic NGS® (TN) whereas "Raf" mour grades (specifically grade 2 from grade 3) as well as tomatoes only in soil (RS). different tumour types (Type 2 from Type 4 demonstrated the best discrimination). In addition we have used several 1H HRMAS NMR spectra were measured on tomato purée different data fusion approaches to elucidate the combined samples from fresh tomatoes. The data were analyzed by a potential of the multiple profiling techniques for the diagnosis combination of PLS and integration of selected signals (As- and classification of breast cancer. We systematically tested signed Signal Analysis, ASA). Compositional differences be- correlation based approaches, orthogonal projections multi- tween the two varieties grown in soil can be associated to variate data analysis and multi-block principal component genetic variations that are also reflected in differences in analysis data fusion approaches [1] on a combined dataset of shape and taste. Hydroponic culture affects significantly the 1H HR-MAS NMR, GC-MS and LC-MS measured on a co- metabolic content of the fruits and compositional differences hort of 300 breast cancer patients with regard to various data between TN and RS samples decrease in some extension. The pre-processing schemes and compared the outcomes. On the results obtained also point that malic acid may be a good level of individual platforms it became obvious, that the dif- marker in order to differentiate "Tigre" tomatoes. ferentiation between healthy and diseased samples is often hampered by the heterogeneity of the tissue sample (specif- ically due to the fat content), with individual platforms be- TH186: Metabolomic approach in mapping Huang- ing affected to different extents. In comparison, it showed longbing markers that Multi-Block PCA, as an unsupervised technique aiming 1William Ohashi, 1Ana Paula Espindola, 2Helvecio Della Co- to find the common structure inherent across the platforms, letta Filho, 1∗Ljubica Tasic was able to differentiate between healthy and diseased sub- 1DQO, IQ, UNICAMP, Campinas, SP, Brazil, 2IAC, SP, jects. Inspecting the individual block loadings, revealed that Brazil choline and phosphocholine metabolites measured by NMR spectroscopy demonstrated similar trends to PC 16:0-18:1 by Key words: metabolomics, oranges, huanglongbing, biomark- LC-MS. Both metabolites contributed most to the observed ers separation of cancer from normal tissue, which is in accor- dance with current findings in the literature [2, 3] Brazil is the major orange producer and exporter in the world. Unfortunately, Brazilian production suffers millions [1] Bro, R., F. van den Berg, et al. (2002). "Multivariate of dollars in damage annually due to phytopatogenic mi- data analysis as a tool in advanced quality monitoring in the croorganisms. One of the major diseases to affect Brazilian food production chain." Trends in Food Science & Technol- orchard is Huanglongbing. This disease caused trough in- ogy 13(6-7): 235-244. fection with the bacterium Candidatus Liberibacter asiaticus was first detected in Sao Paulo in 2004 and is transmitted [2] Katz-Brull, R., R. Margalit, et al. (1998). "Choline 13 31 by insects. Herein, we are looking for biomarkers of the early metabolism in breast cancer; 2H-, C- and P-NMR studies disease onset. At this stage of our research, we are comparing of cells and tumors." MAGMA 6(1): 44-52. young and adult leaves of healthy, symptomatic and asymp- tomatic plants. The leaves extracts were obtained using dif- [3] M. Hilvo, et. al, "Novel theranostic opportunities offered ferent solvents: (i) phosphate saline buffer, (ii) methanol, (iii) by characterization of altered membrane lipid metabolism 1 CHCl3/Methanol (1:1 v v-1), and (iv) CHCl3. H NMR spec- in breast cancer progression", Cancer Res. (2011). doi: tra were obtained using 600 MHz Brucker spectrometer at 10.1158/0008-5472.CAN-10-389 equal samples conditions, such as, concentration, acquisition parameters and the same spectra processing. NMR data were TU185: Impact of nutrient solution recycling on then analyzed with chemometrics tools (Pirouette). Principal the metabolic content of tomato fruit: a HRMAS component analysis (PCA) and hierarchical cluster analysis NMMR study (HCA) were employed to reduce dimensionality and investi- ∗ gate which variables should be more effective for classifying Maria José Iglesias Valdés-Solis, Jesús García López, three different samples classes. Generated PCA and HCA Juan Fernando Collados Luján, Fernando López Ortiz results show that healthy, symptomatic and asymptomatic Almería University plants could be divided in three groups with clear influence

124 Poster Abstracts – Chemometrics and metabolomics of sugars, amino acids and aromatics compounds concentra- 2s; AQ: 4.7s; TD: 64k and SW: 25 ppm. The 1H NMR spec- tions. tra were treated by chemometrics, using principal component analysis (PCA). FAPESP and CNPq The PCA showed the differentiation in PC1 (83.71%) be- tween inoculated and control samples already in the period MO187: Distinction of Tropical Brazilian Wines by 1 of 4 days after inoculation due to changes in production of Chemical Profile Through Association of H NMR primary metabolites. According to disease progression, we with Principal Component Analysis have observed that the greatest change in the metabolites 1,2R. H. S. Andrade, 2L. S. Nascimento, 3G. E. Pereira, production occurred in the beginning of the disease, without 1A. P. S. Paim, 1∗Fernando Hallwass visible symptoms. Throughout the development of disease 1Universidade Federal de Pernambuco, 2Instituto Federal de we have observed the reduction of metabolic changes, lead- Educação, Ciência e Tecnologia de Pernambuco, 3Embrapa ing to the separation of groups of samples in PC2 axis (13.53 Uva e Vinho/Semiárido %) after 20 days. The detection of citrus canker before the manifestation of disease is the principal advantage of this It is essential to take great care with grape growth and the preliminary study using NMR and chemometric methods. maturation process for winemaking. Thus, particular atten- tion must be paid to the rootstock, preventing pest damage, Acknowledgment: CAPES, UFMS, IAPAR. in order to guarantee the healthy development of the fruit. The selection of the type of rootstock suitable for wine culti- TH189: HLB in Citrus leaves: evaluation of the vation, however, depends heavily on the environmental con- metabolic changes by NMR and chemometrics ditions of the vineyard. The means of transplantation can 1Deisy dos Santos Freitas, 2Eduardo Fermino Carlos, intervene with the chemical composition of the fruit, thus 1∗ affecting the chemical composition and sensory characteris- Glaucia Braz Alcantara tics of the wine. Towards understanding this better, we con- 1Universidade Federal de Mato Grosso do Sul, 2Instituto ducted a study of tropical wines made from grapes growing Agronômico do Paraná in the São Francisco Valley (SFV) in the Northeast of Brazil. HLB (Huanglongbing) is a disease that affects the citricul- 1103 P IAC 313 1 Two different rootstocks were used: and . H ture in several countries and has been responsible for large NMR spectroscopy associated with the chemometric method losses in the production. Typical symptoms include the pres- of Principal Component Analysis (PCA) was used to study ence of yellowish spots on leaves, defoliation, sharp drop in the influence of the rootstocks on the composition of the wine. production and fruit dropping. Thirty-three samples from SFV bottled wine were rota- HLB is associated with Candidatus Liberibacter sp. bac- evaporated, lyophilized, dissolved in a D2O/TMSP (Sodium terium and its transmission occurs through contaminated 1 3-Trimethylsilylpropionate) solution, and underwent H plant material and by action of psyllid insects. There is not NMR spectra analysis. For statistical analysis we selected the cure for HLB and the only way to control the disease is the spectral range of interest and generated a matrix of chemi- elimination of the contaminated plants. In this context, we cal shift (in ppm) X signal intensity. The data were mean- have evaluated the metabolic changes caused by HLB in aque- centered and exported to the software Unscrambler 9.7, with ous extracts of leaves of Citrus sp. by 1H NMR spectroscopy which the PCA study was conducted. and chemometric analysis. Our results showed an efficient characterization of the sam- The NMR measurements were obtained on a Bruker DPX ples by the type of rootstock. Samples of the rootstock 1103 300 spectrometer (7.05 T). The spectra were acquired with P had higher levels of lactic acid, proline and fructose. Sam- 128 scans (NS), 64k points (TD), 3.64s acquisition (AQ) and ples of the rootstock IAC 313 presented higher levels of suc- relaxation delay of 2s (d1). 1H NMR spectra was process- cinic acid and glycerol. ing using 64k points (SI), exponential multiplication 0.30 Hz (LB) and manual phase and baseline corrections. Data treat- ACKNOLEDGEMENTS: FACEPE, CAPES, CNPq, IFPE, ment was performed using the Principal Component Analysis UFPE, EMBAPA, (PCA).

TU188: 1H NMR spectroscopy and chemometrics for In PCA, there was a clear distinction between HLB symp- comparative study of metabolic changes in Citrus sp. tomatic and asymptomatic leaves from branches contain- caused by different stages of citrus canker ing fruits. The loadings responsible for grouping of asymp- tomatic leaves in positive values of PC1 were the signals at 1Caroline Silva de Oliveira, 1Rafael do Prado Ap- 3.10 and 3.30 ppm, corresponding to proline-betaine singlets. parecido, 2Eduardo Fermino Carlos, 3Luciano Morais Lião, The signals at 3.66, 3.82 and 5.42 ppm were responsible for 1∗Glaucia Braz Alcantara grouping of symptomatic leaves in negative values of PC1, 1Universidade Federal de Mato Grosso do Sul, 2Instituto corresponding to the region of sugars. 3 Agronômico do Paraná, Universidade Federal de Goiás On the other hand, asymptomatic leaves in plants with HLB Brazil is the highest worldwide citrus producer. Nevertheless, showed a similar metabolic composition with healthy leaves. citrus canker, disease caused by Xanthomonas axonopodis pv. The results indicate that these metabolites may be related citri bacterium (Xac), is potentially considered a devastate to the defense mechanism of Citrus when affected by HLB, threat to many producer regions of citrus in our country. Al- which are detected by NMR in the specific site where the though recent studies have indicated metabolic differences in Candidatus Liberibacter sp. bacteria are acting. Citrus sp. attacked by citrus canker, the knowledge about Acknowledgments: CAPES, IAPAR. the variations in different periods of Xac infection was not obtained. MO190: Salivary metabolite signatures of children In this context, NMR associated to chemometric analyses was with and without dental caries lesions. employed to comparative study of metabolomics of Citrus sp. 1Tatiana Kelly S. Fidalgo, 1Liana B. Freitas-Fernandes, for inoculated with Xac bacterium and non-inoculated leaves 2 3 4 (control) in different periods of citrus canker evolution (0, Renata Angeli, Adriane M. S. Muniz, Elicardo Gonsalves, 1Raquel Santos, 3Jurandir Nadal, 2Fabio C. L. Almeida, 4, 12 and 20 days). Extracts from leaves was obtained us- 2∗ 1 1 Ana Paula Valente, Ivete P. R. de Souza ing buffered D2O/TMSP-d4. The measurements of H NMR were performed on a Bruker Avance III 500 spectrometer, 1School of Dentistry, Federal University of Rio de Janeiro, equipped with a 5 mm TBI probe. The 1H NMR spectra 2National Center for Nuclear Magnetic Resonance, Fed- were acquired using the CPPR pulse sequence, NS: 64; d1: eral University of Rio de Janeiro, 3Biomedical Engineering

125 Poster Abstracts – Chemometrics and metabolomics

Program, Federal University of Rio de Janeiro, 4School of cellular environment those mitochondrias that do not prop- Physics, Federal University of Rio de Janeiro erly metabolize pyruvate received from the glycolytic path- A metabolomic approach was used to analyze endogenous way ("deficient" mitochondria). metabolites and to correlate with a specific biological state. The analysis of salivary metabolites is a growing area of in- TH192: Identification of urinary metabolites to dis- vestigation with potential for basic and clinical applications. criminate between HCV patients and healthy volun- Analyses of children’s saliva in different dentitions and with teers using metabolomics or without caries could potentially reveal a specific profile 1∗Carlos Jonnatan Pimentel Barros, 1Ronaldo Dionísio related to oral disease risk. Nuclear Magnetic Resonance 2 2 (NMR) is well suited for mixture analysis followed by Prin- da Silva, Michele Maria Gonçalves de Godoy, Edmundo Pessoa de Almeida Lopes, 3Paulo Renato Alves Firmino, cipal Component Analysis combined with Linear Regression 1 (PCA-LR) statistics and was used to identify differences in Ricardo Oliveira da Silva the salivary metabolites. The classificatory analysis was per- 1Departamento de Química Fundamental, Universidade Fed- formed using PCA-LR based on 1,000 cross-validation boot- eral de Pernambuco, Recife, Brazil, 2Departamento de strap runs from both classifiers in order to increase the data Medicina Clínica, Hospital das Clínicas, Universidade Fed- information from a small sample size. The PCA-LR pre- eral de Pernambuco, 3Departamento de Estatística e Infor- sented a statistically good classificatory performance for chil- mática, Universidade Federal Rural de Pernambuco, Brazil P dren with and without caries with an accuracy of 90.11 % ( Hepatitis C is an infectious disease that may evolve into P < 0.001), 89.61 % sensitivity ( < 0.001), and 90.82 % speci- cirrhosis or cancer. Since the liver is the main organ of P ficity ( < 0.001). Children with caries lesions presented metabolic control in mammals, an HCV infection may change higher levels of several metabolites, including lactate, fatty the profile of endogenous metabolites, which can be observed acid, acetate and n-butyrate. Saliva from subjects with dif- through metabonomics. Recently, our group has demon- ferent dentition stages was also analyzed. Although the sali- strated that it is possible to classify urine samples supplied vary samples were poorly classified, permanent dentition pre- for healthy or infected HCV volunteers, using 1H NMR and sented increased levels of acetate, saccharides and propionate. metabonomics strategy[1]. In the present report, we indicate The NMR data and PCA-LR were able to classify saliva from the possible metabolites and metabolic pathways that are re- children with or without caries, with performance indexes sponsible for this classification. We used 61 samples, 30 from comparable to the partial least-squares regression discrimi- patients with hepatitis C and 32 from healthy volunteers. nant analysis (PLS-DA) results also performed. Our data Different forms of preprocessing of the data were used and also showed similar salivary metabolite profiles for healthy for each one, multiple hypothesis tests were used to identify subjects despite the differences in their oral hygiene habits, the features that differ significantly between the two groups. socioeconomic status and food intake. From the metabolomic study, we identified 12 spectral re- gions associated with endogenous metabolites, which are re- ACKNOLEDGEMENTS: FAPERJ, CNPq, CAPES, UFRJ. sponsible for the classification. These metabolites are: L- valine, L-isoleucine, L-leucine, glycine, 2-hydroxy-isovalerate, TU191: Metabolic profile of murine melanoma stud- 3-aminoisobutanoic, dimethylglycine, argininosuccinate and ied by Nuclear Magnetic Resonance citrate, found in higher concentrations in samples from pa- tients with hepatitis C, and trigonelline, carnosine and creati- 1Pedro Bacchi, 1Roger Chammas, 1Silvina O. Bustos, nine, found in higher concentrations in samples from healthy 2 , 3Antonio C. Bloise, 3∗Said Claudia M. G. de Souza volunteers. Based on a pathway enrichment analysis software R. Rabbani (http://metpa.metabolomics.ca),[2] we obtained the most 1Faculdade de Medicina, Universidade de São Paulo, Brazil, significant metabolic pathways are involved in the discrim- 2Instituto de Pesquisas Tecnológicas, São Paulo, Brazil, ination found. They are: (1) Valine, Leucine and Isoleucine 3Instituto de Física daUniversidade de São Paulo, Brazil biosynthesis; (2) Valine, Leucine and Isoleucine degradation; Using murine melanocytes, a new model of murine melanoma, and (3) Aminoacyl-tRNA biosynthesis. Tm-1 called , was produced by repeated cycles of cell disad- Acknowledgments: CNPQ, CAPES, FACEPE and gal-3 hesion. In this strain, galectin-3 ( ), a multifunctional PRONEX. endogenous lectin which seems to act by modulating the mi- tochondrial response to different types of stress and condi- References: tioning the cell death, is silenced. Through transfection of original Tm-1, we produced two new strains of melanomas, [1] Godoy, M. M. G.; Lopes, E. P. A.; Silva, R. O.; Hall- one expressing gal-3, and the other not. In order to evaluate wass, F.; Koury, L. C. A.; Moura, I. M.; Gonçalves, S. M. C.; the changes in glucose metabolism induced by gal-3, these Simas, A. M.. J. Viral Hepat., 2010, 17, 854-858. cells were cultivated in different conditions of oxygen tension 7 [2] Xia, J.; Mandal, R.; Sinelnikov, I.; Broadhurst, D; and glucose level. Approximately 10 cells were collected Wishart, D. S.. Nucl. Acids Res., 2012, 1-7. from each culture. These were sonicated 5 times for 1 minute with intervals of 10 seconds between sonication, centrifuged MO193: Hepatitis C and B discrimination using uri- at 4000 rpm for 20 minutes, the supernatant were collected nalysis metabonomics and lyophilized. The lyophilized extracts were diluted in D2O and finally their NMR spectra were obtained. These spectra 1∗Carlos Jonnatan Pimentel Barros, 1Érika de Almeida were sectioned in small intervals and the integrals of these sec- Leite, 2Michele Maria Gonçalves de Godoy, 2Edmundo tions, representing the concentration of different metabolites Pessoa de Almeida Lopes, 3Paulo Renato Alves Firmino, present in the liquid, were analyzed by multivariate meth- 1Ricardo Oliveira da Silva ods. The modifications in concentration of certain metabo- 1Departamento de Química Fundamental, Universidade Fed- lites were attributed to the impact of gal-3 in the mitochon- eral de Pernambuco, Brazil, 2Departamento de Medicina drial homeostasis process. Among all metabolites pointed Clínica, Hospital das Clínicas, Universidade Federal de Per- out by multivariate analyses, the most relevant were lactate, nambuco, Brazil, 3Departamento de Estatística e Infor- alanine, free choline, GPC, PC, acetate, 3-hydroxybutyrate mática, Universidade Federal Rural de Pernambuco, Brazil and taurine. The results suggest that gal-3 gene seems to act in the mitochondrial homeostasis only in the specific case The aim of this preliminary study was to build a metabo- where tumorigenic cell are exposed to stress, such as hypoxia. nomics model for distinguishing urine samples supplied from In addition, when the homeostasis is triggered by hypoxia it patients infected with hepatitis B virus (HBV) and from pa- is only carried out if there is an excess in glucose levels. A tients infected with hepatitis C virus (HCV). We collected possible explanation for the homeostasis relies on the fact urine samples from 31 patients who were classified into two that cells expressing the gal-3 gene are able to remove from groups: 13 patients with HBV infection and 18 patients in-

126 Poster Abstracts – Chemometrics and metabolomics fected with HCV. 1H NMR spectra were obtained using 400 study to identify in saliva changes of those individuals that µL of urine in natura added to 200 µL of a buffer solution may indicate the dynamic process of healthy or unhealthy (Na2HPO4/NaH2PO4 - 0.2 mol/L). We used the follows ex- state. perimental parameters: RF pulse of 45◦, acquisition time equal to 3.1 s, delay 1.0 s, 16 repetitions and presat pulse se- TH195: Metabolic profile of murine melanoma stud- quence. All the spectra were binned into 154 regions of 0.05 ied by Nuclear Magnetic Resonance ppm, excluding the region between δ 4.2 and 6.4 ppm. The cases were normalized by sum in row and auto-scaled in col- 3Pedro Bacchi, 3Roger Chammas, 3Silvina O. Bustos, umn. Statistical analyses were performed by PCA, LDA and 2Claudia M. G. de Souza, 1Antonio C. Bloise, 1∗Said PLS-DA. The PCA was unable to separate the two groups, R. Rabbani while the PLS-DA and LDA both showed adequate separa- 1São Paulo University/Physics Institute, 2Instituto de tion between the groups, but the validation of models were Pesquisas Tecnológicas, 3São Paulo University/ Faculty of not satisfactory. We decided to perform the LDA with the Medicine matrix of PC’s score and, after a variables selection, a better model was obtained. In the cross-validation, 85% and 83% of Using murine melanocytes, a new model of murine melanoma, Tm-1 the HBV and HCV samples, respectively, were classified cor- called , was produced by repeated cycles of cell disad- gal-3 rectly. The chemical shifts responsible for discrimination are hesion. In this strain, galectin-3 ( ), a multifunctional d 3.32 and 3.17 ppm, which are assigned to trimethylamine endogenous lectin which seems to act by modulating the mi- N-oxide (TMAO), which was found in higher concentrations tochondrial response to different types of stress and condi- in samples from patients with hepatitis B. tioning the cell death, is silenced. Through transfection of original Tm-1, we produced two new strains of melanomas, Acknowledgments: CNPQ, CAPES, FACEPE and one expressing gal-3, and the other not. In order to evaluate PRONEX. the changes in glucose metabolism induced by gal-3, these cells were cultivated in different conditions of oxygen tension References: and glucose level. Approximately 107 cells were collected from each culture. These were sonicated 5 times for 1 minute Godoy, M. M. G.; Lopes, E. P. A.; Silva, R. O.; Hallwass, F.; with intervals of 10 seconds between sonication, centrifuged Koury, L. C. A.; Moura, I. M.; Gonçalves, S. M. C.; Simas, at 4000 rpm for 20 minutes, the supernatant were collected A. M.. J. Viral Hepat., 2010, 17, 854-858. and lyophilized. The lyophilized extracts were diluted in D2O and finally their NMR spectra were obtained. These spectra TU194: NMR analysis of salivary metabolites of lac- were sectioned in small intervals and the integrals of these sec- tating women with chronic hypertension tions, representing the concentration of different metabolites present in the liquid, were analyzed by multivariate meth- 1∗Carla Martins, 1Livia Roberta P. de Oliveira, 1Luciana 1 2 ods. The modifications in concentration of certain metabo- Pereira, Tatiana Kelly S. Fidalgo, Luciana Pomarico, lites were attributed to the impact of gal-3 in the mitochon- 1Valéria Abreu, 3Romeu Ricardo da Silva, 1Liana B. Freitas- 4 4 1 drial homeostasis process. Among all metabolites pointed Fernandes, Fabio C. L. Almeida, Ana Paula Valente, Ivete out by multivariate analyses, the most relevant were lactate, P. R. de Souza alanine, free choline, GPC, PC, acetate, 3-hydroxybutyrate 1Departamento de Odontopediatria e Ortodontia, Univer- and taurine. The results suggest that gal-3 gene seems to sidade Federal do Rio de Janeiro., 2Departamento de act in the mitochondrial homeostasis only in the specific case Odontopediatria Universidade Federal Fluminense. (Nova where tumorigenic cell are exposed to stress, such as hypoxia. Friburgo), 3COPPE, Universidade Federal do Rio de In addition, when the homeostasis is triggered by hypoxia it Janeiro., 4CNRMN, Universidade Federal do Rio de is only carried out if there is an excess in glucose levels. A Janeiro. possible explanation for the homeostasis relies on the fact gal-3 Chronic hypertension during the pregnancy is appointed as that cells expressing the gene are able to remove from one of the most dangerous intercurrent diseases which can cellular environment those mitochondrias that do not prop- pyruvate lead to women’s death and contributes significantly to neona- erly metabolize received from the glycolytic path- tal morbidity and mortality. This indicates the importance way ("deficient" mitochondria). of prevention and early diagnosis of chronic hypertension. 1 ACKNOWLEDGMENTS: FAPESP, São Paulo University, The aim of this study was to identify, by H NMR, sali- CNPq vary metabolites in lactating women with chronic hyper- tension and compare with lactating healthy women. The MO196: 1H-NMR-Based Metabolomics of Chronic Hypertension Group (CHG) was composed by 12 Methotrexate Resistance in Acute Lymphoblastic lactating women with diagnostic of chronic hypertension and Leukemia Cell Lines the Healthy Group (HG) by 9 clinically healthy lactating women with no history of chronic illness - in particular, no 1,2Rafael Renatino Canevarolo, 1,2Carolina Pereira de history of chronic hypertension. Unstimulated whole saliva Souza Melo, 1José Andrés Yunes, 2∗Ana Carolina de Mattos was collected and the salivary samples were centrifuged at Zeri 4◦C and 10,000g for 60 minutes. The NMR spectra acquisi- 1Centro Infantil Boldrini, 2Laboratório Nacional de Bio- tion was performed by a Bruker 400MHz Advance spectrom- ciências eter equipped with a 5 mm high-resolution probe and operat- ing at a frequency of 400.13 (1H) MHz. Statistical analyses Methotrexate (MTX), a folic acid antagonist used against were carried out applying MATLAB software for the anal- acute lymphoblastic leukemia (ALL), prevents cell division ysis of metabolites data. The PCA and k-means methods by inhibiting nucleotide synthesis, therefore causing cell were applied. The PCA showed that at PC1 samples from death. In this work, intracellular metabolic concentrations CHG and HG were positioned separately, revealing a ten- were associated with MTX-resistance in a series of ALL cell dency of different profiles of salivary metabolites between the lines. MTX-resistance (or susceptibility) was determined groups. However, the sample of three subjects from HG were by the MTT method in six B- and eight T-derived cell found along the CHG. The application of k-means method lines. Five cell lines were classified as MTX-resistant and (which is based on grouping data searching for similarities the remaining cell lines, as MTX-sensitive. The intracellular between these) revealed that from the three samples from metabolic content was assessed by proton nuclear magnetic HG found between the samples from CHG, two samples were resonance (1H-NMR) of cells after 24h culture with MTX or considered (by similarities) as belonging to CHG. This means control. In total, 84 metabolites were quantified, 72 of which that, although these women are considered as healthy ones, were also identified. Multivariate analysis by partial least maybe they present a tendency to develop chronic hyperten- square discriminant analysis (PLS-DA) highlighted the main sion. These results motivate further longitudinal following up metabolites that were most associated with MTX-resistance

127 Poster Abstracts – In-vivo and in-cell NMR or -susceptibility, from which the receiver operating char- more, the binding of these effector molecules may alter the acteristic (ROC) curves evidenced carnitine, CB-MTX (un- pathway through which proper, biologically active confor- known compound), cholate, glycocholate, malate and suc- mations are achieved. It is not clear a priori that the fi- cinate as good metabolic biomarkers of MTX-susceptibility, nal conformation and commensurate activity of the complex whereas phosphocholine and sarcosine have proved to be will be different due to this temporal control. The Mycobac- good biomarkers of the MTX-resistant phenotype. The com- terium tuberculosis proteasome is required for maximum vir- bination of carnitine, sarcosine and succinate showed a 100% ulence and to resist killing by the host immune system. The sensitivity (15 out of 15 samples) and 92.3% specificity (24 prokaryotic Ubiquitin-like protein, Pup-GGE, targets pro- out of 26) in classifying untreated cell lines. In MTX-treated teins for proteasome-mediated degradation. We demonstrate cells, glycocholate, CB-MTX, sarcosine and succinate exhib- that Pup-GGQ, a precursor of Pup-GGE, is not a substrate ited 100% sensitivity (15 out of 15) and 85.2% specificity (23 for proteasomal degradation. Using STINT-NMR, an in- out of 27). Despite the necessity of validation in patients’ cell NMR technique, we studied the interactions between samples, these results pointed to a promising tool for the Pup-GGQ, mycobacterial proteasomal ATPase, Mpa, and 1.2 early detection of MTX-resistance in ALL. megadalton Mtb proteasome core particle (CP) inside a liv- ing cell at atomic resolution. We showed that under in-cell in vitro TU197: NMR-based metabolomics profiling to de- conditions, contrary to results, in the absence of the velop prognostic and diagnostic models for an au- proteasome CP, Pup-GGQ interacts with Mpa only weakly, toimmune disease primarily through its C-terminal region. When Mpa and non-stoichiometric amounts of proteasome CP are present, 1Marcos Rodrigo Alborghetti, 2Maria Elvira Pizzigatti both the N-terminal and C-terminal regions of Pup-GGQ Correa, 1Mauricio Luis Sforça, 1∗Ana Carolina de Mattos bind strongly to Mpa, commiting a pupylated substrate for Zeri degradation. This suggests a mechanism by which transient 1Brazilian Biosciences National Laboratory, Brazilian Cen- binding of Mpa to the proteasome CP controls the fate of ter for Research in Energy and Materials, 2Hematology and Pup. Hemotherapy Center, State University of Campinas. ACKNOLEDGEMENTS: This work is supported by NIH Background: we are studying, in patients from Hemocen- grant R01GM085006 to A.S.. tro, an autoimmune disease that causes high morbidity and mortality. Many tissue injuries due to immune response af- MO199: Ex vivo NMR during human cell cycle con- fects skin, salivary glands, eyes, GI tract, lung and liver. Ob- trol jective: the aim of this work is to develop prognostic and ∗ diagnostic models for this disease based on the metabolomics Jochen Balbach profile of the serum blood of this patients. Methodology: Institute of Physics, Biophysics, Martin-Luther-University time series serum blood collection was performed in periods Halle-Wittenberg, Germany during the patient’s hospitalization and after discharge. The Cyclins and cyclin-dependent kinases (CDKs) are key regu- samples were filtrated at 3kDa filter, buffered with phosphate lators of the human cell cycle. CDK4/6 assemble with D- buffer, referenced with D2O and DSS. The acquisition of 1D type cyclins for progression from the G1 to S-phase and are spectra was performed on a Agilent/Varian INOVA spec- negatively controlled by CDK inhibitors of the INK4 fam- trometer operating at 1H resonance frequency of 600MHz. ily. P19INK4d belongs to this inhibitor family, which gets The spectra processing, identification and quantification of posttranslationally phosphorylated. In earlier studies [1-3] metabolites were performed by using the application pack- we could show in vitro by glutamate substitutions, mimicking age Chenomx NMR Suite and the target profiling approach phosphorylation, local unfolding of 2 of the 5 ankyrin repeats (Chenomx, Inc). The PCA and PLS-DA statistical analy- of p19INK4d. This prevents CDK4/6 inhibition. Now, we ses were performed at Pirouette 4.0 (Infometrix, Inc) and succeeded to follow this regulation in much more detail and MetaboAnalyst 2.0. Results: we were able to develop pre- very close to the in vivo situation by simply adding crude cell liminary prognostic models at T1, T3 and T4 times com- extracts from various cell lines (HeLa, HEK-293, MDA-MB- paring patients that will develop the disease (T4, n=9) with 231 etc.) to the isotope labeled p19INK4d NMR samples. patients that will not develop the disease (T4, n=8) and a di- The NMR read out directly allows to reveal, which serine agnostic model comparing T5 (with the disease, n=10) with residues get phosphorylated and consequently which ankyrin T4 (without the disease, n=17). Here we show the model repeats locally unfold. By synchronizing the cells and by for the time T4 (figure 2-IV). The PLS-DA statistical model employing CDK inhibitors, the so far unknown kinases of with 7 metabolites shows accuracy = 1.0, Rcal (R2) 0.86529 p19INK4d could be identified. Dephosphorylation by sub- and Rval (Q) 0.68104. The permutation test shows p=0.017. sequent addition of a phosphatase confirms the integrity of Conclusions: this pilot study suggests that use of time- p19INK4d in the cell extracts by fully native NMR spectra. series models based on metabolic profile could be usefull in These high resolution data furthermore revealed, which ser- the medical routine and stimulate a populational study. ine has to become phosphorylated to induce ubiquitination ACKNOLEDGEMENTS: FAPESP and subsequent proteasomal degradation of CDK inhibitor p19INK4d and thus progression of the cell cycle to the next phase. These ex vivo NMR methods correlate cell biology In-vivo and in-cell NMR methods with structural biology. REFERENCES:

TH198: In-cell fate of Prokaryotic ubiquitin-like pro- 1. Löw C, Homeyer N, Weininger U, Sticht H, Balbach J. tein, Pup, inside the Mycobacterium proteasome. (2009), ACS Chem. Biol. 4, 53-63.

∗Alexander Shekhtman, Andres Maldonado, David Burz, 2. Löw C, Weininger U, Neumann P, Klepsch M, Lilie H, Sergey Reverdatto Stubbs MT, Balbach J. (2008), PNAS 105, 3779-3784. State University of New York at Albany, Albany, NY, USA 3. Löw C, Weininger U, Zeeb M, Zhang W, Laue ED, Schmid Inside a cell, macromolecular complexes are assembled along FX, Balbach J. (2007), J. Mol. Biol. 373, 219-231. specific pathways necessary to carry out biological functions in the presence of a crowded cytosol. Often, during assem- TU200: In-vivo NMR- Acetate Metabolism in C. bly, effector molecules such as ligands or substrates are also reinhardtii present. The presence of these molecules prior to or follow- ∗ ing the expression of components of the complex can play Himanshu Singh, K. V. R. Chary a regulatory role in the assembly of that complex. Further- TIFR

128 Poster Abstracts – In-vivo and in-cell NMR

In recent times, Chlamydomonas reinhardtii a unicellular Department of Electronics Science, Xiamen University, Xi- algae has been a model organism for studying metabolic amen, China C. rein- changes associated with biofuel production. Besides, Localized magnetic resonance spectroscopy (MRS) allows hardtii offers duality of metabolism being both phototrophic noninvasive in vivo analysis of metabolites. Due to the nar- and hetrotrophic in nature. The major metabolic flux it row chemical shift range and complexity caused by the reso- follows in various conditions is not yet clearly understood. nances of metabolites with coupled spin systems, the spectral On the other hand, live cell NMR, a non invasive technique, resolution of one-dimensional MRS is often low. Therefore has been extensively used in the past to detect metabolites two-dimensional (2D) MRS was introduced to alleviate the in the cells. Against this backdrop, we set out to monitor spectral overlap in one-dimensional MRS [1]. Unfortunately, C. reinhardtii live metabolic changes in cells during light numerous indirect dimension increments have to be collected (mixotrophic) and dark (heterotrophic) phases of growth as for a conventional 2D spectrum with good resolution, which well as following the UV/nutritionally stressed stationary leads the scan fairly time-consuming. This greatly hinders in in-cell C. rein- phase cultures. The metabolic changes in vivo application of localized 2D MRS. In this work, a new hardtii 13 cells were followed by monitoring [1, 2- C]-labelled scheme for ultrafast 2D spatially-encoded localized COSY is acetate assimilation over a period of 8 days. For the initial designed based on spatially encoded technique [2]. In this 24 hours of detailed kinetic measurements, the assimilation in sequence, the usual indirect dimension encoding is replaced dark phase of growth was faster than light phase of growth, by a spatial encoding, which is decoded by an echo planar reflecting higher heterotrophic metabolic efficiency in dark imaging alike detection scheme during the detection period. over mixotrophic metabolism in light. While bicarbonate was It can be executed for a 2D localized COSY spectrum in the predominant metabolite observed both in light and dark the order of sub-second. This enables the study of short phases of growth, Carbon dioxide was observed only during timescale phenomena via 2D MRS and may greatly reduce the dark phase of growth, reflecting a differential metabolic the motion effects. Theoretical analysis and experiments are flux in growth. UV stress decelerated acetate assimilation performed to show the performances of the new sequence. and metabolite formation in cells. The UV effect also re- The method opens important perspectives for fast in vivo sulted in an increased bicarbonate accumulation perhaps due analysis of metabolites in living organisms and may promote to reduced efficiency of carbon concentrating mechanisms op- the wide in vivo application of 2D localized MRS for more erating in the cell. When the cells were incubated in light or information which is not available in one-dimensional MRS. dark for up to eight days and monitored at intervals of about 48 hours, further remodeling in metabolism took place where ACKNOWLEDGMENTS: This work was supported by the in bicarbonate and Carbon dioxide were routed towards li- NNSF of China under Grant 11174239. pogenic pathway leading to lipid body production containing triacyl glycerol(TAG). We explain this metabolic flow by a [1] Lin, Y. Q.; Gu, T. L.; Chen, Z.; Kennedy, S.; Jacob, M; model for acetate assimilation in the cell. Zhong, J. H. Magn. Reson. Med., 2010, 63, 303-311. ACKNOLEDGEMENTS: TIFR NMR facility-Mumbai, In- [2] Tal, A.; Frydman, L. Prog. Nucl. Magn. Reson. Spec- dia trosc., 2010, 57, 241-292.

TH201: Baseplate protein organization of chloro- TU203: Analysis of proteins inside human cultured some mutant from green-sulfur bacteria Chlorobium cells using 19F-NMR Tepidum, observed with atomic resolution in vivo 1Syuhei Murayama, 2Kohsuke Inomata, 3Ayako Ohno, 4 1∗ 1 1Natalia Kulminskaya, 1∗Niels Christian Nielsen, 1Jakob Kenichi Aakagi, Hidehito Tochio, Masahiro Shirakawa 1 1 2 Nielsen, Morten Bjerring, Karen Thompson, Martin Lin- 1Deptatment of Molecular Engineering, Graduate School of dahl Engineering, Kyoto University, 2Quantitative Biology Cen- 1Aarhus University, 2Lund University ter, RIKEN, 3Division of Nutritional physiology, Institute of Health Biosciences, The University of Tokushima Graduate In order to obtain high resolution structure information for School, 4National Institute of Biomedical Innovation, Japan proteins in native heterogeneous environment, one should choose either cryo-electron microscopy[1] or solid-state NMR In-cell NMR is an isotope-aided NMR technique that en- spectroscopy[2]. However, combinations of these two tech- ables observations of structure of proteins in living cells. In niques provide even more powerful instrumentation for pro- mammalian in-cell NMR, the method has mostly relied on 2D 1 15 15 tein research. In this work we present organization of the H- N correlation spectra of uniformly N-labeled proteins. baseplate in the photoreceptor from Chlorobium Tepidum However, by employing amino acids specific labeling, in-cell with atomic resolution. Even thought the baseplate was ear- NMR spectroscopy of proteins can be substantially simplified 19 lier observed with freeze-fracture electron microscopy tech- and thus facilitated. In this study, in-cell F NMR spec- 19 nique, high resolution of the baseplate has been never troscopy of F-labeled proteins in HeLa cells was examined. achieved. We compare solid-state NMR protein structure FKBP12, a target protein of immunosuppressants, was em- with the 3-dimentional cryo-EM reconstruction of the pro- ployed as a model system. The p-fluoro-L-phenylalanine- tein baseplate and also demonstrate abundance of new in- incorporated FKBP12 was prepared and delivered into HeLa formation in the supramolecular organization of the protein- cells using the CPP (Cell Penetrating Peptide) -mediated pigment composition. protein transduction method. 19F NMR spectra of the cells [1] a) G. Zanetti, J. A. Briggs, K. Grunewald, Q. J. Satten- were acquired in the presence or absence of FK506 in the cell tau, S. D. Fuller, PLoS pathog. 2006, 2, e83; b) F. Brandt, culture. The obtained 19F NMR spectra showed that the spe- L. A. Carlson, F. U. Hartl, W. Baumeister, K. Grunewald, cific complexes between the protein and FK506 was formed Mol. Cell 2010, 39, 560-569. in the cells. Our data demonstrates that in-cell 19F NMR provides an efficient way to monitor proteins functioning in [2] a) A. C. Sivertsen, M. J. Bayro, M. Belenky, R. G. Grif- living HeLa cell. fin, J. Herzfeld, J. Mol. Biol. 2009, 387, 1032-1039; b) A. Goldbourt, B. J. Gross, L. A. Day, A. E. McDermott, J. Am. 1 Chem. Soc. 2007, 129, 2338-2344; c) R. Fu, X. Wang, C. TH204: H-NMR metabolic profiling of breast can- Li, A. N. Santiago-Miranda, G. J. Pielak, F. Tian, J. Am. cer cell lines as a tool for understanding the process Chem. Soc. 2011, 133, 12370-12373. of tumor metabolism adaptation. ∗Melissa Quintero Escobar, Rafael Renatino Canevarolo, MO202: Ultrafast Localized Two-Dimensional Cor- Kaliandra Gonçalves, Sandra Martha Gomes Dias, Ana Car- related Spectroscopy olina de Mattos Zeri ∗Zhong Chen, Yanqin Lin, Shuhui Cai Laboratorio Nacional de Biociencias, LNBIO.

129 Poster Abstracts – Magnetic Resonance Imaging

The analysis of metabolites by nuclear magnetic resonance the origins of presbyopia - the loss of the ability to focus on can be used to define the metabolic phenotype of cells, tis- near objects (i.e. to "accommodate") with age. sues or organisms.The metabolic phenotype of a cell is not static, but emerges from the coordinated and dynamic net- TU206: Spatial Resolution Enhancement in MRI work of interactions between genes, proteins and metabo- lites. Given the complexity of these relationships, the ap- ∗Daniel Fiat plication of metabolomics has the potential to contribute University of Illinois at Chicago to the understanding of biological mechanisms of tumors. The ’Metabolomic’ approach provides important informa- We report here the development of a novel method for en- tion about tumorigenesis, with the potential of revealing hancement of the spatial resolution in MRI. new therapeutic targets and identifying pathways that are perturbed in cancer. Apart from that, Metabolomic has Subpixel-shifted MR images were taken at several fields of been also broadly employed as a mean of finding diagnostic view to reconstruct the high-resolution image. The experi- and prognostic metabolic markers. Tumor cells have altered mental method, theory and method of data analysis will be metabolism compared to quiescent cells, needing constant described. supplements of macromolecular precursors for their growth Experimental results that demonstrate significant enhance- and proliferation. The glutaminolytic and glycolytic path- ment of the spatial resolution for 1H MRI studies of a phan- ways are the main generators of these metabolic precursors, tom, carried out with GE clinical scanner, will be presented. which will contribute to the synthesis of proteins, nucleic acids and lipids. The goal of this project was use 1H NMR to analyze the metabolic profiles of triple negative breast cancer TH207: Intra-aneurysmal flow determination by 3D cell lines along with other breast cancer cells lines displaying velocity maps using low field NMR tomography variable phenotypes regarding to the presence of progesterone 1∗Josefina Perlo, 2Emilia V. Silletta, 1Ernesto Danieli, (PR), estrogen (ER) and the membrane receptors Her2. The 2 1 1 triple-negative phenotype is well known as a difficult to treat Rodolfo Héctor Acosta, Bernhard Blümich, Federico type of breast cancer due to its resistance to the hormonal Casanova therapies. We identified and quantified in the triple-negative 1Institute of Technical and Macromolecular Chemistry, cell line MDA-MB231 several compounds related to the gly- ITMC, RWTH Aachen, Germany, 2Instituto de Física En- colytic pathway, such as lactate and ATP, as well as com- rique Gaviola, CONICET - Córdoba, Argentina pounds such as glutamate, glutamine and glutathione, all Dynamic NMR imaging is the only method capable of mea- precursors / products of the breakdown of glutamine. Fi- suring velocity patterns within flowing systems in a complete nally, we found high levels of O-phosphocholine, proline and non-invasive way. This makes MRI velocimetry an attrac- taurine, previously known biomarkers already described in tive technique to study flow phenomena in many areas of the literature, validating our methodology. Our perspectives research, in particular medical systems. Aneurysms, for in- are to extend the approach to other cell lines to look for spe- stance, are originated by a weakness of the vessel walls, lo- cific triple-negative biomarkers. cally increasing the normal size of the artery or vein. The Supported by: São Paulo Research Foundation (Fapesp). two events that dominate the evolution of an intracranial aneurysm are growth and rupture, being both dependent on intra-aneurysmal flow. Decrease of intra-aneurysmal flow is considered an alternative for treating intracranial aneurysms. Magnetic Resonance Imaging Such modification can be achieved by inserting stents or flow diverters. The determination of velocity patterns in such systems promises to be a powerful tool to prevent abnormal MO205: Measuring Optical and Refractive Proper- behaviors which can have severe health consequences. ties of the Human Eye with In Vivo MRI In this work we present low field NMR imaging for the study 1,2∗James M. Pope, 2Catherine E. Jones, 1Sanjeev of intra-aneurysmal velocity patterns under steady flow con- Kasthurirangan, 1David A. Atchison ditions before and after the insertion of a stent. A spin-echo 1 Institute of Health & Biomedical Innovation, Queensland pulse sequence was implemented for velocity map measure- 2 University of Technology, Brisbane, Australi, Faculty of ments in a portable low-field tomograph (0.22T) to study the Science & Technology, Queensland University of Technol- flow behavior in phantom systems resembling arteries with an ogy, Brisbane, Australia aneurism. 3D velocity maps were measured in liquids with We have employed MRI to study optical properties of the hu- symilar rehological properties as blood. It was posible to de- man eye that are not measurable by standard techniques of termine the influence of the local geometry of the artery in optometry. Unlike a conventional glass or plastic lens, where the internal flow of the aneurism. In absence of flow divert- refraction of light takes place only at the surfaces, the eye ers, a rotational vortex in the flow pattern could be observed. lens exhibits a refractive index distribution (RID), so that The flow effects due to the insertion of the stent provoked a refraction occurs continuously through the lens. To date it complete alteration of the flow patterns and a reduction of has not been possible to measure the refractive index distri- velocity magnitudes. bution non-invasively by optical techniques, without making assumptions about its shape and form. MO208: High throughput screen of mouse cardiovas- cular functions with MRI and Echocardiography We have developed an MRI technique that for the first time allows the refractive index distribution of human eye lenses to 1∗Luciana Caminha Afonso, 1Kristin Moreth, 1Valérie be measured and mapped non-invasively both in vitro and in Gailus-Durner, 1Helmut Fuchs, 1,2Martin Hrabě de Angelis vivo. We have used it to investigate changes in the distribu- 1Institute of Experimental Genetics, 2Technical Univeresity tion with age and the state of accommodation of the eye. The Munich method relies on the fact that the refractive index of the lens is determined by the concentration of proteins (crystallins) in The rapid increase in the development of mouse models is the lens tissue. This in turn determines the NMR transverse resulting in a growing demand for non-invasive physiological relaxation time (T2) of water protons. Consequently there is monitoring of large quantities of mice. Accordingly, we have a direct correlation between T2 and refractive index (n) that implemented an experimental set up, which enables us to per- can be exploited to map the refractive index distribution us- form high throughput non-invasive transthoracic echocardio- ing MRI. The results demonstrate that existing models of the graphy and electrocardiography on mice. For functional test- human eye lens are inadequate to describe changes that occur ing of the vascular system and detection of cardiac patholo- with aging of the lens. They also provide new insights into gies in mice, innovative techniques and miniaturized classical

130 Poster Abstracts – Magnetic Resonance Imaging physiological procedures are applied by us to achieve non- 1Alberto Tannús, 1∗Tito José Bonagamba invasive analysis of cardiovascular structure and function. 1Instituto de Física de São Carlos, Universidade de São 2 An electrocardiogram (ECG) is a recording of the electrical Paulo, Schlumberger Brazil Research and Geoengineering activity of the heart with surface electrodes. We recorded Center ECG’s to analyze cardiac rhythms, conduction and repolar- NMR relaxation and MRI are currently used as tools to study ization patterns within control wildtypes and mice suffering both the structure of porous media and the fluid dynamics oc- from cardiomyopathy. curring within, allowing the determination of characteristics Echocardiography is a non-invasive method to assess left ven- such as porosity and permeability. Carbonate rock cores are tricular systolic, diastolic, regional and vascular function. highly soluble in some acids, so reservoir matrix acid stim- Transthoracic echocardiography was performed in awake con- ulation provides a cost-effective means to enhance well pro- trol wildtypes and mice suffering from cardiomyopathy to sys- ductivity, where acid action forms special paths called worm- tematically evaluate heart rate, respiration rate, stroke vol- holes. These matrix acidizing experiments combined with ume, left ventricular mass, left ventricular contraction and visualization techniques as MRI and X-ray tomography are performance, systolic- and diastolic left ventricular interior commonly used to study wormhole networks. diameter, systolic- and diastolic thickness of the interventric- In this work, NMR relaxation and MRI were employed to ular septum and left ventricular posterior wall. characterize these materials and follow the wormhole forma- Cardiac magnetic resonance imaging is an important method tion. NMR relaxation experiments were performed using a TECMAG Redstone console with a 0.05 T permanent mag- to diagnose cardiomyopathy. Besides being able to assess di- 1 mensions of the left ventricle (at systole and diastole), cardiac net (2.64 MHz for H). Fast MRI sequences based on gradient MRI allows the analysis of the right ventricle, which is not echo, such as FLASH and UTE were used to characterize a 1" achievable with echocardiography. diameter and 3" length cylinder sample of Indiana limestone, before and after the acidizing treatment. MRI experiments Black-blood short-axis images were acquired with a fast low- were performed using a wide-bore (30 cm diameter) 2 T su- angle shot (FLASH) multi-slice sequence employing a self- perconducting magnet (85 MHz for 1H), interfaced with a gated protocol that incorporates a navigator echo to gener- Bruker Avance AVIII console. In order to perform the exper- ate cardiac triggering and respiratory gating signals retro- iments, a crossed saddle radiofrequency coil together with a ™ spectively (IntraGate ). The application of this technique gradient coil system was used, which was able to provide a reduces considerably the time for preparation of the animal, maximum gradient of about 15 G/cm. as the use of ECG leads and calibration of ECG signal for gating are unnecessary. Before the acid treatment of the Indiana samples, the UTE images showed that even pores with diameters of about 100 With the combination of these techniques we found signifi- mm can be visualized. Using FLASH technique, the images cant differences between control mice and mice developing a showed pores with diameters higher than 300 µm. Despite cardiomyopathy. Further on, this simple and fast protocol this lack of resolution for the micro- and meso-porosity sizes with its high sensitivity and specificity permits us to perform fraction, compared to the low-field NMR relaxation results, primary screening of the murine cardiovascular system in a it is enough to check for fractures and any related effect high throughput setting. that could influence the acidizing experiment results. The cores studied did not show any problem that could influence TU209: Developing an API and an IDE to Creating the wormhole pathway formation. After the acid treatment, and Managing Pusle Sequences for Nuclear Magnetic wormholes were easily observed using the FLASH MRI tech- Resonance nique, allowing the estimation of their diameters (around 1 mm). This information, associated with the acid preferred Daniel C. Pizetta, Danilo M.D. Delfino da Silva, Gustavo pathway visualization, could be used to characterize and op- V. Lourenço, Guilherme M. Freire, Felipe B. Coelho, Mateus ∗ timize the porosity and permeability gain observed after the Martins, Edson Luiz Gea Vidoto, Alberto Tannús acidizing treatment. University of São Paulo The next step is to apply and develop new techniques to imag- Due to the logistic importance of the Magnetic Resonance ing this media, especially those based on single point images Imaging (MRI) technology, the CIERMag - through the as SPRITE, which permits fast images, free of artifacts due ToRM-15 Project - is continually developing projects to com- to magnetic field inhomogeneities and magnetic susceptibility plete the domain of the technology of Nuclear Magnetic Res- contrast effects. onance. This paper will discuss the new tools adopted for building a fully Digital Spectrometer in its more complex Acknowledgments: IFSC/USP, FAPESP, CNPq, CAPES form, an MRI scanner. The effort is in particular on the de- velopment of the Application Programming Interface (API) MO211: In-vivo Controlled Deposition of Superpara- and Integrated Development Environment (IDE) to create magnetic Nanoparticles and manage pulse sequences and ancillary procedures as well 2 2 2 as the configuration of the spectrometer as a functional, Mayara Klimuk Uchiyama, Sergio Hiroshi Toma, Koiti Araki, 3Stephen Fernandes de Paula Rodrigues, 3Rodrigo friendly and free of commercial dependency of software and 1 1∗ hardware. Preliminary trials were performed in the spec- Azevedo Loiola, Hernán Joel Cervantes, Said R. Rab- trometer with digital technology entirely developed by the bani group, in order to validate the project, and some results have 1Instituto de Física da Universidade de São Paulo, 2Instituto already shown its ability to generate complex MRI sequences. de Química da Universidade de São Paulo, 3Faculdade de Ciências Farmacêuticas da Universidade de São Paulo Keywords: resonance magnetic imaging, free software, graph- ical pulse sequence editor, application programming inter- Superparamagnetic iron oxide nanoparticles (SPION) offers face, integrated development environment. many applications in biomedicine, such as the magnetic sep- aration of target cells, hyperthermia, magnetic resonance Acknoledgements: FAPESP: 2005/5.6663-1 e CNPQ: imaging (MRI) contrast enhancement and controlled depo- 565.047/2010-8, CAPES, FINEP, FNS. sition of stem cells or drugs.

TH210: Using fast MRI techniques to study acid- The superparamagnetic nanoparticles generate a spatial vari- treated rock cores ation of the magnetic susceptibility of the medium and, there- fore, the effect of the presence of SPIONs in the volume being 1 2 2 Roberson Saraiva Polli, Manuel Krebs, Andre Souza, scanned is mainly decrease the spin-spin relaxation time (T2) 2 2 2 Bernhard Lungwitz, Alexandre Pepin, Austin Boyd, and the decay time of the FID (T2*). As a consequence, the 1Edson Luiz Gea Vidoto, 1Fernando Fernandes Paiva, affected region becomes hypointense, i.e. there is a decrease

131 Poster Abstracts – Magnetic Resonance Imaging in the pixels intensity in the T2-weighted images. The pixel and have been characterized their T1 magnetic resonance reduction level depends on the nanoparticles quantity allow- imaging (MRI) properties and fluorescent signatures. Com- ing, thus, the determination of local concentration. paring with commercial contrast agent such as Omniscan®, 6-Gd3+-QD showed high relaxivities of 11.7 ± 0.1 mM−1 One wistar male rat was cannulated in the left femoral vein s−1 and 10.2 ± 0.1 mM−1 s−1 per Gd at 36◦C in phos- and a 1 ml suspension of SPIONs 10mg/mL with 5 nm of di- phate buffered saline (PBS) at 60 MHz and 200 MHz, re- ameter was injected directly into the vein. In the same paw, spectively. Also, in the cell uptake investigation, 6-Gd3+ QD µ 300 L of the same solution was injected in the muscle. At was successfully taken to the RAW264.7 cells. This nanopar- the opposite paw, one magnet was placed subcutaneously in ticle type of contrast agent can easily label cells for MR the thigh of the animal. The magnet stayed during 12 hours, imaging-tracking and have dozens of Gd3+ per particle which and 2 hours later, the rat was scanned. At the paw where it increases efficacy of labeling cells. In summary, a bimodal was affixed, due to the presence of the magnet, it was possi- fluorescent MR contrast agent, 6- Gd3+-QD, has been devel- ble to see an accumulation of nanoparticles, as the image of oped by conjugation of CdSe/ZnS QDs with a uridine-based the right thigh became black. paramagnetic complex (6-Gd3+). The rat was followed-up for more than 138 days and the clearance of deposited SPIONs was studied. The agglomera- MO214: Real-time visualization of myocardial in- tion of the nanoparticles remained visible in the image with flammatory evolution using magnetic nanoparticle- slow reduction over time. At the opposite paw, where the combined cardiac MRI SPIONs were injected directly into the muscle, the reduction 1,2Hyeyoung Moon, 1,3Jongeun Kang, 4Hyo Eun Park, was slower when compared with the first paw. Other organs 4 1,2,3∗ (liver, kidneys) were also observed. Kiyuk Chang, Kwan Soo Hong 1Divison of MR Research, Korea Basic Science Institute, 2 TU212: Exploring microscopic iDQC MR imaging Cheongwon, Korea, University of Science and Technology, 3 technique for iron-labeled single cell detection Daejeon, Korea, Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Ko- 1Jee-Hyun Cho, 1Kwan Soo Hong, 1,2Janggeun Cho, rea, 4Department of Internal Medicine, Catholic University, 1Cheajoon Cheong, 2Sangdoo Ahn, 1∗Chulhyun Lee Seoul, Korea 1Division of Magnetic Resonance Research, Korea Basic Sci- Clinically, myocardial inflammation, a critical pathogenic fac- ence Institute, Ochang, Republic of Korea, 2Department of tor in myocarditis, cannot be directly visualized by conven- Chemistry, Chung-Ang University, Seoul, Republic of Korea tional CMR. In our study, we investigated whether PEGy- Recently, to create magnetic resonance contrasts different lated fluorescent magnetic nanoparticles (MNP) could detect from those in conventional techniques, new magnetic reso- the inflammatory areas in experimentally induced autoim- nance imaging methods based on intermolecular double quan- mune myocarditis (EAM) rats, and compared the detectabil- tum coherences (iDQC) have attracted considerable atten- ity of focal inflammation area between MNP-enhanced CMR tion. Owing to its intrinsic sensitivity to changes in the and conventional CMR. MNP-CMR was also employed to susceptibility structures, iDQC imaging has the potential to track the evolution of inflammation in myocarditis. provide a fundamentally different contrast from that in con- We performed in vivo CMR in EAM and control rats using a ventional MRI. 4.7 T MRI system. At first, T2W, and pre-MNP MRI were In this study, we compared iDQC MR images with conven- performed, and then early gadolinium enhancement and late tional ones (e.g. GE, Spinecho EPI) acquired in various ex- gadolinium enhancement MRI were performed after tail vein ® perimental conditions to evaluate the contributions of exci- injection of Gd-DTPA (Magnevist , 0.1 mmol/kg). Then, tation and readout methods in image contrasts for detecting after 24 hrs of tail vein injection of home-made MNPs (10 iron-labeled single cells. mg Fe/kg), post-MNP MRI was performed. To observe the inflammatory evolution, we acquired the pre-MNP CMR im- ages at 15 day (15D) post-immunization (PI), and then 5 mg TH213: Uridine-based Paramagnetic Amphiphilic Fe/kg of MNP-FITC was intravenously injected. Post-MNP T1 MRI Contrast Agents and Nano-assembled Bi- CMRs were acquired at 16D and 20D PI. After acquiring modal Contrast Agent post-MNP CMR at 20D PI, same dose of MNP-RITC was 1Hyunseung Lee, 1,2Hyeyoung Moon, 1Sankarprasad Bhu- injected in the same rats. Then, post-MNP CMR imaging niya, 3Junwon Park, 3Sumin Lee, 3Jong Seung Kim, was performed at 21D PI. 1,2∗ Kwan Soo Hong Relatively large area of myocardial inflammation in EAM rats 1Division of MR Research, Korea Basic Science Institute, were detected by conventional T2W-, EGE-, and LGE-MRI, 2Department of Bio-analytical Science, University of Science while small-sized inflammation regions consistently failed to and, 3Department of Chemistry, Korea University be detected. The superiority of MNP-CMR to conventional The chelated Gd3+ metal ion improves imaging contrast by MRI was clearly shown for focal inflammation. Also, MNP- CMR with two times’ MNP injection could visualize the in- increasing the longitudinal relaxation time (T1) of proximal flammatory evolution . water protons, which appear brighter in the T1-weighted im- 3+ age. The current Gd -based contrast agents (CAs) ligated From our study, we could verify that MNP-CMR could effec- with polyamino carboxylate are incapable of meeting require- tively visualize myocardial inflammatory cellular infiltrates, ments as they do not have optimal relaxivity profiles at high and monitoring the dynamic evolution of myocardial inflam- magnetic fields. This requirement drives the research for mation in a preclinical model of EAM. We showed that MNP- smart contrast agents with high relaxivities (r1) for better CMR could be a highly efficient tool for visualizing myocar- tissue contrast at high magnetic fields. We have chosen a nu- dial inflammation in the early course of the disease with a cleoside as a molecular platform with Gd-DTPA to generate a potential for quantifying the response to specific therapies MRI contrast agent. The relaxation efficiency of these newly for myocarditis. synthesized amphiphilic MR CAs was determined by measur- ing longitudinal relaxivity (r1) and transverse relaxivity (r2) at 20 (0.47 T) and 60 MHz (1.41 T) in phosphate buffered TU215: High Performance of Magneto-fluorescent saline (PBS) at 36◦C The highest relaxivities achieved were Nanogels for Visualizing Migration of Dendritic Cells 30.3 and 23.4 mM−1 s−1 in PBS (pH 7.4) at 0.47 and 1.41 by both Magnetic Resonance and Optical Imaging T, respectively, for LGd3 (chain length = 10). 1Hyun Min Kim, 1Hyunseung Lee, 2Young-Woock Noh, 2 1,2∗ Uridine-based paramagnetic molecule (6-Gd3+) coated quan- Yong Taik Lim, Kwan Soo Hong tum dots (QDs) for multimodal imaging were synthesized 1Division of MR Research, Korea Basic Science Institute,

132 Poster Abstracts – EPR/ENDOR

Cheongwon, Republic of Korea, 2Graduate School of Analyt- tune the electronic structure of the center reducing by the ical Science and Technology, Chungnam National University, energy gap between σµ* and πµ electronic levels. Daejeon, Republic of Korea [1] Bertini, I.; Bren, K. L.; Clemente, A.; Fee, J. A.; Gray, Cell-based therapies have been focused on attention in re- H. B.; Luchinat, C.; Malmstr"m, B. G.; Richards, J. H.; generative medicine and cancer trials. Magneto-fluorescent Sanders, D.; Slutter, C. E. J.Am.Chem.Soc. 1996, 118, nanoparticles have a great potential for cell tracking that 11658. can possibly monitor labeled cells by MR/optical dual mode imaging. Such probes incorporate fluorescent quan- [2] Abriata, L. A.; Ledesma, G. N.; Pierattelli, R.; Vila, A. tum dots or organic fluorophores into iron oxide nanopar- J. J Am Chem Soc 2009, 131, 1939. ticles or gadolinium chelates. However, fluorescent com- [3] Abriata, L. A.; Alvarez-Paggi, D.; Ledesma, G. N.; Black- pounds incorporated to the magnetic nanoparticels could burn, N. J.; Vila, A. J.; Murgida, D. H. Proc Natl Acad Sci change intrinsic properties such as size and surface charge U S A 2012, 109, 17348. of nanoprobes. The ideal nanoprobes should be self- fluorescent without the need for conjugation of external MO217: Tuning the Electronic Structure of Electron fluorophores. We described synthesis of a self-fluorescent Transfer Copper Proteins Through Loop Engineer- high-relaxivity T2-weighted MRI contrast agent as a novel ing type of MR/optical multimodal imaging nanoprobe via elec- trostatic assembly and crosslinking of biocompatible poly- Andrés Espinoza Cara, Luciano A. Abriata, ∗Alejandro electrolytes and MnFe2O4 nanoparticles without fluorescent J. Vila nanocrystals or organic fluorophores. Multi-core property Instituto de Biología Molecular de Rosario (IBR-CONICET) of ionic assembled magnetic nanogels showed high relaxiv- ity, which r2 value of the self-fluorescent nanogels was 382.6 Nature employs metal ions for a wide variety of biological (Fe+Mn) mM−1s−1 , and fluorescence maximum peak at functions. Copper proteins play an important role in elec- λem = 553 nm and smooth shoulder peak at λem = 510 tron transfer processes. There are two types of copper elec- nm. The MR/optical multimodal imaging modalities of the tron transfer sites in nature: (a) mononuclear type I cen- γ-PGA/MnFe2O4/PLL(PEG) nanogels used for molecular ters (T1), also known as blue sites, and (b) the dinuclear 1 imaging of dendritic cells, which are commonly used in clin- CuA center . Proteins with T1 and CuA centers share a ical trials to induce tumor-specific immunity. We confirmed conserved fold (cupredoxin fold) despite being from different 2 that a bright fluorescence signal (in optical) and darken- organisms, revealing a common evolutionary origin . The ing effects (due to the shortening of T2 in MRI) in cellular functional properties of these redox sites cannot be compared level with a small amount of nanogels. The novel type of directly because of being in different protein matrixes. The MR/optical dual-modality nanoprobe also showed high per- T1 and CuA centers presents and ideal situation for loop formance in the labeling and monitoring of dendritic cells changing as they share the same protein fold and most of migrated to lymphnode in vivo. Crosslinking of the ionic the metal ligands are present in loops. There are few ex- assembled polyelec­trolyte magnetic nanogels with glu- amples using this strategy, but all of them suggest that the taraldehyde not only stabilized the nanogels but also gen- ligand-containing loops gather most of the structural infor- 3,4 erated a strong magnetic and self-fluorescence signals. The mation that regulates the function of the redox site . In novel multimodal imaging nanoprobes based on MnFe2O4 this project we employ loop directed mutagenesis in order nanoparticles/polyelectrolyte nanocomposites were used to to introduce different T1 loops in the soluble fragment of label and monitor therapeutic cell both in vitro and in vivo. the subunit II of the cytochrome ba3 oxidase from Thermus thermophilus, naturally harboring a CuA site (TtCuA). Two T1 variants were constructed, employing the loops from am- Paramagnetic NMR icyanin (Ami-TtCuA) and azurin (Az-TtCuA). These pro- teins have distinct characteristics from the native sites. Using distinct biophysical approaches such as optical spectroscopy, TH216: NMR characterization of an eukaryotic-like EPR and NMR we determined that both proteins have prop- paramagnetic CuA center erties that vary along those of rhombic T1 sites. Both pro- teins can also bind exogenous imidazole in a reversible way ∗ Marcos N. Morgada, Luciano A. Abriata, Alejandro J. giving rise to a copper site similar spectral features as those Vila found in nitrosocyanin5. Taken together, these observations Instituto de Biologia Molecular y Celular de Rosario (IBR- discard the "loop defines everything" hypothesis and show CONICET), Facultad de Ciencias Bioquimicas how reversible binding of small molecules can be elicited in an otherwise occluded metal site. The dinuclear copper center CuA is the electron entry point of cytochrome c oxidase (COX). CuA funnels the electrons References: from reduced cytochrome c to the CuB center at subunit I 1. Solomon, E. I.; Szilagyi, R. K.; DeBeer, G. S.; Ba- of COX where O2 is reduced to water molecules. These elec- sumallick, L. Chem.Rev. 2004, 104, 419-458. 2. Randall, tron transfer reactions are highly efficient and this efficiency D. W.; Gamelin, D. R.; LaCroix, L. B.; Solomon, E. I. is given by the characteristic coordination chemistry of this J.Biol.Inorg.Chem. 2000, 5, 16-29. 3. Hay, M.; Richards, center. J. H.; Y Lu, Y. PNAS, 1996, 93, 461-464 4. Li, C.; Banfield, Two alternative electronic ground levels, with different or- M. J.; Dennison, C. J.Am.Chem.Soc., 2007, 129, 3, 709–718 bital symmetries, have been established for the CuA center 5. Basumallick, L. Sarangi, R.; DeBeer George, S.; Elmore, +1.5 +1.5 in its oxidized form (Cu -Cu ) σµ* and πµ. NMR B.; Hooper, A. B.; Hedman, B.; Hodgson, K. O.; Solomon. studies of COXII from Thermus thermophilus (TtCuA) re- E. I. J.Am.Chem.Soc., 2005, 127, 10, 3531–3544. vealed the presence of signals with Non-Curie temperature dependence behaviour, indicating that these levels are very close in energy[1]. We have shown previously that the en- EPR/ENDOR ergy gap between these levels can be tuned by mutations of residues of the first coordination sphere of the CuA center in TtCuA[2,3]. TU218: EPR and ED ESE study of the nitroxide rad- icals confined in breathing MIL-53(Al) nanochannel Now we have replaced three loops in TtCuA by the ones system present in the human homologue. This substitution preserves 1,2∗ 2 2 both CuA center ligand identity and the loops length present Elena Bagryanskaya, Alena Nishchenko, Matvey in the native protein, introducing only second sphere pertur- Fedin, 3Alexander Stepanov, 3Daniil Kolokolov, 3Anton bations. Using NMR we have found that these mutations Gabrienko, 4Sergey Gromilov, 1Inna Shundrina

133 Poster Abstracts – EPR/ENDOR

1N.N. Vorozhtsov Novosibirsk Institute of Organic Chem- for nitric oxide [1] and spirocyclic substituted pyrroline ni- istry, Novosibirsk, Russia, 2International Tomography Cen- troxides as spin labels to study structure and functions of ter, Institutskaya, Novosibirsk, Russia, 3Boreskov Institute proteins [2]. The series of new spirocyclohexane substituted of Catalysis, Novosibirsk, Russia, 43Nikolaev Institute of In- nitroxides wew synthezised including thiol-specific methane organic Chemistry, Novosibirsk, Russia thiosulfonated spin label for site-directed spin labelling [2]. Metal-organic frameworks (MOFs), a novel class of micro and CW and pulse EPR was used to study the effect of spiro- meso-porous materials, have attracted much attention due cyclohexane moities on nitroxide’s chemical and spectral to their high practical importance in industry and medicine. properties. The obtained temperature dependencies of elec- Adsorption of radical molecules in MOFs could provide novel tron relaxation times demonstrate that the new nitroxides materials with new catalytic or magnetic properties. The may be suitable for PELDOR distance measurements at MIL-53 type (MIL: Materials of Institute Lavoisier) has a 80-120 K. Moreover, the new nitroxides demonstrate much remarkable feature. Transition from large pore (LP) to nar- higher stability towards reduction by ascorbic acid than row pore (NP) in MIL-53(Al) can occur both by influence spirocyclohexane-substituted nitroxides of piperidine series of guest and by changing the temperature. Moreover, this and showed 3-5 times lower reduction rates compared to cor- material is stable at up to 500◦C. 2H nuclear magnetic reso- responding 2,2,5,5,-teramethyl substituted nitroxides. The nance (NMR)[1-2] as well as continuous wave (CW) and echo first examples of application of new nitroxides to measure detected (ED) electron paramagnetic resonance (EPR) spec- distances in real biological systems will be shown. troscopy [3] are reliable tools to extract information about the Nitronyl nitroxides specifically react with NO to form imino dynamics of molecules confined in porous media. Since EPR nitroxides (IN), with comparatively high reaction constants is only sensitive to the paramagnetic guest it provides the of about 104 M−1 s−1. This reaction has been used for NO tool of choice to study the guest properties under modified detection in a number of biological applications, based on host conditions. We performed first EPR study of nitrox- significant differences in the electron paramagnetic resonance ide radicals (TEMPO and di-tert-butylnitroxide) adsorbed (EPR) spectra of NN and IN. New water soluble non-toxic inside MIL-53(Al). The temperature dependence of CW and nitronyl nitroxides (NN) as well as their fluorine substituted ED X-band (9 GHz) and Q-band (34 GHz) EPR spectra analogs were used as a spin probes for nitric oxide detection has been measured for polycrystalline MIL-53(Al) contain- in vitro and in vivo. Stability of these nitroxides was inves- ing probe radicals with various ratios per MIL-53(Al) unit tigated and possibility of their application as NO probes in cell. CW and ED EPR for samples with 1/1000 ratio of EPR imaging was studied. Experiments in vivo using EPR TEMPO demonstrated a temperature driven phase transi- imaging revealed that entire radical injected in a mouse in- tion from paramagnetic to diamagnetic state of absorbed travenously accumulates in urinary bladder. Injection of the molecules with pronounced structural hysteresis, in agree- nitronyl nitroxide while NO expression in mouse’ vessels in- ment with neutron diffraction and scattering experiments [4]. duced by nitroglycerin and observed subsequent slow down of It was shown observed that the EPR line shape is sensi- the nitronyl nitroxide accumulating in bladder and no EPR tive to the temperature prehistory. Moreover, it has been signal of imino nitroxide was detected. demonstrated that the reversible phase transition of MIL- 53 from LP crystalline state to the NP crystalline state is [1] R. Strizhakov, E.Fursova, N.Kolosova, L.Shundrin, accompanied by the reversible changes in the second inte- V.Ovcharenko, E.Bagryanskaya, Free Rad. In Biol.and Med. gral of the CW EPR spectra by a factor of three. This fact 2013, (to be submitted). was explained by partial complexation of TEMPO molecules [2] I.A. Kirilyuk, Y.F. Polienko, O.A. Krumkacheva, R. K. with OH groups of MIL-53(Al). The motion of the probe Strizhakov, Y.V. Gatilov, I.A. Grigor’ev, and E.G. Bagryan- molecules revealed by EPR reflected structural changes of skaya, J. Org. Chem., 2012, 77 (18), pp 8016–8027 Mil-53(Al). Temperature dependent CW EPR and ED ESE spectra were obtained and simulated using Easy Spin pro- Acknowledgment: This work was supported by the Ministry gramm.The X-ray and FTIR data confirmed the H-bonding of education and science of Russian Federation, projects 8456 between TEMPO and µ2-OH bridging hydroxyl group for the and 8456 and Russian Science Foundation, project 12-04- sample with 1/1 ratio of radical per unit cell. 01435. This work was supported by the Russian Foundation for Ba- sic Research, grant no.12-03-33010, 12-03-31329; RF Ministry MO220: Magnetic Transition of the Mineral for Education and Science no.8456 Goethite by Electron Paramagnetic Resonance ∗ [1] A.M. Nishchenko, et al. J.Phys. Chem., 2011. 115(26): Daniel Farinha Valezi, Eduardo Di Mauro p. 7428-7436. Universidade Estadual de Londrina [2] A.M. Nishchenko et al. , Journal of Physical Chemistry Synthetic and natural samples of the mineral goethite were C, 2012.116(16): p. 8956-8963. characterized by EPR, with the main objective of studying the magnetic transition from the antiferromagnetic state to [3] E.G. Bagryanskaya et al., Phys. Chem. Chem. Phys., the paramagnetic state, that this mineral undergoes upon 2009. 11(31): p. 6700-6707. reaching a certain critical temperature (Néel temperature). [4] Y. Liu, et al J.Amer.Chem. Soc. 2008. 130(35): p. 11813- EPR experiments were performed at microwave frequency 11818. of X-band (9.3 GHz) on a JEOL spectrometer (JES-PE-3X) in LAFLURPE Laboratory at the State University of Lon- drina. Experiments with temperature variation were per- TH219: New spin probes for spin-site labeling and formed with variable temperature controller (JES-VT-3A) in EPR imagine a range of 300 to 444K. Synthetic samples were measured in 1,2∗Elena Bagryanskaya, 1Igor Kirilyuk, 2Olesya powder form and natural samples were measured in powder Khrumkacheva, 2Rodion Strizhakov, 2Matvey Fedin, 1Igor and flakes form in quartz tubes of 4mm in diameter in case of Grigorjev, 1Denis Morozov, 1Yulija Polienko, 2Elena room temperature measures, and in tubes of 3mm in diame- Fursova, 2Evgeny Tretyakov ter in case of variable temperature. Although it will be not expected EPR signal in samples of goethite, due to its antifer- 1N.N. Vorozhtsov Novosibirsk Institute of Organic Chem- romagnetic arrangement, the resonance lines were observed istry of the SB RAS, Novosibirsk, Russia, 2International at room temperature, and this behavior was attributed to Tomography Center, SB RAS, Novosibirsk, Russia the existence of a weak ferromagnetism. When increasing of In this presentation we report the CW and pulse X-band and the temperature is above 348K for the natural sample, and Q-band EPR study of newly synthesized imidazoline, imida- 351K for the synthetic sample, goethite spectrum started to zolidine and fluorine substituted imino-nitroxide nitroxides present a new line of EPR, which grows in intensity until [1,2], in particular nitronyl nitroxides radical as spin probes it stabilizes at 440K. The appearance of this resonance line

134 Poster Abstracts – EPR/ENDOR was attributed to the magnetic transition that the mineral 1∗D. Kaminski, 1A. L. Webber, 1J. Liu, 1P.A. Goddard, goethite suffered with the increase in temperature above the 2J. L. Manson, 1A. Ardavan Néel temperature. The magnetic transition did not occur 1Centre for Advanced Electron Spin Resonance, Claren- at a certain temperature, this fact was attributed to non- don Laboratory, Department of Physics, University, uniformity of the samples in both particle size and vacancy 2Department of Chemistry and Biochemistry, Eastern Wash- concentration. For the first time were performed the sim- ington University, Cheney, WA, USA ulation of the EPR line of the mineral goethite due to the paramagnetic state, above the Néel temperature, as well as, In both 1D and 2D systems, the formation of long-range mag- the variation of signal intensity with increasing temperature. netic order is impossible at temperature T > 0 K. However, attempts experimentally to realise such systems are imper- fect, resulting in ’quasi’ 1D (Q-1D) and 2D (Q-2D) systems. TU221: Polymerization Process Interpretation of These have a small but finite coupling across the remaining Dental Resins via Electron Paramagnetic Resonance dimensions, meaning magnetic ordering at T > 0 K is a pos- (EPR) in X, Q and W Bands. sibility. 1Adriana Da Silva Fontes, 3Bruno Luiz Santana Vi- 2 3∗ Polymeric magnets are quasi-low dimensional and consist of centin, Walter Sano, Eduardo Di Mauro magnetic ions linked by organic groups, forming chains and 1Federal Technological University of Paraná, Campo layers. With the ability we have to fine-tune their magnetic Mourão, 2Physics Institute, University of São Paulo, São parameters, they provide a fertile test ground for aspects of Carlos, 3LAFLURPE, CCE, State University of Londrina, the quantum theory of magnetism [1, 2]. Londrina We here explore, via X- and D-band CW-EPR at variable Many studies have been done on photopolymerizable dental temperature, a group of coordination polymers which form resins, used on tooth restoration, because its properties, like antiferromagnetic states at T < TN where TN is on the or- color, hardness and resistance, shows that they are the best der of a few Kelvin. Through careful extraction of linewidth substitutes for the lost part of tooth. The most part of those and principle g-tensor components, we are successful in prob- studies have been done by Electron Paramagnetic Resonance ing the temperature region above TN . We therefore propose (EPR) experiments, which analysis in X band reveals a nine use of this method across a wider range of compounds to gain lines spectrum, due to free radical formation on the polymer- insight into the ordering process itself. ization process, initiated by radiating the sample with blue visible light. However, many authors still disagree about the [1] P. A. Goddard et al., New J. Phys. 2008 (10) 083025. formation of EPR spectrum. Some authors state that the [2] P. A. Goddard et al., Phys. Rev. Lett. 2012 (108) spectrum is formed by a sum of three or more radicals, oth- 0077208. ers says it is a superposition of two radicals, one with five lines and the other one with four lines. In this work, we uti- lized commercial resins Z100 (3M) to obtain EPR spectrum MO223: Chemical Tuning of Molecular Magnets for in X, Q and W bands in order to identify the free radicals in- Quantum Information Processing volved on the polymerization process, and also theoretically 1∗D. Kaminski, 1A. L. Webber, 1C. J. Wedge, 2E. T. Spiel- simulated the spectrums with computation resources. These berg, 2G. A. Timco, 2F. Tuna, 2E. J. L. McInnes, 2R. E. P. resins are composed essentially by a mixture of dimethacry- Winpenny, 1S. J. Blundell, 1A. Ardavan late monomers (Bis-GMA and TEGDMA), initiating agents 1Centre for Advanced Electron Spin Resonance, Claren- (Camphorquinone and Amine) and inorganic charge parti- 2 cles, zirconia and silica (ZrO2/SiO2). As blue visible light don Laboratory, Department of Physics, University, School source we used a LED (ULTRA BLUE-DABI ATLANTE) of Chemistry and Photon Science Institute, University of with intensity of 492 mW/cm2. The EPR experiments in X- Manchester, United Kingdom. band were performed on the JEOL spectrometer (JES-PE- As classical computational devices head towards the atomic 3X) at room temperature, at LALFURPE, in University of scale, they approach their technological limit. Quantum me- Londrina (UEL). A g marker of MgO:Mn2+ (g=1.981 in the chanical effects are coming into play and the world is seeking fourth line of the spectrum) was maintained in the cavity of a new way forward: the ’quantum computer’. A quantum the spectrometer, so that the data were obtained simultane- computer stores data as ’qubits’. These are two level sys- ously with the samples spectra. The samples were prepared tems that, as well as either state, can occupy a superposition in siliconia molds of 2x2mm, irradiated for 40 seconds and im- of the two. A system of n coherent qubits would allow us to mediately analyzed. EPR spectrum in Q-band were obtained form a superposition of 2n states and perform certain oper- in Biophysics Laboratory, at Physics Institute in University ations in fewer computational steps than is necessary with a of São Paulo (USP) at São Carlos, using a VARIAN (E-109) classical machine. spectrometer, having chrome as standard. Spectrum in W- band was obtained by a BRUKER (Elexsys E 680) spectrom- When in 2001 the theoretical possibility of using EPR on high eter, with a TerraFlex probe, in Bruker Laboratory at Rhe- spin molecular magnets to implement elementary quantum instettern, Germany, using samples with 1x1mm. We sim- algorithms was shown, research in the area exploded [1]. In ulated experimental results considering the spectrum been order to be useful in a quantum computer, the coherence time formed by the sum of three different radicals (named radicals of a qubit must exceed substantially the time required for I, II, and III) with distinct life-time. The radical II was con- single-qubit manipulations. At low enough temperature, this sidered having a very short life-time. The simulation showed is true of a Cr7Ni molecular nanomagnet, whose exchange that the spectrum is formed by a superposition of a weak coupled magnetic core can be treated as an effective electron nine lines spectrum of radical I with a strong five lines spec- spin-1/2 system [2]. By varying key structural components, trum of radical III, in perfect agreement with experimental Wedge et al. have studied the dominant mechanisms of de- results. The EPR spectrum of the composite resin has been coherence in the Cr7Ni nanomagnet family [3]. In particular, interpreted throughout the analysis performed in X-, Q- and a comparison of electron spin coherence times observed for 1 I 2 I W-band, with their simulations, concluding that for EPR are protonated ( H, = 1/2) and deuterated ( H, = 1) Cr7Ni proposed two methacrylate radical species: (-CH2-C.-CH3 - molecules has suggested that the nuclear magnetic moments ) and (-CH2-C.-CH2 -), called in this work of RI and RIII, in the extensive network of protons are key in limiting the respectively, which are present in concentrations very close. spin coherence times in these systems. These species are responsible for the continuity of the resin The present work aims to further isolate the sources of elec- polymerization process after photopolymerization. tron spin decoherence in molecular magnets, initially by chemical substitution of the proton network with halogen el- TH222: Antiferromagnetic ordering in quasi-low di- ements [e.g. 35Cl and 19F]. Coherence lifetimes are presented mensional polymeric magnets: a CW-ESR study for dilute, frozen solutions measured using a 2-pulse Hahn-

135 Poster Abstracts – EPR/ENDOR echo EPR sequence at X-band. In addition, the electron spin linearly independent planes gives us a linear set of equations. properties of Cr7Ni dimers are investigated as potential two- The solution of this linear system are principal values and di- qubit systems capable of more complex quantum algorithms. rections of the g(A)-tensor. Our method contains the Schon- land method as a particular case. We present here the ap- [1] M. Leuenberger and D. Loss, Nature 2001 (410) 789. plication of our method to LiMnPO4 single crystals. In this [2] A. Ardavan et al., Phys. Rev. Lett. 2007 (98) 057201. case, three planes were polished in the sample, and the ori- entation of them respect to the crystal axes obtained from [3] C. J. Wedge et al., Phys. Rev. Lett. 2012 (108) 107204. X-ray diffraction experiments.

TU224: ESR Study of Hybridization Effects in Some MO226: Light-Induced Synthesis and Paramagnetic New Dense Posphides Properties of Phenothiazine Nanoparticles in the Presence of Polyethyleneglycol Eduard M. Gataullin, ∗Vladimir A. Ivanshin Kazan Federal University 4Carolina G. dos Santos, 1André L. Silva, 2Alexandre J. C. Lanfredi, 3Otaciro R. Nascimento, 1∗Iseli L. Nantes The explanation of transformation of localized electrons at 1 2 high temperatures into itinerant quasi-particles with an en- CCNH - Universidade Federal do ABC, CECS - Univer- 3 hanced mass at lower temperatures belongs to the most im- sidade Federal do ABC, Universidade de São Paulo, São 4 portant challenges in the physics of the solid state. The Carlos, Universidade de Mogi das Cruzes mechanism of this evolution depends on the Kondo interac- Literature data report that phenothiazines can be converted tions between the CE and the localized d or f electrons. ESR to cation radical by enzymatic, chemical and and photochem- probes microscopically both the local moment (LM) spins and ical reactions. Previously, our research group had character- CE in different strongly correlated electron systems (SCES) ized the photochemical behavior of the antipsychotic drugs such as high-temperature superconductors, pnictides, heavy thioridazine (TR), trifluoperazine (TFP), and fluphenazine fermion systems. As a rule, LM ESR is studied in compounds (FP) influenced by the aggregation state of the molecules. with doped paramagnetic ions and localized electrons. ESR These compounds are converted to stable cation radicals in from CE can be detected in metallic systems with an en- high concentration in mild conditions, i.e., aqueous buffered hanced Pauli susceptibility. Surprisingly, ESR could be also solutions, pH 5.0. The stability of phenothiazine cation radi- observed in some dense SCES without any kind of param- cals in the phenothiazine aggregates at room temperature has agnetic doping (s. [1] and references therein). According to been assigned to the stacking of the thiazine phenyl moieties. several theories, a measurable ESR signals in these experi- Therefore, stable cation radicals of phenothiazines have been ments are caused by an effective hybridization between lo- described for aggregates of the drugs and for dimeric and calized f electrons and conduction bands in conjunction with oligomeric derivatives produced by chemical synthesis. How- ferromagnetic (FM) correlations which reduce usually a very ever, water-dependent dimerization of phenothiazines from fast spin-lattice relaxation [1, 2]. In the present contribu- chemically produced cation radicals was also described but tion, we report the temperature-dependent ESR spectra in the magnetic properties of these dimers as well as possi- two ternary recently synthesized phosphides YbRh6P4 and ble formation of nanostructures with these compounds re- CeIr2P2. A relatively weak ESR signals in both compounds mained to be investigated. In the present study, the synthesis reflect the extremely weak f-p hybridization effects in con- of polyethyleneglycol (PEG)/phenothiazine nucleus (PHT) trast to the case of silicides YbRh2Si2 and YbIr2Si2. Our nanoparticles (NP) using the photochemical properties of results in YbRh6P4 reveal temperature dependences of the PHT is characterized by electronic absorption (EA), elec- ESR g-factor and peak-to-peak linewidth for an Orbach-like tronic paramagnetic resonance (EPR), infrared spectroscopy 3+ ESR relaxation of the Yb LM which can be related to the (ATR/FTIR), atomic force microscopy (AFM) and scanning first excited crystal-field state at 82.1 K [3]. electronic microscopy (SEM). In the studied system, the UV light-induced formation of NPs is dependent of the forma- [1] P. Schlottmann, Phys. Rev. B 86, 075135 (2012) tion of PHT cation radical, the presence of water and acetate [2] D. Huber, Mod. Phys. Lett. B 26, 1230021 (2012) as the counterion. In the presence of PEG, the formation of NPs was modulated by the PHT/PEG ratio that influ- [3] V.A. Ivanshin et al., Journal of Phys.: Confer. Ser. 391, ences PHT aggregation and oligomerization. The aggrega- 012024 (2012) tion of PHT molecules stabilizes the cation radical and the photo-oligomerization is dependent of the access to bulk wa- TH225: The g-tensor for ESR in single crystals: a ter modulated by the microenvironment provided by PEG. In new approach for non-perpendicular faces PEG/PHT NPs, the photochemically produced cation radi- 1∗ 2 1 cals are stable but the distance between the radical center Cesar José Calderon Filho, David Vaknin, Gaston Bar- prevents they are coupled in a triplet state. beris 1IFGW, Unicamp, Campinas (SP) Brasil, 2Ames Lab, IO, ACKNOLEDGEMENTS: FAPESP, CAPES and CNPq USA TU227: Effects of KL4 on LIpid Bilayer Dynamics The electron paramagnetic resonance (EPR) spectra of mag- and Fluidity Properties netic ions in anisotropic single crystals are characterized by a g-tensor (and hyperfine tensor, if exists) that cannot be Otonye Braide, ∗Joanna Long, Gail E Fanucci obtained directly by a few measurements, except in the case University of Florida that directions of principal axes of the crystal respect to the macroscopic directions in the sample are known. To obtain KL4, a 21 residue mimetic of surfactant protein B, is effec- the parameters in the general case, Schonland proposed a tive in the treatment of infant respiratory distress syndrome method for obtaining the g-tensor but only for certain par- (RDS) and functions by lowering aveolar surface tension and ticular cases. This excludes single crystals without growing promoting oxygen exchange. We utilized a pyrene phospho- faces related to the principal axes of the unit cell. This work lipid analog to investigate the effect of KL4 on lipid orga- proposes a general method to obtain the principal values of nization and acyl chain dynamics by monitoring changes in the g- (and A) tensors, that can be used in any single crys- excimer-to-monomer (Ie/Im) ratio. This experiment probes tal, where it is known the orientation of three independent the environment of the hydrophobic core of DPPC/POPG planes respect to the crystal axes. Using the fact that the and POPC/POPG liposomes. An average decrease of 27- g- (and A) values in a plane can be written as g2 = A+B 40% and 0-10% in Ie/Im was observed in the DPPC/POPG cos(2 β) + C sin(2 β), where heta is the rotation angle and and POPC/POPG LUVs, respectively, with increasing pep- the parameters A, B and C are functions of the elements of tide concentration (0.5 to 5 mol%). This decrease is directly the g-tensor, the measurements of the spectra in the threee proportional to a lowered probability of excimer formation,

136 Poster Abstracts – EPR/ENDOR which is highly dependent on proximal interactions of an ex- in the the z axis. The evaluation of the Cu(II) bonding pa- cited monomer with a pyrene moiety at ground state. The rameters indicates that the in-plane sigma-bonding between ability of the peptide to modify membrane fluidity properties ethylenediamine and the copper becomes slightly more co- was studied via anisotropy measurements of a rhodamine- valent when the Cu(en)2 complex is introduced in the ben- labeled phospholipid. A steady increase in the order was tonite clay. Partial support of Capes, CNPq and Fapesp observed in the DPPC/POPG liposomes with relatively con- (Brasil) and of Chilean agencies FONDECYT (1090282), stant fluorescence intensity, while collisional quenching was CONICYT, FB0807 (CEDENNA), and ICM and Innovation observed in the POPC/POPG liposomes. These observations Fund for Competitiveness, Ministry of Economy, Develop- agree with NMR and EPR observations, and proposed mech- ment and Tourism Project (FP10-061-F-FIC), are gratefully anisms of peptide-mediated lipid trafficking. acknowledged. [1] A. Kaya, A.H. Oren, J. Hazardous Mat. B125 (2005) 183. TH228: Topological Games and Spin Gymnastics: a Systematic Approach to ENDOR Pulse Sequences [2] L. Chen et al., J. Radioanal. Nucl. Chem. 292 (2012) 1181. Daniel Finkelstein-Shapiro, ∗Vladimiro Mujica [3] L.F. Gonzalez Bahamon et al., Chemosphere 82 (2011) Arizona State University 1185. Pulsed electron-nuclear double resonance (ENDOR) tech- niques reveal the nuclei coupled to an electron spin. The [4] M. Lezhnina et al., Chem. Mater. 19 (2007) 1098. two fundamental sequences, Mims and Davies, come in sev- [5] S. Celedon et al., Materials Research Bulletin 44 (2009) eral flavors: hyperfine selective ENDOR, sequences indepen- 1191. dent of spectrometer deadtime, optimized polarization trans- fer just to name a few. An understanding of these sequences [6] C.J. Magon et al., J. Magnetic Resonance 184, 176 (2007). via the product operator formalism reveals which coherences contribute to the final electron magnetization carrying the TU230: Deconvolution of the EPR spectra of vana- electron-nuclear coupling information. dium oxide nanostructures In this work, we propose a classification of ENDOR experi- 1∗Cláudio José Magon, 1J. F. Lima, 1J. P. Donoso, 2V. ments based on the structure of the pulse sequence itself. We Lavayen, 3E. Benavente, 4D. Navas, 4G. Gonzalez decompose the pulse sequence in the basis of superoperators, 1Universidade de Sao Paulo, Sao Carlos, Brazil, where a pulse sequence is naturally represented by a path 2Universidad Tecnica Federico Santa Maria, Valparaiso, of vectors whose lengths correspond to the pulse duration Chile, 3Universidad Tecnologica Metropolitana, Santiago, or the time during which the system evolves. By restricting Chile, 4Universidad de Chile, Santiago, Chile our analysis to pulses along the x-axis, the pulse sequence is fully described in the basis (Sx, Ix, H), and as a conse- Vanadium oxide (VOx) nanostructures have attracted con- quence admits an intuitive representation in R3. Using this siderable attention in the last years because of their potential geometrical representation and a few constraints on starting importance for catalysis and electrochemical applications [1- and ending points, we can generate candidate sequences for 3]. This work reports an electron paramagnetic resonance ENDOR in a systematic way. We discuss new possible se- (EPR) study of two VOx nanostructures: nanotubes and quences, and begin to explore the properties of this space in- nano-urchins. Nanotubes consist of vanadium oxide sheets cluding its analogy with moduli spaces. We believe that this with alternating layers of long-chain alkyl amines, which new representation, which has applicability beyond ENDOR, bend to form one-dimensional tubular products [4,5]. The will provide a more transparent intuition and a systematic urchin-like nanostructure consists of high-density spherical way to explore new sequences. radial arrays of nanotubes [6]. The layers of the nanotubes are constituted by vanadium ions in two different oxidation 4+ states, magnetic V with spin S = 1⁄2 and non-magnetic MO229: EPR study of copper ethylenediamine in- V5+ with S = 0. The observed X-band EPR spectra of tercalated in bentonite clay VOx nanotubes consist of a hyperfine structure arising from 4+ 1∗Cláudio José Magon, 1J. P. Donoso, 1I. D. A. Silva, the isolated V ions superposed on a broad resonance line, 1 1 2 2 which is attributed to spin-spin exchange interactions be- Otaciro R. Nascimento, J. F. Lima, M. Moreno, E. Be- 4+ navente, 3G. Gonzalez tween neighbouring V sites [7,8]. In this work we applied the deconvolution approach based on the Krylov basis diago- 1Universidade de São Paulo, Brasil, 2Universidad Tecnológ- nalization method (KBDM) to separate the contributions of ica Metropolitana, Santiago, Chile, 3Universidad de Chile, isolated ions (sharper lines) and clusters (broader lines) in the Santiago, Chile experimental EPR spectra of the VOx nanotubes and nano- Bentonites are rocks dominated by smectites layer clay miner- urchins. The objective is to correlate the spin Hamiltonian als, which has attracted great interest in environmental pollu- parameters and simulation data with the physical structures tion treatment due to its remarkable properties [1-3]. Layered of different VOx nanostructures. Acknowledgments: Partial clays offer a rich inclusion chemistry, which allows to mod- support of Capes, CNPq and Fapesp (Brasil) and of Chilean ify and functionalize materials at nanoscale level, exhibiting agencies FONDECYT (P. 1090282), CONICYT, FB0807 new physical and chemical properties [4,5]. In this work we (CEDENNA), and ICM and Innovation Fund for Compet- report results from electron paramagnetic resonance (EPR) itiveness, Ministry of Economy, Development and Tourism spectroscopy of Cu(II) exchanged in bentonite and Cu(II) Project (FP10-061-F-FIC), are gratefully acknowledged bis(ethylenediamine), Cu(en)2, exchanged in bentonite. The observed X-band EPR spectra consist of a hyperfine structure [1] W. Jin, et al. Applied Surface Science 257 (2011) 7071. arising from the isolated Cu(II) ions superposed on a broad [2] S.D. Perera et al., Advanced Energy Materials 1 (2011) resonance line, which is attributed to spin-spin exchange in- 936. teractions between neighbouring Cu(II) ions.The observation of a single Cu(II) isolated species with well resolved hyper- [3] C. Wu, Y. Xie, Energy and Environment Science 3 (2010) fine structure in the EPR spectra of the Cu(II) exchanged 1191. bentonite suggests that copper ions preferentially occupy the [4] C. O’Dwyer et al., J. Electrochemical Society, 154 (2007) interlayer regions. The analysis of the spin Hamiltonian pa- K29. rameters suggests that paramagnetic Cu(II) ions are in ax- ially distorted sites. The analysis of the spin Hamiltonian [5] J. Livage, Materials 3 (2010) 4175. parameters indicates the presence of the Cu(en)2 complex in [6] C.O’Dwyer et al. Chemistry of Materials 18 (2006) 3016. the interlayer region of the bentonite, with the cooper coordi- nated to four nitrogen in the equatorial plane and to oxygen [7] M.E. Saleta et al., J. Applied Physics 109, 093914 (2011).

137 Poster Abstracts – Dynamic Nuclear Polarization

[8] C.J. Magon et al., J. Magnetic Resonance 222 (2012) 26. been to correlate the magnetic and optical properties of the materials in question, and elucidate the electronic structure TH231: Biophysical studies on the orientation of responsible for the unusual colors. This may result in prac- coiled-coils of septin peptides tical industrial applications since historically and currently used blue pigments all show certain deficiencies (e.g., in terms 1∗Edson Crusca, 1Patricia S. Kumagaia, 1Claudia Elisa- of toxicity and/or stability). Both Mn(III) and Fe(III) com- beth Munte, 2Antonio José Costa-Filho, 1Richard Charles pounds gave high-quality HFEPR spectra from low to room Garratt temperature showing a variety of spin concentration- and 1Grupo de Biofísica Molecular “Sérgio Mascarenhas”, In- temperature-dependent phenomena that will be discussed stituto de Física de São Carlos, Universidade de São during the presentation. 2 Paulo, Laboratório de Biofísica Molecular, Departamento References de Física, Faculdade de Filosofia, Universidade de São Paulo [1] Smith, A.E., et al., J. Am. Chem. Soc., 2009, 131, 17084- Mammalian septins form a subfamily of GTP-binding pro- 17086. teins conserved from eukaryotic protists to mammals that are involved in various cellular processes as well as in di- [2] Mizoguchi, H., et al., Inorg. Chem., 2011, 50, 10-12. verse neuropathological conditions. Specific combinations of septins form hetero-oligomeric complexes that polymerize in TU233: EPR and ENDOR Studies of Two-Iron vivo and in vitro into non-polar filaments. Septins share a Ferredoxins, [2Fe-2S]+, Isolated from the Hyper- conserved GTP-binding domain and a predicted coiled-coil thermophile Aquifex aeolicus domain at their C-terminus. Although the crystal structure 1∗Joshua Telser, 2George Cutsail III, 2Peter E. Doan, of the complex formed by human septins SEPT2, SEPT6 and 2 3 SEPT7 has been determined by Sirajuddin et al. (2007), no Brian M. Hoffman, Jacques Meyer electron density was observed for the C-terminal domains. 1Roosevelt University, 2Northwestern University, Therefore, the structure provided no information about how 3BMC/DRDC CEA these domains associate to assemble the hetero-oligomeric We have employed EPR and a set of recently developed complexes. A model for the formation of septin complexes Electron Nuclear DOuble Resonance (ENDOR) spectro- was postulated by Kinoshita et al. (2003): SEPTX/6/7, scopies to characterize a suite of [2Fe-2S] ferredoxin clusters where X could be SEPT1, 2, 4 or 5 (members of the SEPT2 from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5), which were group). Here we present biophysical studies on a series of grown on 57Fe-enriched media.[1] Antiferromagnetic coupling peptides derived from SEPT2 group proteins, which corre- between the FeII , S = 2, and FeIII , S = 5/2, sites of the [2Fe- spond to the central region of the septin C-terminal domain. 2S]+ cluster in these proteins creates a total spin, S = 1/2 The four peptides have been manually synthesized by Fmoc- ground state. Complete 57Fe hyperfine coupling (hfc) tensors SPPS strategy with an additional cysteine residue at the C- for both iron sites, with respective orientations relative to terminus region in order to allow for the selectively label- the g matrix have been determined by the use of "stochastic" ing with the spin-label 1-oxyl-2,2,5,5-tetramethylpyrroline-3- continuous wave and/or "random hopped" pulsed ENDOR, methyl-sulfonate (MTSSL). Samples were purified by HPLC- with the relative utility of the two approaches being empha- RP (>95% of purity). The peptides have been analyzed by sized. The reported hyperfine tensors include absolute signs 2D-NMR and showed a helical conformation. CD measure- determined by a modified Pulsed ENDOR SaTuration and ments indicated a high helical content and a typical coiled- ◦ REcovery (PESTRE) technique,[2] RD-PESTRE, a post- coil signal for temperatures below 10 C. For each peptide processing protocol of the ’Raw Data’ that comprises an the coiled-coil orientation was determined by CW-EPR ex- ENDOR spectrum. The 57Fe hyperfine tensor components periments, where the interaction of the MTSSL spin label found by ENDOR are nicely consistent with those previously yielded large dipolar broadening in the cases where the pep- found by Mössbauer spectroscopy, while accurate tensor tides adopted a parallel orientation. Our results show that orientations are unique to the ENDOR approach. These peptides SEPT1CC, SEPT4CC and SEPT5CC are antipar- measurements demonstrate the capabilities of the newly de- allel, whereas SEPT2CC is parallel. Thus, despite belonging veloped methods. The high precision hfc tensors serve as a to the same group, the peptides can form coiled-coils with benchmark for this class of FeS proteins, while the variation distinct orientations, which may have crucial implications in in the 57Fe hfc tensors as a function of symmetry in these the formation of septin complexes. small FeS clusters provides a reference for higher nuclearity 1 2 Supported by FAPESP, CNPq and CAPES. FeS clusters, such as those found in nitrogenase. H and H (of protein in D2O buffer) ENDOR studies of these systems will also be described. MO232: High-Frequency and –Field EPR (HFEPR) Studies of New Magnetic Materials with Unusual Op- References tical Properties that Contain High-spin Mn(III) and [1] Mitou, G, et al., Biochemistry, 2003, 42, 1354-1364. Fe(III) [2] Doan, P. E., J. Magn. Reson., 2011, 208, 76-86. 1∗Joshua Telser, 2J. Krzystek, 2Andrew Ozarowski, 3Peng Jiang, 3M. A. Subramanian 1Roosevelt University, 2NHMFL Florida State University, Dynamic Nuclear Polarization 3Oregon State University The Subramanian group at Oregon State University has re- TH234: Designing Radicals and Surfaces for Ef- cently prepared a variety of new oxide-based materials based ficient Dynamic Nuclear Polarization Surface En- on YInO3 [1-2]. In these materials, the In(III) ions are substi- hanced NMR Spectroscopy. tuted by either Mn(III) or Fe(III), which each find themselves in a trigonal-bipyramidal (tbp) environment. While YInO3 1∗Alexandre Zagdoun, 2Olivier Ouari, 2Gilles Casano, is white, and YMnO3 and YFeO3 are black, systems such 1Aaron J. Rossini, 3Maxim Yulikov, 3Gunnar Jeschke, as YIn1−xMnxO3 and YIn1−xFexO3 where 0 < x < 1 dis- 3Christophe Copéret, 1Anne Lesage, 2Paul Tordo, 1Lyndon play intense colors; in the case of Mn(III) the color is bright Emsley blue for 0.02 < x < 0.25. Since both Mn(III) and Fe(III) 1Centre de RMN à Très Hauts Champs, Université de Lyon, ions are magnetic (respectively, S = 2 and S = 5/2), and Villeurb, 2Aix-Marseille Université, Institut de Chimie Rad- are in an uncommon coordination environment, we initiated icalaire, Marseilles, 3ETH Zürich, Department of Chem- a High-Frequency and -Field Electron Paramagnetic Reso- istry, Zürich, Switzerland. nance (HFEPR, using frequencies above 90 GHz, B0 up to 25 T) study of these materials. The aim of this project has Dynamic nuclear polarization (DNP) is attracting more and

138 Poster Abstracts – Porous Media more interest as a method to increase the sensitivity of NMR PEDRI experiments. EPR transmit system, surrounding experiments. In 2010 our research group described the ap- NMR resonator, based on a modified Alderman-Grant design. plication of DNP enhanced solid-state NMR spectroscopy Also, specially design loop-gap double frequency resonator for the characterization of surfaces, a method dubbed DNP with coaxial B1 fields was successfully tested for PEDRI. SENS. However the enhancements routinely obtained at 9.4 Studies performed with phantoms and mammary tumor bear- T and 100 K are still far from the theoretical maximum (70 ing living mice at fields of about 200 G (EPR frequencies vs 660), and sensitivity remains an issue for challenging sur- 560 MHz, MRI - 784.9 kHz). pH-sensitive nitroxide with face experiments. Here we show how enhancements up to 200 enhanced stability towards reduction, narrow linewidth and can be obtained through controlled design of radicals and the extracellular targeting was used for in vivo studies. pH map surface. was extracted using only two PEDRI images acquired at pre- selected 8 sec EPR excitations providing pH resolution of 0.1 Biradicals have been demonstrated to be the most efficient pH unit and a spatial resolution of 0.5 mm. Acidosis of tumor polarizing agents at high magnetic fields. The efficiency of tissue was observed. these polarizing agents has been shown to depend on many parameters, such as the relative orientation of the electron ACKNOWLEDGEMENT: This work was supported by NIH g tensors and the strength of the electron-electron dipolar Grant R01EB014542 coupling. We show that the electron longitudinal relaxation times are also an important factor for obtaining high .[1] A TU236: Quantum Rotor induced Polarization new series of bTbK derivatives is developed and investigated with the aim of producing radicals with very long electron ∗Ulrich L. Günther, Christian Ludwig  relaxation times, and the observed dependence of on elec- University of Birmingham tronic relaxation properties is discussed. In the best case,  of 200 are obtained, unprecedented at this field and temper- Among the currently known NMR polarization methods, ature. quantum rotor polarization (QRP) represents the most re- cent addition. QRP utilizes highly non-linear changes that We also discuss the impact of nuclear relaxation properties take place in the populations of the energy levels of tun- and the spin density of the surface groups on both the en- nelling methyl groups when subject to rapid changes in tem- hancement and the strength of the paramagnetic effects in- perature. In fact the latter exploits effects associated with duced by the radicals at the surface.[2] Here we show that spin angular momentum, through the influence of the Exclu- when a substrate possesses methyl groups that have short sion Principle on the space-spin symmetry of the molecular T1’s, these protons act as relaxation sinks and dramatically states. Analogous polarisation effects occur in PHIP (para- reduce DNP enhancements. We then illustrate how the DNP hydrogen induced polarization), in fact QRP can be seen as enhancement can be restored by replacing these fast relax- a C3 symmetry analogue of PHIP. ing protons with deuterons, or with functionalities containing slowly relaxing protons.[2] While QRP has been known for many years, it has been thought to be limited to few molecules, primarily γ-picoline. [1] A. Zagdoun et al., J. Am. Chem. Soc., 2012, 134, 2284. This effect is often been called the Haupt effect [1,2]. We [2] A. Zagdoun et al., Angew. Chem. Int. Ed., 2013, in press, have recently observed QRP for a series of molecules at tem- DOI:10.1002/anie.201208699 peratures below 1.5K [3]. The polarization generated by this mechanism is efficiently transferred via protons. We observe MO235: Pedri-Based Functional Measurements – such effects for a much broader range of substances with very Concept, Probes, and Instrumentation different polarization rates at low temperatures. Moreover, transfer of quantum rotor polarization across a chain of pro- 1,2∗Alexandre Samouilov tons is possible. The observed effect not only influences the 1The Ohio State University, USA, 2Wexner Medical Center polarization in low-temperature DNP experiments but also opens a new independent avenue to generate enhanced sen- Overhauser enhancement is used for numerous biomedical sitivity for NMR. and clinical applications but capacity to map chemical en- vironment (pH, O2, redox status, etc.) is still limited. EPR [1] Haupt J. A new effect of dynamic polarization in a of exogenous paramagnetic probes has the unique advantage solid obtained by rapid change of temperature. Phys Lett of functional specificity due to the absence of overlap with 38A:389–90 (1972). endogenous EPR signals. However EPR-based techniques are far from attaining their maximum potential due techni- [2] Horsewill A. Quantum tunnelling aspects of methyl group cal limitations of low field EPR imaging (EPRI) resulting in rotation studied by NMR. Prog Nucl Mag Res Sp 35:359–389 longer acquisition times and lower functional and spatial reso- (1999). lution. Proton-electron double-resonance imaging (PEDRI) [3] Ludwig C, Saunders M, Marin-Montesinos I, Günther UL. represents an alternative NMR-based approach for imaging Quantum Rotor Induced Heteronuclear Hyperpolarization. of paramagnetic probes based on the Overhauser enhance- Proc Nat Acad Sci USA, 107(24), 10799-10803 (2010). ment of the proton MRI after EPR irradiation. It inherently offers high resolution, plane selectivity and rapid image data collection. Concepts of functional PEDRI, namely Variable Porous Media Field (VF) and Variable Radio Frequency (VRF) PEDRI are proposed for functional mapping (e.g. pH mapping) using specially designed paramagnetic probes. TH237: 1H NMR transverse relaxation as a probe PEDRI oriented paramagnetic probes are required to have to investigate water accessibility in cellulose in pre- functionally-dependent ratiometric spectral parameters (e.g. treated sugarcane bagasse ratio of intensities measured at two pre-selected fields). With 1∗ 2,3 these probes, spectral parameters at each pixel can be ex- Jefferson Esquina Tsuchida, Camila Alves Rezende, 1 1 1 tracted from a limited number of PEDRI acquisitions ac- Rodrigo de Oliveira-Silva, Marisa Aparecida Lima, Marcel 1 1 quired at pre-selected EPR excitation fields or frequencies. N. D’Eurydice, Igor Polikarpov, Tito José Bonagamba Number of probes has been developed and under the devel- 1Instituto de Física, Universidade de São Paulo, São Carlos, opment for PEDRI studies of biologically relevant parame- 2Instituto de Química, Universidade Estadual de Campinas ters of the medium such as pH, redox state, concentrations Biorefining of the lignocellulosic biomass for the production of of oxygen or glutathione. bioethanol has become a promising research area. Enzymatic Home-made imager/spectrometer based on iron core Resonex hydrolysis is a determining step to the biomass conversion 5000/Paradigm resistive magnet has been used for functional into biofuels and different pretreatment strategies have been

139 Poster Abstracts – Porous Media proposed to improve efficiency of this process. Among the dif- for the characterization of nanoporous systems, and as such ferent factors affecting hydrolysis yields of the biomass sam- also a useful tool in bio-nanotechology. ples, porosity is highlighted due to its intimate relation with enzyme accessibility to cellulose fraction of the biomass. An- TU239: Evaporation kinetics in macroporous poly- other important factor is related to the surface area available meric supports evaluated by relaxation measure- for hydrolysis also related with biomass porosity. In this way ments the use of Nuclear Magnetic Resonance (NMR) techniques is necessary because information regarding of the pore size 1Emilia V. Silletta, 2C.G. Gomez, 2M.C. Strumia, distribution and chemical characteristics of the pore surface 1Gustavo Alberto Monti, 1∗Rodolfo Héctor Acosta can be obtained. In this work, sugarcane bagasse samples 1Universidad Nacional de Córdoba, CONICET, Córdoba, treated by acid and alkaline pretreatments were prepared. Argentina, 2Departamento de Química Inorgánica, Univer- The changes due to the pretreatments in the total surface sidad Nacional de Córdoba, area, hydrophilicity and water accessibility in cellulose were investigated by nitrogen adsorption, Scanning Electron Mi- Macroporous polymer beads present a permanent well- croscopy (SEM), and NMR techniques (relaxometry in low developed porous structure in the dry state, and have a wide magnetic field and spectroscopy in high magnetic field). With range of applications such as support for catalysts, immo- the use of SEM images the transversal relaxation rates, T2- bilization of enzymes, HPLC columns, liberation of active distributions, were associated to three different water affinity substances, or adsorbents (1). Their porous structure im- sites along the sample, corresponding to long and intermedi- proves the diffusion of different solutes through the network. ate as well as short T2M ’s, which were assigned to wide and These networks have particular properties as high degree of narrow vessels in addition to cell wall surfaces, respectively. crosslinking and rigid structure both in swollen and dry state, turning the study of their synthesis conditions of consider- NMR relaxometry revealed that with application of pretreat- able interest. The properties of the polymer mesh change ment, the removal of lignin promotes increase of the water radically due to swelling, a process that strongly depends on accessibility in cellulose, ie there is an increase in the in- the mesh structure; therefore, changes in the content of a se- teraction between water and cellulose. NMR spectroscopy lected cross-linker (ethylene glycol dimethacrylate-EGDMA) showed the presence of water with different mobilities (free, percentage are studied (3). Evaporation kinetics of swelling restricted and rendered) which can be respectively associ- liquid molecules (L) distributed in the network show a dif- ated with amorphous, semi-crystalline and crystalline cellu- ferent interaction with itself (L-L) compared to those liquid lose. From the standpoint of NMR, the pretreatment with molecules adsorbed to the polymer chain (L-P). In the first 1% NaOH showed the better results of the water accessibility case the evaporation kinetics present a linear decay; while in cellulose and therefore more efficiency for hydrolysis. This in the second an exponential decay is present (2). Therefore, result is in agreement with the study by Rezende (Rezende, monitoring the kinetics of evaporation renders direct informa- CA et al. Biotechnol. Biofuels. 2010, 4, 1-18). tion of the swelling process (4). We use 1H-CPMG sequences ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq to measure the distribution of transverse relaxation times of polar and non-polar solvents contained in the matrices. As non-polar solvents do not interact with the network, informa- MO238: Self-diffusion in nanopores studied by the tion of the unperturbed porous system is obtained. On the NMR pulse gradient other hand, polar solvents present the L-P interactions that 1,3∗Janez Stepišnik, 2Bernd Fritzinger, 2Ulrich Scheler, compared to L-L provide information on the swelling process. 1Aleš Mohorič Kristensen TE, Vestli K, Fredriksen KS, Hansen T, Org. 1 University of Ljubljana, FMF, Ljubljana, Slovenia, EU, Lett. 2009, 11: 2968-2971. 2Leibniz-Institut for Polymerforschung , Dresden, Germany, EU, 3Institute Jozef Stefan, Ljubljana, Slovenia, EU Errede L., Kueker MJ, J Polym Chem Ed 1988; 26:3375-3380. With the proper interpretation of experimental data, the C.G. Gomez, M. Strumia;Journal of Polymer Science: Part method of NMR pulse gradient spin echo provides informa- A: Polymer Chemistry 46, 2557–2566(2008). tion about the molecular self-diffusion in nanopores. We demonstrate it by the analysis of restricted self-diffusion C.G. Gomez, G. Pastrana, D. Serrano, E. Zuzek, M.A. Villar, measurement of water molecules trapped in a porous M.C. Strumia, Polymer 2012, 53, 2949-2955. poly(amide)-6 (PA-6) membrane. The PGSE sequence with fixed gradient pulse width, but variable interval between pulses and by changing the magnetic field gradient from zero TH240: NMR Relaxation Study of Ionic Liquids Un- to 11.6T/m at temperature of 22 ◦C we observed the spin der Confinement in Porous Media echo dependences on the gradient that decay with small un- ∗ Carlos Mattea, Amin Ordikhani Seyedlar, Siegfried Stapf dulations. These "diffraction" patterns prevent the use of the inverse-Laplace transform method for the extraction of Department of Technical Physics II / Polymer Physics, In- pore size distribution. However, the shape of the diffusion stitute of Physics, Ilmenau University propagator obtained by q-space cosine Fourier transform of The investigation of low melting organic salts commonly re- experimental data can be perfectly fitted the multi-normal ferred to as ionic liquids, constitutes one of the most fascinat- distributions. Their second moments remain fixed as the in- ing and rapidly developing areas in physical chemistry and terval between gradient pulses changes. This is a clear indi- engineering. Ionic liquids have been discovered in the last cation of a motional narrowing regime, in which the second decades and since then received much attention as "green" moments of propagators are proportional to the fourth power replacement of common solvents in chemical technology [1]. of pore size. Results are presented on the 3-D plot of the spin-relaxation distribution and the pore radius distribution, They are liquid below 100 ◦C, and are composed of an or- which shows the prevailing share of spins in the pores with ganic cation and organic or inorganic anion presenting nor- the radius r = (100±10) nm (70%) and pores with the ra- mally negligible vapor pressure. They are benign alternatives dius (175±30) nm (20%). Water in these two types of pores to the usual organic solvents for synthesis, liquid/liquid ex- has almost identical spin relaxation T2 =(10±2) ms, while tractions and heterogeneous catalysis. They reveal different water in the pores with the radius of r = (282±7) nm (5%) properties from the common molecular liquids mainly due to have a broader distribution of relaxation times, T2 =(14±5) their ionic character. There is much interest in the study of ms. These experiments provide information on the nanopore their molecular motions that control transport properties in structure in the polyamide membrane not observed in the these materials [1] both in bulk and under confinement. Re- previous investigation by the method of stimulated gradient orientation and translational dynamics can be suitably stud- spin echo. The results confirm the potential of NMR PGSE ied by Nuclear Magnetic Resonance (NMR) [2].

140 Poster Abstracts – Porous Media

From both, the fundamental research and for applications, ACKNOLEDGEMENTS: CONICET, SECyT-UNC, FON- it is important to know the dynamical properties of ionic CYT, FONTAR liquids in porous media, where the transport properties are predominantly affected by the geometrical restrictions and TU242: Chromatographic NMR, complement and wall interactions. prediction of LC? T In this contribution, the spin-lattice relaxation ( 1) of two 1Caroline Carrara, 1Claire Lopez, 1,2Manjunatha Reddy ionic liquids, emim(+)-Tf2N(-) and bmim(+)-Tf2N(-), bulk 3 3 1,2∗ G.N, Rafael R. Ballesteros, Jérôme Lacour, Stefano and confined in porous media are presented. The confine- Caldarelli ment is done in porous silica glasses with mean nominal pore 1 2 sizes of 4 nm and porosity of 28%. T1 relaxation times were Aix Marseille Université,Marseille, France, CNRS UPR measured at different magnetic fields and temperatures, cov- 2301 ICSN, Antenne Ecole Polytechnique, Palaiseau, 3 ering a range from 0.24 mT to 0.5 T and from 210K to 300K. France, Department of Organic Chemistry, University of The different ions can be independently assessed by NMR Geneva, Geneve, Suisse 1 19 selecting either H-NMR (for the cations), or F-NMR (for Chromatographic NMR, introduced in 2003, [1] is a variant the anions). of DOSY (diffusion ordered spectroscopy). [2,3] This method allows you to create charts NMR spectra of molecules ordered The relaxation experiments reveal strong changes in the dy- in relation to their average molecular mobility. The addition namics when the ionic liquids are under confinement. The of a stationary phase in a mixture analyzed by DOSY can results show however, that different ions exhibit similar re- induce changes in the distribution of components which can laxation mechanisms. This is attributed to a strong coop- be correlated to their affinity for the solid. [1,4] In this work, erative dynamics in these kinds of complex liquids. Further we first show how differences observed between the NMR and studies in order to discriminate whether this influence is a LC Chromatography [5] can be explained on the basis of a consequence of the physical confinement and/or interaction strong impact of solution-to-solid phase ratio, possibly due to with the walls are discussed. Furthermore, at low tempera- slow exchange between intra- and interparticle porosity. [6] tures, evidence of decoupling of translational diffusion from A correct choice of this parameter provides excellent model- structural relaxation has been found in agreement with recent ing tests for shape selectivity.[7] A further aspect that must light scattering experiments [3]. be included in comparisons between NMR and LC chromato- References graphic is their different timescale. This will be illustrated by an example of separation of labile diastereoisomeres con- [1] P. Wasserscheid, T. Welton (Eds.), Ionic Liquids in Syn- sisting of chiral ion pair. [8] thesis, VCH-Wiley, Weinheim, 2003. ACKNOWLEDGEMENTS The stationary phases were [2] R. Kimmich, NMR Tomography, Diffusometry and Relax- kindly provided by Dionex. Funding from ANR (ANR-08- ometry, Springer, 1997. BLAN-273) and Region PACA (APO-G 2009) are gratefully [3] P. Griffin et al., J. Chem. Phys, 135, 114509 (2011) acknowledged. [1] C. S. JohnsonJr, Progr. NMR Spectr. 34, 203-256 (1999) MO241: Characterization of Compacted Silty Soils by Means of Nuclear Magnetic Resonance [2] K. F. Morris, P. Stilbs, C. S. Johnson, Anal Chem. 66, 211-215 (1994) 2,3∗Marcos Montoro, 1,3Lucas Matías Cerioni, 3Daniel José Pusiol [3] S. Viel, F. Ziarelli, S. Caldarelli, PNAS 100, 9696-9698 1Spinlock SRL, 2Universidad Nacional de Córdoba, (2003) 3CONICET [4] G. Pages, C. Delaurent, S. Caldarelli, Anal. Chem. 78, Compacted soils are widely employed as construction ma- 561-566 (2006) terial for different facilities such as highways, dams and landfills. The aim of soil compaction procedure consists in [5] G. Pages, C. Delaurent, S. Caldarelli, Angew. Chem. Int. obtaining the maximum dry unit weight of a particular soil. Ed. 45, 5950-5953 (2006) Resulting dry unit weight depends on the compaction en- [6] C. Carrara, S. Caldarelli, J. Phys. Chem. C, 116, 20030- ergy and the water content. Maximum dry density can be 20034 (2012) reached only at the optimum water content, in consequence it can be possible to have compacted soils with the same [7] C. Carrara, C. Lopez, S. Caldarelli, J. Chromatogr. A dry unit weight (same porosity) compacted with different 1257, 204-207 (2012) moisture. Soils compacted dry from optimum use to present flocculated fabric characterized by open and big pores, while [8] Manjunatha Reddy G.N., R. Ballesteros , J. Lacour and soils compacted wet from optimum use to present dispersed S. Caldarelli Angew. Chem. Int. Ed. 1 52, 3255–3258 (2013) fabric characterized by small pores. In this work we present a study of soil compaction using Time Domain Nuclear TH243: Artificial ceramic porous media preparation Magnetic Resonance (TD-NMR). We measured samples of and their characterization by NMR compacted silty soils with different molding water content 1 1 1 using a low field (12 MHz) spectrometer (SLK-100). We mea- Elton Tadeu Montrazi, Roberson Saraiva Polli, Edson Luiz Gea Vidoto, 2Sérgio Rodrigues Fontes, 2Carlos Alberto sured the transversal relaxation times (T2) using a CPMG 1∗ pulse sequence (inter-echo spacing time 400 µs, 1200 echoes Fortulan, Tito José Bonagamba and 160 repetitions) in samples with moisture content be- 1Instituto de Física de São Carlos - Universidade de São tween 10 and 22%. The amplitude of the obtained signal was Paulo, 2Departamento de Engenharia Mecânica - Escola de correlated with the sample moisture content and comparing Engenharia de São Carlos - Universidade de São Paulo cross correlations between the different signals with the one In this work we present a method for preparing ceramic ma- registered for the sample compacted at the optimum mois- terials with controlled porosity and their characterization by ture content assessed the compaction degree. A high linear NMR relaxation, MRI FLASH, mercury intrusion porosime- correlation between the signal amplitude and moisture con- try (MIP), and scanning electron microscope (SEM). tent was determined (r2 = 0.9537). The relation between the cross-correlations of the signals and the dry unit weight of Artificial alumina ceramics porous medium was manufac- the samples presented a non-linear behavior. T2 distribution tured by a sintering process, which preserves its intrinsic curves allowed to identify different pore sizes distribution in porosity. Additionally, it was added to the manufacturing sample with the same porosity but compacted either dry or process a porogenic agent in order to induce bigger pores un- wet from optimum. der a controlled way. Sucrose crystals were chosen as poro-

141 Poster Abstracts – Porous Media genic agents and their sizes were carefully selected by using AKNOWLEDGEMENTS: IFSC/USP, FAPESP, CNPq, and sieves. CAPES. During the sintering process, these crystals are burned off, creating along the ceramic material induced pores intercon- TU245: Direct Evidence of the Nanopore Inner- nected by the intrinsic pores. Sphere Enhancement (NISE) Effect by NMR and EPR Spectroscopy Three groups of samples were prepared, presenting intrinsic pore sizes of about 0.5 µm and induced pores sizes in the 1∗Daniel R. Ferreira, 2Marcus V. Giotto, 3Nadine J. range of 10 - 100 µm (sample S), 30 to 300 µm (sample M), Kabengi, 4James E. Amonette, 4Eric D. Walter, 2Cristian and 300 to 1800 µm (sample B), which were estimated by P. Schulthes MIP, SEM and MRI. 1Southern Polytechnic State University, 2University of Con- necticut, 3University of Kentucky, 4Pacific Northwest Na- By the use of CPMG, the following average T2 values 0.05, 1.2, 1.7, and 1.9 s were observed for water within the intrinsic tional Laboratory and induced pores in samples S, M, and B, respectively. From The adsorption mechanisms of mono- and divalent cations the point of view of T2 distributions, the results observed are in zeolite nanopore channels can vary as a function of their very consistent with sample preparation, being the average pore dimensions. The nanopore inner-sphere enhancement T2 values directly proportional to the size of the intrinsic and (NISE) theory predicts that ions may dehydrate inside small induced pores. nanopore channels ≤ 0.5 nm in order to adsorb more closely to the mineral surface if the nanopore channel is sufficiently Due to the bigger pore sizes observed for samples M and B, small. Both sets of results from 23Na MAS Nuclear Magnetic only these samples allowed getting more precise pore size in- Resonance (NMR) spectroscopy and Electron Paramagnetic formation by using MRI FLASH technique, with a minimum Resonance (EPR) spectroscopy of Mn and Cu adsorption resolution of about 250 µm. However, due to this resolution on the zeolite minerals zeolite Y (large nanopores), ZSM-5 the smaller pores in sample M were not properly observed. (intermediate nanopores), and mordenite (small nanopores) The obtained results validate the manufacturing process and support the NISE model. The Na cation adsorbs via an outer- the prepared artificial porous media are excellent samples to sphere mechanism on zeolite Y, via an inner-sphere mecha- be used as model for testing traditional and under develop- nism on ZSM-5, and via a mixture of the two mechanisms ment NMR pulse sequences. New manufacturing procedures on mordenite. The Cu and Mn cations both adsorbed via are being developed in order to introduce well distributed an outer-sphere mechanism on zeolite Y based on the sim- paramagnetic impurities along the porous ceramic material. ilarity between the adsorbed spectra and the aqueous spec- tra. Conversely, Mn and Cu adsorbed via an inner-sphere ACKNOWLEDGEMENTS: IFSC/USP, FAPESP, CNPq, mechanism on mordenite based on spectrum asymmetry and CAPES. peak broadening of the adsorbed spectra. However, Mn ad- sorbed via an outer-sphere mechanism on ZSM-5, whereas MO244: Simulation of Transverse Relaxation in Re- Cu adsorbed on ZSM-5 shows a high degree of surface inter- alistic Porous Media Based on Random Walk and action that indicates that it is adsorbed close to the mineral Monte Carlo Methods surface. Evidence of dehydration and immobility was more readily evident in the spectrum of mordenite than ZSM-5, Everton Lucas de Oliveira, Mariane Barsi Andreeta, indicating that Cu was not as close to the surface on ZSM-5 Arthur Gustavo de Araujo Ferreira, Marcel Nogueira as it was when adsorbed on mordenite. Divalent Mn cations D’Eurydice, ∗Tito José Bonagamba are strongly hydrated and are held strongly only in zeolites Instituto de Física de São Carlos - Universidade de São with small nanopore channels. Divalent Cu cations are also Paulo strongly hydrated, but can dehydrate more easily, presum- NMR transverse relaxation time (T2) distribution of fluids ably due to the Jahn-Teller effect, and are held strongly in in porous media, such as rock cores, ceramics, bones and ce- zeolites with medium sized nanopore channels or smaller. ments, is considered one of the most important information ACKNOLEDGEMENTS: United States Department of Agri- to characterize these materials, allowing understanding, for culture (USDA-Hatch No. CONS00864) and United States example, permeability, wettability and porosity distribution. Department of Energy’s (DOE) Office of Biological and En- Great efforts have been made to simulate these properties and vironmental Research. associate them with T2 distributions obtained from CPMG (Carr-Purcell-Meiboom-Gill) decays in a more detailed way. Usually, it is possible to track and classify the interaction TH246: NMR Study of Diffusion and Relaxation in of spins with porous media surfaces based on Monte Carlo Micro-porous Glass Beads and random walk methods, in order to reproduce the liq- 1∗Giovanna F. Carneiro, 2Lawrence Schwartz, 2Yi-Qiao uid Brownian motion. The purpose of this work is to com- Song, 3Bernardo Coutinho, 3Edmilson Rio, 3Willian An- bine two random walk approaches, over the space and phase- drighetto Trevizan space, in order to simulate the relaxation behavior of fluids 1 within porous media, taking into account the dephasing due Schlumberger Brazil Reserach and Geongineering Center, 2 3 the interactions of the nuclei with the pore walls and among Schlumberger-Doll Research, Centro de Pesquisas e De- themselves. The relationship between T2 distributions and senvolvimento Leopoldo Américo Miguez de Mello pore properties, e.g. morphology and S/V ratio distribution, In this study, micro-porous borosilicate glass beads[1] were is well-established and can be analyzed under slow and fast evaluated since they are a good representation of bimodal translational diffusion regimes. Considering these properties, porosity systems. They consist of of spherical grains with a computer simulations were developed in order to describe the mean diameter of 150 µm and each of these grains is, itself, fluid dynamics inside such materials. The pore morphology porous with an average micro-pore diameter of 0.3 µm. The is computationally represented by a 3D voxel-based descrip- system’s total porosity is 80% and is divided equally between tion, in which both real (obtained by x-ray microtomogra- the micro- and macro-pores. phy) and virtual idealized models can be used as boundary conditions. The simulated decays obtained by the developed Pulsed-field gradient stimulated echo techniques were used to method can be compared to the CPMG experimental results, measure the time (t) dependent diffusion coefficient, D(t), in where the correlation between collision rates and T2 values a Magritek 2 MHz Rock Core Analyzer. For long diffusion can be analyzed. The next step is to apply the proposed com- times, the limiting value of D(t) is directly related to electri- putational method to study these materials with well-known cal transport.[2] Our experiments are supported by numerical porosities and compositions, including computationally con- simulations in which we compare two geometrical models for structed and artificial porous media. the structure of the micro-pores. We looked first at a model in

142 Poster Abstracts – Porous Media which grains of diameter 150 µm are arranged on an ordered 3Martin D. Hurlimann, 1Tito José Bonagamba lattice. Each of these grains is comprised of an array of much 1Instituto de Física de São Carlos, Universidade de smaller spherical grains.[2] In the second model, the same São Paulo, São Carlos, Brazil, 2Schlumberger Brazil Re- ordered packing of 150 µm grains defines the macro-pores search and Geoengineering Center, Rio de Janeiro, Brazil, but their micro-structure is represented by a Swiss cheese ar- 3Schlumberger-Doll Research Center, Cambridge, USA rangement of (slightly) overlapping spherical pores. For both models the micro-pore diameter is 30 µm and the net micro- Magnetic susceptibility contrast (δχ) between the rock and porosity is 40%. Indeed, we saw that only the Swiss cheese the saturating fluid can cause distortions in the local mag- model properly described the long time behavior of D(t). The netic fields of the pores in a Nuclear Magnetic Resonance lower values of D(t) reflect the fact that, at a given porosity, (NMR) experiment. This effect is described as an inter- diffusion through an array of overlapping spherical pores is nal magnetic field gradient, and scales with the pore size considerably more difficult than diffusion through a network distribution, matrix composition and pore geometry. Hür- of spherical grains. At short times the two models yield re- limann[1] studied in great detail the effect of translational sults that are quite similar since both have the same total diffusion through those internal gradients on T2 relaxation surface area. Unfortunately, the short time regime is difficult time, a mechanism that can influence and even dominate the to probe experimentally because of limitations on the speed T2 distribution of porous media. He was able to obtain a with which gradient pulses can be switched on and off. distribution of internal gradients using a variable echo time CPMG methodology. Sun et al.[2] studied the δχ effect in [1] http://www.millipore.com/techpublications/tech1/ T2 relaxation spectra for two sandstone rocks, showing that ds1007en00 the pronounced effect for the small pores. Mitchell et al.[3] performed an extensive NMR study on a sandstone rock core [2] Zhang Z., Johnson D, and Schwartz L, Phys. Rev. E84, at several magnetic field strengths, showing that the internal 031129 (2011). gradients are nonuniform across an individual pore. [3] Sen P., Schwartz L., Mitra P., and Halperin B., Phys. In this work, the contribution of such effects in transversal Rev. B49, 215 (1994). (T2) and longitudinal (T1) relaxation times was studied in a [4] Feng S., Sen P., and Halperin B., Phys. Rev. B35, 197– set of 4 sandstone and 7 carbonate samples. Magnetic fields 214 (1987) ranging from 0.047 - 9.4 T were used to characterize the δχ effects. Also, thin section images, mercury intrusion, mag- MO247: Connectivity and permeability in complex netic susceptibility measurements and electron paramagnetic carbonate rocks, through diffusivity measurements. resonance experiments were performed, in order to correlate them with NMR results. 1∗Bernardo Coutinho Camilo dos Santos, 1Willian An- drighetto Trevizan, 1Vinicius de França Machado, 3Andre The relaxation spectra of carbonates have a low contribution Souza, 2Giovanna F. Carneiro, 2Austin Boyd, 3Edmilson of paramagnetic elements, and so the main magnetic effect Helton Rios, 4Rodrigo Bagueira de Vasconcellos Azeredo arise from the pore shape heterogeneity. In contrast, the sandstones have shown higher magnetic effects due mainly 1 2 PETROBRAS-CENPES, Schlumberger - BRGC, to the presence of paramagnetic species (e.g. Fe3O4 and 3 4 Observatorio Nacional, Universidade Federal Fluminense FeSO4). In sequence, two-dimensional NMR T1-T2 exper- - Instituto de Química iments, capable of investigating molecular dynamics details In this study we explored techniques uni-dimensional and of the confined saturating fluid and differences in the physico- two-dimensional Nuclear Magnetic Resonance (NMR) mea- chemistry properties of pore sites, were performed and corre- surements of diffusion coefficient (D), and its relationship to lated with the magnetic susceptibility findings. properties such as connectivity, tortuosity and permeability [1] Hürlimann M.D., Journal of Magnetic Resonance, Volume of porous media. The information is usually extracted from 131 (1998), 232-240. measurements of low field NMR T2 spectra, which are asso- ciated with a pore size distribution. However, when a fluid [2] Sun B., Dunn K., Physical Review E, 051309 (2002). (hydrocarbon or water) is placed in a porous medium the re- laxation time changes pore to pore. The restriction imposed [3] Mitchell, J., Chandrasekera, T.C., Johns, M.L., Gladden, by the rock causes to the diffusion coefficient changes depend- L.F., Fordham, E.J, Physical Review E, 026101 (2010). ing on the pore structure. Thus, smaller pores and more tor- ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq, Schlum- tuous paths decreases the diffusion coefficient, whereas larger berger. pores or less tortuous paths do not restrict dramatically the diffusion. The ability to measure both D and T2 allows us TH249: Evaluation of uncertainty in petrophysical to construct two-dimensional maps (D vs.T2) and estimate properties derived from the profile NMR due to ac- besides the pore size distribution the value of D in each quisition noise pore size. Measurements over 15 complex carbonate samples from well cores showed the possibility of inferences about ∗Eduardo Barreto Oliveira, Marcelo Torrez Canaviri the connectivity of the porous medium, and quantify the Petrobras interaction of water with the rock matrix. Through the two- dimensional maps D-T2 were obtained the values of surface Signal / noise ratio noise in NMR logging is significantly lower relaxivities of each samples and compared with the mercury than most other logging tools. In the case of Schlumberger injection method. These values, when incorporated into the CMR tool, each echo has Gaussian noise of zero mean and Ksdr permeability model, improves by almost 30% of per- standard deviation of approximately 3.5 pu. During logging, meability estimates compared with standard petrophysical a moving average of 3 levels is performed to reduce the stan- measurements. There was also a good correlation between dard deviation for close to 2 pu, resulting in a decrease of the values of D (obtained by NMR) and the permeabilities resolution (Freedman & Morriss, 1995). Obtaining the T2 k, obtained from standard petrophysical assays, for specific distribution from the decay is an ill-posed problem, since it diffusion times. Therefore, there is an optimum measuring allows many solutions and small changes in input data pro- time in which the molecules of fluid diffusing through the duce significant changes in the solution. Thus, the presence of porous medium are obtained by NMR and provide informa- noise influences the solution of the inversion process and con- tion about the connectivity of the pores. sequently the petrophysical properties estimated from NMR interpretation, especially the irreducible fluid volume. In or- TU248: Magnetic Susceptibility Effects on NMR Re- der to assess the impact of noise in the measurements of NMR laxation of Sedimentary Rock Cores tool, it was generated multiple realizations of Gaussian noise with standard deviation equal to the real data, which re- 1,2∗ 1 2 Andre Souza, Marcel N. D’Eurydice, Austin Boyd, sulted in multiple T2 distributions which were subsequently

143 Poster Abstracts – Polymers & Materials subjected to the traditional NMR interpretation , being ob- quiry of molecular translational motion by the NMR gradient tained total porosity, free fluid, irreducible fluid and perme- spin echo is unique, because its signal is the characteristic ability estimated by Timur-Coates model. The main result of functional of stochastic process, if the molecular displace- this work was to obtain distributions of petrophysical proper- ments are treated as a random motion. Thus, the theory of ties rather than a single value. The curves generated showed random processes provides the relation of spin echo attenua- higher resolution compared to that provided by the logging tion to VAS. New MGSE technique, which permits the VAS company, indicating a likely application of a moving average measurement in the frequency range of 100 Hz-100kHz, was procedure in order to increase the signal / noise ratio. used to study the motion in the mono-disperse poly(isoprene- 14) using the NMR mouse. Although, the obtained fre- MO250: MRI spatial CPMG in the study of rock quency dependence of VAS for the molten polymers with the heterogeneity and capillary pressure curves molecular weights: Mn=704, 1610 and 3920 at Mw/Mn 1.05, were expected to reflect the segmental tube/reptation mo- 1,2∗Edmilson Helton Rios, 1Irineu Figueredo, 2Bernardo tion, they fit well to the spectrum of the Rouse model current Coutinho Camilo dos Santos, 2Willian Andrighetto Trevizan, for diluted solutions. However, the extracted parameters do 2Vinicius de França Machado not completely agree with this model. The deduced coil dif- −2 1National Observatory, 2Cenpes, Petrobras fusion coefficient scales with the molecular weight as N as predicted with the tube/reptation model, the mode ampli- In special core analysis (SCAL) programs, it is very im- tude as N according to the Rouse model, but the relaxation portant to consider sample heterogeneity since many petro- time independence on N disagrees with both models. Pre- physical measurement techniques are based upon models ho- liminary studies give a polymer like VAS for some hydrogen- mogeneity assumptions. We present some results of 2MHZ bonded liquids (water, glycerol) as well. MRI phase encoding spatial CPMG measurements along the length of totally and also partially brine saturated core plugs (1.5’ diameter per 2’ long). With a single fully saturat- TH252: Structural characterization and molecular ing fluid, the inverse Laplace transform obtained for each dynamics in the solid-state in Poly(ethyleneimine) core slice represents the core variation in its pore size dis- polymers tribution. This ’heterogeneity probe’ can be promising for 1,2Juan Manuel Lázaro Martínez, 2,3Gustavo Alberto many applications such as: representativeness verification of Monti, 2,3∗Ana Karina Chattah core end pieces for MICP analysis and posterior compari- 1IFEG-CONICET, 2FaMAF-Universidad Nacional de Cór- son with NMR bulk core measurements; selection of homoge- 3 neous samples for relative capillary pressure analysis; mon- doba, FaMAF-Universidad Nacional de Córdoba itoring of perm porosity changes after acid injection experi- The linear polymer poly(ethyleneimine) (PEI) has a wide field ments, etc. For partial saturation samples we applied spatial of applications. It has been used as a cosmetic ingredient to CPMG along air-water centrifuged cores and it was verified adjust viscosity, as chelating metal ions to inhibit corrosion, a gradual decreasing in T2 values and in its computed to- and as copolymer to develop energy storage devices. Inter- tal area from outer to inner centrifuge radius as result of a estingly, taking into account the biocompatibility of the PEI, saturation gradient. Despite its practicality, traditional cen- it has been used in biotechnology as non-viral vector to carry trifuge capillary pressure curve (CCPC) experiments have out gene transfection processes. However, no physical char- some drawbacks once they just consider average bulk sat- acterization in the solid state has been done. Therefore, the uration per rotation speed. We plotted the cumulative pore objective of this work is to study the structural and dynamic volume (total area) for each slice against sample length re- homogeneity in three synthetic variants of PEI with differ- sulting in a one dimension saturation profile. With it, dif- ent molecular weights (22, 87 and 217 kDa) through Nuclear ferent capillary pressure per saturation slice was quantified Magnetic Resonance techniques in the solid state (SSNMR). in a single speed rotation not only overcoming the simplified In addition, changes will be explored after the uptake of cop- average consideration but also decreasing tremendously con- per ion (II) by these polymeric matrices, given the impor- ventional CCPC acquisition time. In many other SCAL’s it tance of the heterogeneous catalysts of Cu(II) for the activa- is important to use centrifuged cores at the irreducible wa- tion of hydrogen peroxide and degradation of pollutants with ter saturation. In this sense spatial CPMG was also used to high environmental impact. monitor the fluid spontaneous redistribution along the sam- ple (higher the permeability, faster it was) and also to check The SSNMR proved to be a valuable tool for studying the for eventually trapped water in one of the core sides. All ex- dynamics and homogeneity of the different samples of PEI. periments performed here indicate that MRI spatial CPMG Although the results of X-ray powder crystallography do not is a useful technique to support SCAL programs. show significant differences between different samples. SS- NMR through 13C CP-MAS spectra using different spin time ACKNOLEDGEMENTS: CNPQ, PETROBRAS, GIT dynamics enabled to demonstrate the heterogeneity in each case, as well as 15N CP-MAS spectra. These results were confirmed by measuring 13C relaxation time in the rotating Polymers & Materials frame, 1H-13C 2D-WISE and 1H-13C 2D-HETCOR. Finally, the formation of complexes with copper ions (II) pro- TU251: Segmental dynamics in molten polymers duced a significant reduction in the molecular mobility as a studied by modulated gradient spin echo result of crosslinking between the different polymer chains to coordinate the metal ion, based on the results of 13C relax- 1,3∗Janez Stepišnik, 2Carlos Mateo, 2Siegfried Stapf, 1Aleš ation time in the rotating frame and 1H-13C 2D-WISE. Mohorič 1University of Ljubljana, FMF, Ljubljana, Slovenia, EU, MO253: Proton spin-spin relaxation correlations in 2Technical University Ilmenau, Ilmenau, Germany, EU, glass-forming glycerol 3Institut “Jozef Stefan", Ljubljana, Slovenia, EU ∗Damjan Pelc, Miroslav Požek Although, the polymer has a long-standing records of exper- University of Zagreb, Faculty of Science, Department of imental and simulation investigations, the theoretical models Physics of polymer dynamics still remain questionable. We present the investigation of molten polymers by the NMR technique We present the results of stimulated-echo T2 correlation ex- of modulated gradient spin echo (MGSE), which provides a periments on protons in glycerol, as the liquid approaches new insight into details of polymer dynamic by direct mea- the glass transition at 190 K. The stimulated echo sequence surement of the segmental velocity autocorrelation spectrum has been used on other systems (and nuclei) to quantify dy- (VAS). With regard to other experimental methods, the in- namical heterogeneity [1,2], the sequence acting as a filter

144 Poster Abstracts – Polymers & Materials for slowly moving molecules. However, our results on pro- (0.5% HF) to subsequently evaluate it in hydrogen peroxide tons in glycerol show some unexpected features and a pecu- (H2O2). liar temperature dependence. In addition to the conventional spin-lattice relaxation contribution (in agreement with pre- MO256: 1H NMR Relaxation Time Distributions vious work [3]), we observe an anomalous signal decay, and a Study of Styrenic Polymers crossover temperature T* > Tg at which the decay saturates. We believe this to be a combined effect of several factors, 1∗Naira Machado da Silva Ruiz, 2Luiz S. C. Junior, and the influences of glassy slowing-down (modifying mo- 2Fábio L. L. Farias, 2Grazielle. V. Tiziano, 2Eduardo P. Cas- tional narrowing contributions), dynamical correlations and sus, 2Augusto C. C. Peres, 2Sonia Maria Cabral de Menezes, spin diffusion are discussed. 3Edmilson Helton Rios, 3João M. Costa 1Pontifícia Universidade Católica do Rio de Janeiro, References: 2CENPES-PETROBRAS, 3Fundação Gorceix [1] A. Heuer et al., Phys. Rev. Lett. 75, 2851 (1995) The impact resistance of high impact polystyrene (HIPS) is [2] D. Pelc et al., Phys. Rev. Lett. 109, 095902 (2012) achieved by incorporating a rubber phase into the styrenic rigid phase. The HIPS usually is produced by polymeriza- [3] E. Koivula et al., Phys. Rev. B 32, 4556 (1985) tion of styrene in the presence of varying amounts of polybu- tadiene (usually 5-12 in weight %), resulting in a mate- TU254: Synthesis, structural, and physical charac- rial that can be structurally described as a multiphase sys- terization of new mixed-valent Mn7 clusters contain- tem. This structure gives multiphase improvement of opti- ing 2-hydroxymethylpyridine cal and mechanical properties when compared to polystyrene (GPPS-General Purpose Polystyrene resin) without modifi- 1 1∗ 2 Katye M. Poole, Khalil A. Abboud, Christopher C. cation. In this work the microstructure of different commer- 2,3 1 Beedle, Stephen Hill, George Christou cial rubber-modified polystyrene samples were analysed by 1University of Florida, 2National High Magnetic Field Lab- time-domain low resolution nuclear magnetic resonance com- oratory, 3Florida State University monly reported as one of the most efficient methods for an independent determination of the complex polymers inter- One of the main research interests in our group for a number faces. T1, T2 relaxation time measurements were conducted of years has been the synthesis and characterization of tran- on a Bruker Minispec mq20 (0,47T) low field NMR equipped sition metal compounds such as iron and manganese clusters, with a variable-temperature air-flow probe head. NMR ex- which usually contain chelating ligands to prevent polymer- periments were run at a temperature of 40◦C. Typical pulse ization. These complexes are of general interest for a num- length was 3.2 µs for the 90◦ pulse. The NMR sequences ber of reasons, including bioinorganic chemistry, magnetic used were inversion recovery (IR) for T1 and Carr-Purcell- materials, and oxidation of organic compounds. Manganese Meiboom-Gill (CPMG TE=100 µs) for T2 and the relax- (Mn) is found at the active sites of many metallobiomolecules, ation time distributions were obtained by an inverse Laplace with one of the most important being the water oxidation transformation (ILT) of IR and CPMG data. For all samples center (WOC) of plants and cyanobacteria, which contains T1 and T2 exhibit the typical structure for rubber-modified a tetranuclear Mn cluster. Manganese clusters are also of styrenics and reveal that the rubber phase are poorly de- interest due to interesting magnetic properties, and some scribed by only a single exponential component. In order to Mn clusters have been found to be single-molecule magnets separate the signal contributions from individual phases we (SMM). SMMs are molecules that can function as nanoscale combine the ILT data fitted in a Gaussian model with T1/T2 magnets at low temperatures. This behavior results from a ratio. A quantitative evaluation of individual phases revealed large ground state spin (S) due to unpaired electrons and a the presence of at least two phases in the HIPS sample sug- large and negative (easy axis type) magnetic anisotropy, D, gesting the presence of large PS domains segregated from the which together give a significant barrier for magnetization re- soft phase which is clearly related to the material properties. versal, which is characteristic of magnets. This presentation will describe the use of halides (bromide, chloride, and io- ACKNOLEDGEMENTS: PETROBRAS, PUC-Rio dide) and pseudo-halides (N3−, NCO−, etc) in a previously known reaction, and the characterization of the resulting five TU257: Spectroscopic Characterization of meningo- Mn7 wheel compounds. The Mn7 wheel topology can be de- coccal serogroup C Polysaccharide produced by a scribed as a hexagon with a another Mn ion in the center, new purification process based on chromatographic and is with precedent in the literature, but this work has un- extraction, 1D and 2D-NMR Spectroscopy covered examples with a new intermediate spin ground state, S, for this topology. 1,2Milton Neto da Silva, 1,2∗Elza C.S. Figueira 1Instituto de Tecnologia em Imunobiologicos-Bio- TH255: Synthesis and Electrochemical Characteri- Manguinhos/FIOCRUZ, 2Instituto de Tecnologia em Fár- zation of TiO2 Materials macos Far-Manguinhos/FIOCRUZ

1,2∗Ivan Erick Castañeda Robles, 1Jorge Uruchurtu Neisseria meningitidis is one of the most important pathogens Chavarín, 1E. C. Menchaca Campos, 2Roberto Rodriguez as causes of meningitis and other clinical manifestations. Muñoz Meningococcal disease is primarily caused by only five meningococcal groups (A, B, C, Y and W135) among 13 1Universidad Autónoma del Estado de Morelos, groups described. Meningococcal serogroup C polysaccharide 2Universidad Tecnológica del Estado de Zacatecas (MenCPS) is a homopolymer consisting of a-(2→9)-linked The anodization of titanium is an electrochemical process sialic acid and has been widely used as antigen in polysac- that changes the chemical surface of the metal, via oxida- charide and conjugate vaccines. The MenCPS production tion, to produce an oxide layer. During this self organized and purification processes, acquired from Merieux Institut in process, highly ordered arrays of cylindrical shaped pores 1976, has been used up to date. This old process includes can be produced with controllable pore diameters and den- different steps. Two of them have some problems for large sity distribution. This enables titanium applications, such scale production. The first one is related to use of phenol for chemical sensors, without the need for expensive lithographi- protein removal which is a very corrosive and toxic reagent cal techniques. A oxide (TiO2) to be used in a wide range of and the other is the ultracentrifugation step used for LPS nanotechnological applications or incorporate it into specific elimination be very expensive, since many cycles are needed. simple method was used, electrochemical techniques from The new process proposed in the present study is an alterna- low potential in acidic and basic solutions. Cylindrical pores tive to substitute the old one, in order to have a convenient arrangements of TiO2 were constructed by anodizing the ti- purification for scale the MenCPS production up and also tanium surface in an aqueous electrolyte of hydrofluoric acid increase the process yields.

145 Poster Abstracts – Low field NMR

NMR spectroscopy has been one of most important tool for with resins, which are photo-polymerized by visible light (450 control the identity of bacterial polysaccharides used in vac- nm). In this work, the photo-polymerization of two dental cine manufacture. This technique is powerful and useful to re- resins was studied quantitatively by NMR relaxometry with lease polysaccharide batches. The identity of native MenCPS the help of a single-sided NMR tool, the NMR-MOUSE as a produced using the new purification process was evaluated function of time and depth. With increasing curing time, the by different techniques based on NMR spectroscopy, includ- transverse relaxation time initially increases due to sample ing 1D and 2D-NMR analyses. NMR spectra were acquired heating from the exothermic reaction and then decreases as on a BRUCKER AVANCE (400 and 500MHz) using TSP- the liquid polymerized into a solid. d4 and DMSO with DSS as internal standard. The com- pound carbon and proton spectra contain all the important The curing reaction of a flat sample was initiated by con- species whose resonances have been assigned to individual tinuous irradiation with the light of a blue LED lamp. The atoms in the repeating unit and are consistent with the pub- sample was placed on an NMR-MOUSE with 5 mm depth lished structure. These results confirm the molecule identity access and 18.1 MHz proton frequency. The curing reaction and suggest the new purification process would be used to was studied at depths from 1 to 4 mm in terms of the sum of produce native MenCPS as vaccine component. the firsts 64 echoes of a CPMG train. The curing curves were modeled with the original Kohlrausch Low field NMR function and modified according to Weibull to account for the initial sample heating effect and for the subsequent sig- nal decay from curing. The use of the Kohlrausch function TH258: Flow Regime Analyzer Based on Low- is justified from the first-order polymerization kinetics of the Field Nuclear Magnetic Resonance and Field-Cycling initiation, propagation and termination steps of the reaction. Scheme for Fluid Contrast The depth dependence of the reaction kinetics is accounted for by the law of Lambert and Beer. 1,2∗Lucas Matías Cerioni, 1Cristian Sebastián Moré, 1Julia Inés García, 1,2,3Daniel José Pusiol By fitting the model to the experimental curing curves at dif- 1SPINLOCK SRL, Córdoba, Argentina, 2Argentinian Na- ferent depths, the kinetic constants of the reaction and the tional Research Council, 3Instituto de Física Enrique Gavi- optical extinction coefficient could be determined. The ab- ola, Ciudad Universitaria, Córdoba, Argentina sorption coefficient obtained from the NMR analysis is similar to the one determined by optical transmission experiments. The real-time determination of the flow regime and the com- Given the kinetic parameters of the curing reaction, the cur- position of the complex fluid being extracted through the ing conditions can be optimized. production line is today one of the main challenges in the oil industry. In this work we present a system, based on a ACKNOLEDGEMENTS: Antonio Marchi Netto thanks the Halbach magnet type, designed for determining in real-time, Brazilian National Council for Scientific and Technological mean velocity and fraction of components of complex fluids Development for his PhD fellowship. directly in the production vein by means of low-field Nuclear Magnetic Resonance. This constitutes an extension and im- TU260: Mobile Low-Field 1H NMR Spectroscopy for provement of a previous work. on-line monitoring transesterification reactions and The apparatus includes a main Halbach magnet of 35 cm in biodiesel analysis. length, with a cylindrical region of interest (ROI) of 5 cm 1 , 1,2Mario Henrique Montazzolli Kill- in diameter and 10 cm in length, allowing to measure in a Yamila Garro Linck ner, 1Ernesto Danieli, 2Jarbas José Rodrigues Rohwedder, 5 cm (2 inch) pipe. By means of a method based on the 1∗Bernhard Blümich analysis of the early behavior of the echo amplitudes of a CPMG sequence and without applying any static or pulsed 1Institut für Technische Chemie und Makromolekulare gradients, the cuts and mean velocity of oil and water mix- Chemie, Aachen University, Aache, Germany., 2Instituto tures were measured for flow-rates between 0.2 and 4 m3/h. de Química, Universidade Estadual de Campinas, Brazil, Density contrast between oil and water phases with different Campinas, Brazil longitudinal relaxation times T1 was accomplished varying Biodiesel has gained importance in recent decades because of the pre-polarization field. Unlike a previous work where the its favorable properties. It is defined as alkyl esters of veg- pre-polarization field of variable effective length was achieved etable oils or animal fats, by means of transesterification with by means of Halbach stacks with rotation capabilities, the an alcohol. The quality control of biodiesel is important to prepolarization stage of 200 cm in lenght remains fixed, and the successful commercialization of this fuel and its blends[1]. an electromagnet of 30 cm in lenght before the main mag- net provides the fluid contrast. A better contrast is achieved We show the use of a new mobile low-field 1H NMR device, using a field-cycling type scheme on the pre-polarizing mag- based on permanent magnets arranged in a modified Halbach netic field, taking advantage of the greater difference in oil geometry[2], for the real time study of transesterification re- and water relaxation times at Larmor frequencies ranging in actions. Low-field 1H NMR spectra of the compounds co- low-field values (kHz range). A theoretical modeling using existing in a typical transesterification reaction are first dis- adiabatic processes for the changes in the magnetic field ex- cussed. Then, the online monitoring of the reaction through perienced by the sample while flowing allows optimizing the NMR spectroscopy is presented, which allows the determi- contrast between the two phases. The experimental results nation of the conversion rate of the product. This was de- are in agreement with the theoretical framework. termined by a partial least squares (PLS) calibration model, validated with high field experiments. Additionally, the rela- ACKNOWLEDGMENTS: CONICET, FONTAR, SPIN- LOCK tive concentration values of hydroxilic protons were also an- alyzed, giving a quantitative idea of the alcohols partitioning between the two phases during the reaction. These results MO259: The NMR-MOUSE® as a tool to study are useful to understand the reaction mechanism of biodiesel depth photo-curing reaction. production. A strong shift in the spectral position of the hy- 1∗Antonio Marchi Netto, 2,3Johannes Steinhaus, droxilic protons was observed, which shows good correlation 3Bernhard Moeginger, 2Berenika Hausnerova, 1Bernhard with the conversion ratio of the reaction. Moreover, the de- Blümich gree of saturation of different biofuel samples was estimated by low-field NMR spectroscopy measurements[3], which is of 1RWTH Aachen University, 2Tomas Bata University in great importance because of its direct relation with biodiesel Zlín, 3Bonn-Rhein-Sieg University of Applied Sciences properties such as viscosity, density and higher heating val- A current technique in tooth restoration fills shallow cavities ues.

146 Poster Abstracts – Low field NMR

[1] G. Knothe, J. Van Gerpen, J. Krahl (eds.), The biodiesel ian gas stations, were built. All the samples were previously handbook (AOCS Press, Champaign, 2005). analyzed by standard methods.

[2] E. Danieli et al. Angew. Chem. Int. Ed. 49, 4133 (2010). The developed models were validated with 20 external sam- ples that were not used for development of the calibration [3] Y. Garro Linck, M. H. Killner, E. Danieli and B. Blümich. models. For the properties density, distillation temperature Appl. Magn. Reson. 44, 41 (2013). with 10 and 50% recovered, cetane index and flash point the PLS models showed a high correlation coefficient (R2 > 0.9) and determination errors similar to the standard methods. TH261: Prediction of Sweetness of Papaya, in In- The biodiesel content was directly determined by the inte- tact Green Fruits, Using Online Time Domain NMR gration of the methoxy peak of the fatty acid methyl esters Spectrometer Based on Widebore Halbach Magnet. versus an internal standard. 1André de Souza Carvalho, 2Fernando Henrique Vieira, It is important to highlight that the mobile low-field 1H NMR 3∗Luiz Alberto Colnago device needs only 10 seconds for acquisition of 1H NMR spec- 1 2 3 Universidade Federal de São Carlos, UNICEP, Empresa trum, and applying the developed PLS models the process of Brasileira de Pesquisa Agropecuária diesel fuel quality control can easily work fast (less than 1 Brazil is the major papaya’s producer and exporter with an minute for each sample) and continuous in automated mode. estimated production about 1,5 million tons/year. The need ACKNOLEDGEMENTS: DAAD, CAPES to export high quality fruits is increasing and aim of this work is to predict papaya sweetness using online time do- main NMR spectroscopy. Low field time domain NMR has TU263: Prediction of Brazilian crude oils fractional been evaluated as an industrial sensor for intact measurement composition by low-field NMR relaxometry of internal fruit quality. Here we are showing that this tech- Asif Muhammad, Felipe Silva Semaan, Fabio da Silva Mi- nique can be used to predict the papaya sweetness, expressed ∗ randa, Rodrigo Bagueira de Vasconcellos Azeredo in total content of soluble solids (◦BRIX). The analyses can be performed just after harvesting, approximately 10 days Universidade Federal Fluminense before it is read to human consumption. This procedure can Crude oil fractional composition plays an important role in be used to discard low sugar papayas in the farms before the several aspects of the petroleum industry from oil explo- exportation. The unripe papayas analyzed were purchased in ration and extraction, to its refining processes, transport, São Carlos - SP, Brazil and were measured in a SLK CA02.12 and petroleum derivative marketing. Unlike petrochemicals, spectrometer Spinlock, Cordoba, Argentine, based on B0 = which have a well-defined chemical composition, petroleum 0.21 T Halbach magnet with 10 cm free bore. The fruits is a complex multicomponent system, whose individual com- were analyzed with the CPMG pulse sequence and the Brix ponent characterization is a virtually impossible task. Fac- value of the ripe papayas with a portable refractometer RT- ing this difficulty, group type classification methods based on 30ATC. The monoexponential T2 values obtained from the solubility (SARA fractionation) and thermogravimetric be- CPMG decays varied from 0.9 to 1.1 s in the green papaya havior are frequently used by petroleum industry. However, and show small increased after ripening. The T2 values of these methods not always meet the requirements normally both green and ripe papayas show high and negative correla- demanded for in situ applications, like robustness and speed. tion (-0.9) with Brix. Therefore, these results indicate that In this sense, low-field NMR relaxometry can be an interest- T2 can be used to predict, even in unripe fruit, if the pa- ing alternative to these techniques for oil characterization by paya will be sweet or not. The method is now been tested in well logging, in which measurements are performed downhole large scale and can be a useful tool to help farmers and the into the oil well. Therefore, in the present work we evaluated consumers with high quality fruits. the application of low-field NMR relaxometry, assisted by multivariate data analysis (MVA), to predict the crude oils ACKNOLEDGEMENTS: FAPESP and CNPq fractional composition obtained by Thermogravimetric Anal- ysis (TGA). Fifteen samples of Brazilian crude oils, ranging ◦ ◦ MO262: Application of a Mobile Low-Field 1H NMR from light (38.1 API) to extra-heavy (9.9 API), were as- 1 Device for Quality Control of Diesel Fuel sessed for such purpose. From their respective H T2 dis- tributions, Partial Least Squares Regression (PLSR) models 1∗ 2 Mario Henrique Montazzolli Killner, Ernesto were developed to predict the composition according to main 1 2 Danieli, Jarbas José Rodrigues Rohwedder, Bernhard four groups. All developed PLSR models showed good pre- Blümich dictive performances, as follows: group 1 (light compounds, 1Universidade Estadual de Campinas, 2ITMC-RWTH <100◦C), R2 = 0.93 and RMSEP = 0.92; group 2 (heavy Aachen University compounds, 100 - 300◦C), R2 = 0.94 and RMSEP = 2.31, group 2 (resin, 300 - 450◦C), R2 = 0.90 and RMSEP = 0.20; In 2011, 2.3% of 91,022 diesel samples policed in Brazilian gas and group 4 (asphaltene 450 - 500◦C), R2 = 0.90 and RMSEP stations by ANP (Brazilian National Agency of Petroleum) = 1.27. were nonconforming with the ANP specifications. This finding is mainly attributed to fuel adulteration by diesel TWAS, CNPq, PETROBRAS and ANP (Compromisso com providers to increase its profitability. This fact brings forth Investimentos em Pesquisa e Desenvolvimento) onus to the country specially because of tax evasion. The most important properties to certify diesel quality are: den- TH264: Through-package fat determination of com- sity, cetane index, sulfur content, flash point, biodiesel con- mercial mayonnaises with time-domain nuclear mag- tent and distillation temperature with 10, 50, 85 and 90% netic resonance spectroscopy and chemometrics recovered. Although the determination of these properties is inevitable for ensuring diesel quality, it is time-consuming ∗Fabiola Manhas Verbi Pereira, Luiz Alberto Colnago and laborious, which makes an embracing and efficient con- Embrapa Instrumentação, São Carlos, Brazil trol more difficult. Although fat is an important nutrient in human diet, the In view of this, the present work shows an application of a high fat content food has been the major cause of one of the mobile low-field 1H NMR device, built from permanent mag- serious human healthy problems of modern world, the obe- nets which generates a magnetic field of 1T (42 MHz 1H sity. Therefore, the precise determination of fat content in Larmor frequency, with homogeneity better than 0.04 ppm raw and processed foods has been an important analytical is- for the sample region) to diesel fuel quality control. For that, sue. Here, time domain NMR spectroscopy (TD-NMR) based different PLS calibration models applying low-field 1H NMR on wide bore Halbach magnet can be used as fast through- spectra of 40 diesel fuel samples, collected in different Brazil- package method in quality control and quality assurance of

147 Poster Abstracts – Low field NMR fat content in commercial mayonnaises in sealed flasks. The Júnior, Eustáquio Vinícius Ribeiro de Castro analyses were performed using a CPMG sequence in a Spin- Federal University of Espirito Santo lock (Cordoba, Argentine) TD-NMR spectrometer based on 0.23T Halbach magnet with 10 cm free bore. The compar- In this work was investigated the application of low field isons between univariate model using the discrete values of NMR (LF-NMR) to predict physical and chemical proper- transverse relaxation time (T2) obtained by fitting the se- ties of petroleum products. quence of pulses Carr-Purcell-Meiboom-Gill (CPMG) decays For the tests two Brazilian crude oil were distillated: the to mono-exponential function and multivariate models per- crude oil 1 presented TAN = 1.15 mg KOH/g, density of formed with partial least squares (PLS) were evaluated. The 0.9749 g cm−3 and kinematic viscosity 5800 mm2 s−1 at 40 predictability of the PLS model was tested with 10 exter- ◦C, and the crude oil 2 with TAN = 1.42 mg KOH/g, density nal samples from another lot. The high linear coefficients of 0.9164 g cm−3 and kinematic viscosity 58.115 mm2 s−1 at (>0.9) and low root means square errors (RMSE) for cross 40 ◦C. validation and validation proved the applicability of the PLS model.The finding about this study is the TD-NMR CPMG The NMR experiments were performed using a Maran 2 Ul- 1 relaxation decays successfully predict total fat on sealed pack- tra NMR spectrometer at 2.2 MHz for H. The T2 values ing of commercial mayonnaises varying in fat content up 10.0 was measured using the Carr-Purcell-Meiboom-Gill (CPMG) to 55.8 g 100 g−1. The PLS models combined to these signals pulse sequence at 27.5 0C. The CPMG pulse sequence was shown the best results. The main advantage verified is the applied employing π/2 and π pulses with durations of 8.15 no-invasive measurement of fat content performed for intact µs and 16 µs, respectively. In the CPMG experiments were packing of food products. The high linear correlation coeffi- recorded 32 transient with 8192 echoes in each transient, one cients between the reference values from Bligh and Byer lipids point per echo, echo spacing of 0.2 ms and recycle time of 3s. extraction and those predicted by PLS model evidences the The physical and chemical properties as refractive index, accuracy of multivariate model against the univariate fitting kinematic viscosity, total acid number, boiling point, spe- T with the discrete 2 values. cific gravity were determined directly by standard method- ACKNOLEDGEMENTS: CNPq for the fellowship awarded ologies ASTM D-1218, D 445-06, D-664-04, D-2892 and D to F.M.V. Pereira, FAPESP and Finep. 4052, respectively. The results showed good correlation with the mean values of the transversal relaxation time (T2). MO265: Applying NMR relaxometry procedures to It can be concluded of this work, that the proposed method characterize and classify soil samples for distillated analysis, LF-NMR, presented several advan- tages as non-destructiveness and not require solvents or dilu- 1∗ , 2Rodrigo de Oliveira Silva, Ruben Auccaise Estrada tion. This allows the assessment of several properties simul- 2Tito José Bonagamba, 1Fabiano de Carvalho Balieiro, taneously, based on the output of only one NMR experiment, 1Etelvino Henrique Novotny leading to large economy in terms of energy, time and costs. 1Empresa Brasileira de Pesquisa Agropecuária, 2Instituto de Thus, it is suggested that the LF-NMR technique can be ap- Física de São Carlos, Universidade de São Paulo plied for routine analysis as a guide in the quality control of The characterization of unsaturated hydraulic properties of the distilled products. soils by using Richard’s chamber and van Genuchten phe- ACKNOLEDGEMENTS: CNPq, FAPES, PETROBRAS, nomenology of water retention are very time consuming LABPETRO DQUI/UFES. methods[1]. By this procedure, many researcher groups treat to describe the dynamics of water molecules in the soils sam- TH267: Influence of the incorporation of fibers in bis- ples. In this description, they take advantage about the de- cuit dough. Characterization by time domain NMR pendence of moisture content on different soil water potential, and rheology. which in some kinds of soils samples takes approximately of some days to many weeks[2]. 1M.R. Serial, 2M.S. Blanco Canalis, 1M. Carpinella, 2,3A.E. León, 2,4P.D. Ribotta, 1∗Rodolfo Héctor Acosta An alternative and faster procedure is to apply NMR relax- 1 2 ometry protocols and principal component analysis (PCA) Universidad Nacional de Córdoba, CONICET, Instituto technique to perform the classification and characterization de Ciencia y Tecnología de Alimentos Córdoba, CONICET, 3 in different kinds of soils. The main purpose of this new Facultad de Ciencias Agropecuarias, Universidad Nacional 4 procedure is to diminish the time of extracting information de Córdoba, Facultad de Ciencias Exactas, Universidad Na- about characterization and classification of soils samples. To cional de Córdoba put in practice this idea, we used three different soils with Several epidemiological and experimental studies show that different concentrations of clay, sand and silt[3]. This choice consumption of determined food products can act either as allows us to test the sensibility of this procedure. We com- protective or as risk factors on non-transmissible diseases. pare our results with other well established technique which Recommendations have emerged that aim to reducing the is the inverse Laplace transform[4]. ingestion of sugars and fats, or to increase the fiber content References: (particularly soluble) in the diet. Biscuit or cookie dough is a complex system containing abundant components in differ- [1] M. Th. van Genuchten, Soil Sci. Soc. Am. J., 44, 892- ent states, such as starch, gluten, lipids (flour constituents), 898,(1980). sugars, fats and water. In biscuits, both, the incorporation of fibers or the reduction of sugar and fat, create a number [2] B. Minasny et al., Geoderma, 93, 225-253,(1999). of technological problems in processing and in some cases a [3] F. de C. Balieiro et al., Arvore, Viçosa-MG, 32, 153-162, loss of acceptability [1]. For this reason, the effects on the (2008). physico-chemical, rheological and structural characteristics of biscuit dough upon incorporation or reduction of determined [4] Marcel Nogueira D’Eurycide, PhD Thesis, IFSC-USP, São ingredients is of great interest in food industry. It is generally Paulo Brasil (2011). known that the distribution of water can affect the rheology ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, of the dough machinability [2]. Low resolution NMR is an FAPESP. important tool as it allows the study of water mobility by means of relaxation time measurements (T2) in the sample, TU266: Physical and chemical characterization of in a non-invasively, fast and accessible way. In this work we products petroleum. study the proton water mobility in standard biscuit dough through relaxation profiles obtained from a CPMG sequence ∗Lúcio Leonel Barbosa, Flávio Vinicius Crizóstomo Kock, at 0.5 Tesla. Different populations are assigned to different Vinícius Mansur Dose Lage de Almeida, Valdemar Lacerda ingredients [3]. The dependence of mobility as a function

148 Poster Abstracts – Low field NMR of the dough temperature in a cooking process is correlated 10% (w/w) NaCl 50 g/L solution and applying shear rate of to rheological experiments. We analyzed the quality of the 6500 and 3000 rpm, respectively for 3 minutes. Preliminary final product upon variation of different parameters in the analysis showed unimodal distributions in the two emulsions. biscuit formula, such as reduction of fat, reduction of sugar The first emulsion, NMR provided 3.66 µm as the diameter and incorporation of fiber with flour reduction. of the largest occurrence frequency of the drops and O.M about 4.20 micrometers. In the second, NMR showed drops [1] Maache-Rezzoug et al. Journal of food engineering 35, with 4.40 µm in diameters, while O.M presented 5.10 µm. It 23-42 (1998). was concluded that there was good agreement between the [2] Ruan et al. Cereal Chemistry, 76, 231–235 (1999). techniques, demonstrating the potential of low-field NMR to determine droplet size and type of emulsions, i. e, highly [3] Assifaoui et al. Carbohydrate Polymers 64, 197–204 stable. (2006). ACKNOLEDGEMENTS: CNPQ, FAPES, CAPES, MO268: On-Line Monitoring by NMR in Time LABPETRO-DQUI/UFES Domain of Chitosan in Marine Environment Con- taminetd by Oils TH270: Studies of Magnetoelectrodeposition of Cop- per using time domain NMR. ∗Flávio Vinicius Crizóstomo Kock, Lúcio Leonel Bar- bosa, Valdemar Lacerda Júnior, Eustáquio Vinícius Ribeiro 1,2∗Bruna Ferreira Gomes, 2Luiz Alberto Colnago, de Castro, Eloi Alves da Silva Filho 1Luiza Maria da Silva Nunes, 2Luis Fernando Cabeça Federal University of Espírito Santo 1Universidade de São Paulo, 2Embrapa Instrumentação Petrochemical accidents have attracted great notoriety in the NMR has been used to monitor electrochemical reaction in scientific community due to damage caused in offshore and situ for many years. Although the effect of magnetic field in onshore environments. The present study carried out spread the electrochemical reaction is known for a long time, no in oil in seawater and monitored the action of the biosorbent on situ NMR study has considered it. The effect of NMR mag- 1 line inside a NMR spectrometer of 2.2 MHz for H nucleus. netic field (B0) in presence of electric field has been studied only as a pumping and mixing procedures in microfluidics The biomaterial chosen was chitosan, because of this material devices. Therefore we are investigating the influence of NMR has special features, for example, biodegradability, facility magnetic field during copper electrodeposition reaction. The for realizes complexation with metals and flocculation ability experiments were performed in Spin Lock (0.23T) time do- that makes possible to study their action as bioremediation main spectrometer. BV Palm Instruments potentiostat were agent. used in the electrochemical measurements. The electrochem- 2 The on line results indicated that this biomaterial acts in- ical cell was composed of a silver plate (100 mm ), a counter creasing the viscosity and reducing by more than 30% of the electrode of spiral silver and a silver wire as a pseudo refer- T2 values. From of study with dehydrated oils was possible ence electrode. Calibration curves were made for copper ion infer that this biomaterial can interact with structure more concentration considering T2[1]. The copper electrodeposi- denser, leading to destabilization complexes compounds, for tion reaction was higher when the reaction was performed example, asphaltenes. Moreover, studies only with aqueous in the NMR magnet (M) than without it (W). In one-hour 2+ phase of emulsion, lead to infer that this biomaterial acts reaction, the Cu concentration in solution decaysto 55% forming strong interaction with molecule of water and conse- and 30% in M and W experiments, respectively. The fast re- quently increasing the viscosity of the medium. action in presence of B0 is related magneto-convective force (FB) that acts on the species Cu2+, where FB=ixB (i is the Finally, this unpublished results showed displacement of T2 current density and B0 is the magnetic induction vector). from 63.4ms to 108.3ms without and with application of chi- Therefore, we demonstrate that the strong effect of B0 in tosan, respectively. So, it can be conclude that this biosor- electrochemical reactions must be considered. bent have power of flocculation, coalescence and sedimenta- tion, provoking so destabilization of water droplet in emul- [1] NUNES, L. M., et al. (2012). In situ quantifica- sions water/oil, and this ability can be studied by nuclear tionof Cu(II) during an electrodeposition reaction using time- magnetic resonance in time domain (TD-NMR). domain NMR relaxometry. Analytical Chemistry, 84, 6351- 6354. ACKNOLEDGEMENTS: UFES, CAPES, PETROBRAS, LABPETRO. ACKNOLEDGEMENTS: FAPESP, CNPq

TU269: Determination of Droplet Size in oil emul- MO271: The use of TD-NMR as non-destructive sion by low-field NMR method to analyze mechanical injuries in apple

∗Vinícius Gomes Morgan, Lúcio Leonel Barbosa, Valde- 1Lucinéia Vizzotto Marconcini, 2Douglas William mar Lacerda Júnior, Eustáquio Vinícius Ribeiro de Castro Menezes Flores, 2Marcos Davi Ferreira, 2∗Luiz Alberto Col- Universidade Federal do Espírito Santo nago 1Chemical Institute of Paulista State University , Emulsions appear commonly in oil industry, formed by pres- Araraquara., 2Brazilian Agricultural Research Corporation, ence of water in the reservoir and secondary recovery pro- Instumentation Research Center, São Carlos. cess. Emulsion is a dispersion of water droplets in the oil, stabilized by natural emulsifiers presents in oil phase as as- Harvesting and post-harvesting systems and managements phaltenes, naphthenic acids and others. The most common have a direct effect on incidence and severity of mechanical type of emulsion is water in oil (W/O). An important prop- injuries on fruits, resulting in tissue browning, accelerating erty of this system is the droplet size distribution (DSD). water loss and induce CO2 and ethylene production. Here The DSD alter significantly some physicochemical proper- time domain NMR (TD-NMR) T2 relaxometry was used ties as viscosity and stability, indicates the best treatment to analyze the effect of mechanical injury in apple quality. process for separating water from oil, allows to monitor the The CPMG measurements were carried on Spinlock SLK production according the required specifications and serves CA02.12 NMR spectrometer based on wide bore Halbach as the basis for dimensioning the production equipment and magnet, 0.21 T, with a 10 X 10 cm2 of detection area. The treatment. Several techniques can provide the DSD, includ- CPMG decays were analyzed using inverse Laplace transform ing low-field NMR, which was used together with optical mi- program. The T2 distributions spectrum shows three peaks croscopy (O.M). Two W/O emulsions was produced from oil that have been associated to water in the vacuole ( 1.2s), 1 (◦API 29.6 and viscosity of 44.83 mm2/s at 20o C) and 2 cytoplasm ( 0.3s) and cell wall ( 0.01s). Thirty-six apples (◦API 29.4 and viscosity of 44.15 mm2/s at 20◦ C), adding were subject to different mechanical damage (ie, 3 to 12 falls

149 Poster Abstracts – Low field NMR from 40 cm height). The samples were analyzed before and the seed temperature is an important parameter to agricul- just after the impact, every hour up to 8h, and after 48 ture research. The T2 measurements were carried out in a hours. All mechanical injuries caused almost instantaneous homemade UNMR. The spectrometer uses a 0.6T (24MHz) increases in T2 values, reaching a peak at about 5h, and then home made UNMR sensor and a Tecmag console. slowly returning to initial value in 48 hours. The increase of T2 value after the injury is related to reduction of internal MO274: Estimation of available Water in Soils by oxygen in the fruits, and is direct related to the CO2 produc- NMR Relaxometry tion. When the injured apples were maintained in nitrogen 1 2 atmosphere, the T2 values increased more than three times Rodrigo de Oliveira-Silva, Ruben Auccaise Estrada, when compared to storage in air. The ratio between the 2Fabiano de Carvalho Balieiro, 2Etelvino Henrique Novotny, 1∗ T2 area of vacuolar and cytoplasmatic water decreased with Tito José Bonagamba the severity of injury and it was able to distingue between 1Instituto de Física de São Carlos - Universidade de São apples with moderate and severe injuries from non-injured Paulo, 2Embrapa Solos - Rio de Janeiro ones. Therefore, TD–NMR protocols has the potential to classify apples according to mechanical injuries incidence In soil science it is essential to understand water/soil physico- and severity. chemical interactions. Traditional procedures to understand these interactions are the measurement of water retention curves and the construction of pedotransfer functions. Both TU272: Use of TD-NMR to Measure Water Embebi- methods give accurate information about soil moisture, but tion by Seeds Directly in the Soil they can be very time consuming depending on the compo- 1∗Maria Gabriela Aparecida Carosio, 2Luiz Alberto nents of soil to be analyzed, including clay, silt and organic Colnago matter. Also, normally these measurements offer only bulk data. Soil moisture curves can also be measured by NMR 1Institute of Chemistry of São Carlos, 2EMBRAPA Instru- relaxometry, following wetting and dewetting procedures. In mentation this case, the curves can be obtained in a faster way and the NMR and MRI have been used to measure water content and physicochemical interactions between water and the various water transport in soils as well as the water content and dis- soil components normally result in different average T2 val- tribution in plant roots. Most of these studies are performed ues, allowing obtaining water retention curves for each spe- in synthetic soil (mixture of sand/silt) or real soils with very cific soil component, being more informative when compared low content of magnetic particles. The NMR studies are not with bulk measurements. In order to measure water retention well succeeded when the soil has high content of magnetic curves by NMR, we used three different soils: top soil layer of particles due to the distortion in B0 and the presence of large Abruptic Arenic Ochraquult, as well as top and deeper layers amount of paramagnetic ions that reduces the relaxation time of Abruptic Aquic Arenic Hapludult, that differ in terms of to the same order of receiver dead time. Here we are showing composition and particle size distribution. The samples were that the time domain NMR (TD-NMR) can be used to mea- drained in a controlled way using the moisture pressure plate sure the water uptake by seed in moist soil with very high extractor (Richards chamber), which dries the soil by increas- content of magnetic particles. This process is very important ing the internal chamber pressure. NMR experiments were because the seed germination begins with water uptake and performed at room temperature using a TECMAG LapNMR triggers a series of metabolic changes that culminate in the console, a 620-Gauss permanent magnet, and a homemade emergence of the primary root. The CPMG pulse sequence NMR probe. T2 relaxation times were obtained from CPMG were carried out in a SLK-100 Spinlock spectrometer (model experiments as a function of drained water for all the sam- SL.IM.01. The spectrometer uses a 0.23T permanent mag- ples, using draining pressures of 1, 3, 5, 9, 13, 20, 40, 60, and net. It was used soybean (Glycine max) and garbanzo bean 80 psi. From the T2 curves obtained versus draining pres- (Cicerarietinum) seeds. The soil used was a typical Brazil- sures for each soil, different water retention behaviors were ian soil and it was moistened to field capacity. In this soil, observed. These results reflect the different interactions of the water signal was not observed by NMR. Therefore, when water with the various soil components, showing the appli- seeds were placed in this soil the NMR signals was propor- cability of this method to differentiate soils by their specific tional to the water absorbed by the seed (imbibition process). water retention capability and composition. This work indi- The seeds in the soil absorb water in lower rate than the seeds cates how NMR relaxometry can be used as an alternative in pure water, indicating the influence of the soil ionic force method to obtain water retention curves. in this process. Therefore TD-NMR can be used to follow the imbibition process in intact seed direct in soil which is AKNOWLEDGEMENTS: IFSC/USP, FAPESP, CNPq, and not possible with current technologies. CAPES.

ACKNOLEDGEMENTS: FAPESP (2009/09734-1), EM- TU275: Compact, Cryogen-Free, High-Resolution 60 BRAPA Instrumentation MHz Permanent Magnet NMR Systems for Reac- tion Monitoring and On-Line/At-Line Process Con- TH273: Use of Unilateral NMR sensor to Measure trol Observing 1H, 19F, 31P Temperature in Intact Seeds and Seeds Inside Soils 1∗John C. Edwards, 2David A. Foley, 2Mark T. Zell, 1∗Maria Gabriela Aparecida Carosio, 2André de Souza 2Brian L. Marquez, 3Tal Cohen, 1Paul J. Giammatteo 3 4 Carvalho, Luis Fernando Cabeça, Luiz Alberto Colnago 1Process NMR Associates, Danbury, CT, USA, 2Pfizer Inc, 1Institute of Chemistry of São Carlos, 2Federal University Groton, CT, USA, 3Aspect AI, Shoham, Israel of São Carlos, 3Federal Technological University of Parana, A compact high resolution NMR system will be described 4EMBRAPA Instrumentation that can be situated on the bench-top or in the fume hood to Time domain Nuclear Magnetic Resonance (NMR) has been be used as a continuous or stop-flow detector and/or an "in- used as non-contact, non-destructive, no reagent chemical an- situ" reaction monitoring system. The same system can be alytical method. In this paper we are showing that we can fully integrated into on-line shelters for on-line process con- use time domain Unilateral NMR (UNMR) sensor to mea- trol or utilized by engineers and technicians in an "at-line" sure the temperature of intact oilseeds and oilseed in the environment. The system uses a unique 1.5 Tesla perma- soil. The measurement is based on the T2 dependence of ei- nent magnet that can accommodate sample tube diameters ther oil viscosity or temperature. As oil viscosity decreases of 3-10 mm with half-height spectral resolution (water reso- exponentially with temperature the T2 value is used to mea- nance) approaching 1-3 Hz depending on the sample volume sure temperature in intact oilseeds using a UNMR sensor. size and with excellent single pulse sensitivity. These systems Some seeds may survive in high temperature environment can be utilized in a traditional NMR methodology approach for days, but the seedling may die in few hours. Therefore, or combined with chemometric approaches that allow NMR

150 Poster Abstracts – Low field NMR data to predict chemical and physical properties of materi- cross-links with biodiesel addition, which would also be in ac- als via regression analyses that establish correlations between cordance with the increase of exudation in the biodiesel rich observed spectral variability and sample-to-sample property oil mixtures. Therefore, our results suggest that the main variance [1]. The systems utilized since the early 1990’s are responsible by the deterioration of the mechanical properties capable of single channel operation on higher sensitivity nu- commercial VNR when exposed to biodiesel, as compared to clei (1H, 19F, 31P, 23Na, 7Li, 11B). A new generation of diesel, is an exudation effect. NMR systems are now being manufactured featuring multi- channel operation giving the possibility to monitor two nuclei ACKNOWLEDGMENTS: Schlumberger-Doll for the at once or to perform 1H-13C-DEPT and higher sensitivity Laplace inversion program; K.Saalwachter and W.Chassé for approaches to 13C observation. In pharmaceutical applica- the fitreg2.0 program for analyzing DQ NMR data, FAPESP, tions the Aspect-AI 60 MHz system was utilized in a reaction CNPQ, CAPES. monitoring scenario where a reaction was monitored simul- taneously on a split sample loop by the 60 MHz NMR and MO277: Applying Data Mining Techniques on NMR a 400 MHz Bruker Avance III superconducting NMR spec- Data to Predict Carbonate Rocks Permeability trometer [2]. The results obtained on the two systems were virtually identical indicating that the 60 MHz NMR system Eduardo Corrêa Gonçalves, Pablo Nascimento da Silva, ∗ can be used to transfer PAT knowledge generated on phar- Alexandre Plastino de Carvalho, Rodrigo Bagueira de Vas- maceutical reactions in the research lab to the manufacturing concellos Azeredo areas for production monitoring. Universidade Federal Fluminense [1] "Process NMR Spectroscopy: Technology and On-line Ap- Data Mining can be defined as a process that applies effi- plications", John C. Edwards, and Paul J. Giammatteo, in cient algorithms to discover valuable knowledge hidden in Process Analytical Technology: Spectroscopic Tools and Im- databases. Over the last years, an increasing number of en- plementation Strategies for the Chemical and Pharmaceuti- terprises have been incorporating this technology into their cal Industries, 2nd Ed., Editor Katherine Bakeev, Blackwell- major processes in order to gain competitive advantage. Dif- Wiley, 2010. ferent kinds of data repositories can be analyzed by data min- ing algorithms, such as transactional databases, data ware- [2] "Application of a 60 MHz Permanent Magnet NMR Sys- houses, text files, and image data. This work addresses the tem to Online NMR Reaction Development in the Pharma- application of data mining analysis on an 1H NMR relaxation ceutical Industry", David A. Foley, Mark T. Zell, Brian L. dataset of carbonate reservoir rocks with the goal of accu- Marquez, John C. Edwards, and Paul J. Giammatteo, Pre- rately predict their class of permeability. The petroleum in- sented at PittCon 2013, Philadelphia, PA, March 21, 2013. dustry uses NMR as a noninvasive and nondestructive tool to Poster PDF available at www.process-nmr.com. study reservoir fluids (gas or liquid hydrocarbons and aque- ous solutions) and rock properties, yet offering the advantage TH276: Effects of Biodiesel/Diesel on Vulcanized of measurements being performed downhole into the well. Natural Rubber as Revealed by Relaxometry and Among the important NMR deliverables for reservoir eval- Double Quantum Time-Domain NMR uation we may cite porosity, pore size distribution, fluids saturations, wettability, and, specially, permeability, which 1 2 2 Lorena M. A. Silva, Fabiana Diuk, Eduardo Ribeiro de corresponds to the ability a rock has to allow a fluid to 1 3 Azevêdo, Elenilson G. A. Filho, Marcos Roberto Monteiro, pass through it. The dataset studied in this work consists 1∗ Tiago Venâncio of a matrix X78,101 formed by T2 distributions (with 100 1Departamento de Química, Universidade Federal de São T2 bins each) and the respective porosities from 78 fully Carlos, 2Instituto de Física de São Carlos, Universidade de brine saturated core samples (3.8 × 4.5 cm plugs). We eval- São Paulo, 3Centro de Caracterização e Desenvolvimento de uated the effectiveness of five different data mining algo- Materiais, Universidade Federal de São Carlos rithms (SVM, Multilayer Perceptron, k-NN, Decision Tree, and Naive Bayes) to predict the absolute rock permeability Biodiesel is a promising alternative fuel to meet the energy class according to the following ranges: low (<1mD), fair demand. However, its compatibility with the rubber mate- (1-10mD), good (10-100mD) and excellent (>100mD). These rials employed in automobile enginesis a growing concern. data mining algorithms were able to significantly outperform Thus, we address the effect of biodiesel and diesel in the net- two methods widely adopted by the petroleum industry to work structure of commercial carbon black filled vulcanized estimate permeability (Timur-Coates and Kenyon models). natural rubber (VNR). For this purpose, we conducted static The SVM classifier achieved the overall best result (predic- immersions of VNR in blends of fuels containing different tive accuracy of 84.6%), followed by Multilayer Perceptron amounts of biodiesel:diesel (0:100, 5:95, 20:80, 100:0) at 70 (83.3%) and k-NN (74.4%), against 57.7 % and 55.1% from ◦C. These samples were studied using CPMG and DQ (dou- Timur-Coates and Kenyon models, respectively. ble -quantum) time domain NMR experiments performed in an Bruker mq20 TD-NMR. The Laplace inversion of the ACKNOLEDGEMENTS: BG Brasil, ANP (Compromisso CPMG data revealed that for VNR without contact with fuel com Investimentos em Pesquisa e Desenvolvimento) the decay is comprised by three main components associated to rigidified chains at the filler vicinity (T2 0.4 ms), cross- TU278: Field Cycling NMR Relaxometry of Starch- linked chains (T2 2.0 ms), and less restricted chains in the PLA Blends with Montmorillonite Clay polymer network (T2 13.1 ms). Immediately after exposure to fuel, the average T2 increases for all components due to 2Luciana M. Brito, 1∗Pedro José Sebastião, 2Maria Inês swelling, but stabilizes after few minutes. Over time periods Bruno Tavares of days and for higher amounts of biodiesel, the average T2 1Instituto Superior Técnico - Lisboa, 2Universidade Federal values rise up. The changes were more pronounced for the do Rio de Janeiro shorter component (0.4 ms) and also for immersion periods of 30 to 90 days. This suggests an exudation process, where Low-field NMR spectroscopy has been shown to be a use- the removing of the filler is facilitated in biodiesel rich oil ful method to determine proton relaxation times. Its spec- mixtures. DQ-NMR measurements indicate a progressive re- troscopy determines the values of proton spin-lattice relax- 1 1 duction in the averaged H- H residual dipolar coupling for ation time, which has a time constant T1, and proton spin– increasing amounts of biodiesel. These experiments also re- spin relaxation time, with time constant T2. Both relaxation vealed that the amount of defects (not coupled chains) does times allow evaluating the sample behavior at the molecu- not change significantly with the addition of biodiesel, sug- lar level, because they are sensitive to molecular motions at gesting that the reduction in the 1H-1H residual dipolar cou- the MHz scale. Hence, changes in molecular mobility are pling is not due to swelling. Taken together, this behavior normally detected and can be accompanied by T1 and T2 allowed inferring a decrease in the molecular mass between measurements. The main application fields of field-cycling

151 Poster Abstracts – NMR Theory

NMR relaxometry to be considered in the following are: sur- face related relaxation processes of fluids in porous materials; NMR Theory polymer dynamics, biopolymers and biological tissue, liquid crystals and lipidbilayers. MO280: Coherent and squeezed states in an NMR The field-cycling technique has been applied to melts, solu- quadrupolar system tions and networks of numerous polymer species. The pa- rameters varied in the experiments were the temperature, 1∗Ruben Auccaise Estrada, 2Arthur Gustavo de Araújo the molecular weight, the concentration and the cross-link Ferreira, 2Eduardo Ribeiro de Azevêdo, 3Eduardo Inácio density. Duzzioni, 2Tito José Bonagamba, 2Miled Hassan Youssef Moussa, 4Roberto Silva Sarthour Júnior, 4Ivan dos Santos Starch and PLA hybrids were obtained using clay MMT so- Oliveira, 4Itzhak Roditi lution and PVA. All nanocomposites were obtained by the 1Empresa Brasileira de Pesquisa Agropecuária, Rio de solution intercalation method using chloroform and water as Janeiro, Brazil., 2Instituto de Física de São Carlos, Univer- a solvent. The frequency dependence of spin lattice relax- sidade de São Paulo, São Carlos, Brazil., 3Departamento de ation times measured by means of Fast Field Cycling (FFC). Física, Universidade Federal de Santa Catarina, Florianópo- NMR techniques were used to study the molecular dynamics lis, Brazil., 4Centro Brasileiro de Pesquisas Físicas, Rio de of systems. The renormalized Rouse formalism were applied Janeiro, Brazil. to describe the polymer behavior in the studied samples. The results of the FFC showed that the starch has only one relax- The concepts of coherent and squeezed states are well es- ation time T11, related to amorphous region. PLA hybrids tablished in the context of light and atoms[1]. In quantum presented two distinct spin-lattice relaxation times, T11 e mechanics, the importance of coherent state lies in the fact T12. The mixture of the two polymers showed two relax- to minimize the Heisenberg’s uncertainty principle. Coher- ation times. By adding clay or PVA we can observe differ- ent states describe, accordingly to quantum mechanical laws, ences in relaxation time T12, observing that adding clay and the collective behavior of particle systems as take place, for PVA, the effect that each has on the dynamics of the mixture example, in a Bose-Einstein condensate[2]. Squeezed states is canceled when added separately at the same time in the are generated from a coherent state by unitary transforma- polymer blend. tions, in which the Hamiltonian has a nonlinear dependence about second quantization operators, angular momentum op- TH279: PLSR-based permeability estimations in erators, or so on. This concept was extended to spin sce- carbonates: a combined NMR-Resistivity approach nario, preserving commutation rules of angular momentum operators, and physical properties as occurs for light and in 1,2∗Edmilson Helton Rios, 1Irineu Figueredo, 3Asif atoms[3]. Muhammad, 2Vinicius de França Machado, 4André Alves de Souza, 3Rodrigo Bagueira de Vasconcellos Azeredo The purpose of the present work is to show the possibility of extending this main concept to an NMR quadrupolar sys- 1National Observatory, 2Petrobras Research Center, tem. In order to achieve this objective NMR radiofrequency 3Fluminense Federal University, 4Schlumberger Brazil Re- pulse techniques and deviation density matrix reconstruction search and Geoengineering Center procedures were employed. For implementing the proposed Empirical models used in nuclear magnetic resonance (NMR) NMR experiments, 23Na and 133Cs quadrupole nuclei located well logging capitalize on the correlation between pore-body in different lyotropic liquid crystals samples were used[4,5]. and pore-throat-size to estimate permeability from relax- ation time distributions. However, one of the drawbacks References: of these models is that they summarize all the information contained in the distributions to just one variable ignoring [1] M. O. Scully and M. S. Zubairy, Quantum Optics, Cam- that each relaxation bin may have a complex and singular re- bridge University Press 1997. lationship with pore-throat-size. Recently we demonstrated [2] G. J. Milburn et al., Phys. Rev. A, 55, 4318–4324, (1997). that linear partial least square regression (PLSR) applied in relaxation data can deliver more robust and accurate perme- [3] M. Kitagawa and M. Ueda, Phys. Rev. A, 47, 5138–5143, ability estimations than the classical models which are based (1993). on the relaxation time geometric mean or on the free/bound fluid ratio quantity (function of a relaxation cut-off). The [4] R. Auccaise et al., arXiv 1301.2862 (2013). NMR PLSR-based permeability models were originally ap- plied to brine saturated sandstones and here we generalized [5] A. G. Araujo–Ferreira et al., arXiv 1301.5554 (2013). these models for a set of carbonate samples. Although the acquired performance was still better than classical mod- ACKNOLEDGEMENTS: CAPES, CNPq, FAPESP, els, a poor prediction capability was found compared with FAPERJ, Brazilian National Institute of Science and Tech- siliciclastics. Most probably this is due to the inherent com- nology for Quantum Information (INCT-IQ). plexity and heterogeneity of carbonate pore systems that can present low relaxivity which strengthens bulk relaxation TU281: Theoretical Calculations of Chemical Shifts signal especially in large pores (vugs). Furthermore diffusion (δ) of Two Synthetic Intermediates for Guaianes and coupling between different pore sites can also contribute to nor-Guaianes: A Comparison Between GIAO and reduce sensitiveness of relaxation times to pore sizes. Once CSGT Methods electrical resistivity response, such as formation factor, ce- mentation coefficient and tortuosity, depends on rock perm 1Christiane Feijó de Castro Porto, 1Layla Rosário Bar- porosity properties, we measured and integrated this extra bosa, 2Ygor W. Vieira, 1∗Valdemar Lacerda Júnior, 2Kleber petrophysical information to the previous NMR PLSR-based Thiago de Oliveira, 1Reginaldo Bezerra dos Santos, 1Sandro permeability models. The proposed novel approach delivered José Greco, 1Alvaro Cunha Neto, 2Timothy John Brocksom, an overall best performance, improving a lot the correlation 1Eustáquio Vinícius Ribeiro de Castro between predicted and measured permeability and reducing 1Federal University of Espirito Santo, 2Federal University of expressively the standard error of prediction. These results São Carlos highlighted the potentiality of combined NMR-resistivity models, which are also applicable in well logging data, to Fused five- and seven-membered ring systems show peculiar improve permeability estimation in complex carbonate reser- structural features and these building blocks can be consid- voirs. ered as advanced intermediates for the synthesis of a large number of biologically active compounds (sesquiterpenes, ACKNOLEDGEMENTS: CNPQ, TWAS and PETROBRAS diterpenes, sesterterpenes).

152 Poster Abstracts – HR-MAS

The wide spectrum of biological activities allied with struc- in neurons initial conditions. These obtained results stated tural complexity make these compounds interesting targets this methodology as a promising tool in MRI studies. for the synthetic and NMR studies. ACKNOLEDGEMENTS: CNPq and FAPEMIG Recently, structural assignments using NMR analysis, and supported by sophisticated theoretical calculations, have be- come important for providing a good correlation between ex- HR-MAS perimental and theoretical results. In previous studies, the NMR signals of two synthetical in- MO283: Analysis of magnetic nanoparticle function- termediates of guaianes and nor-guaianes were assigned, cor- alized with oleic acid by 1H HR-MAS NMR relating the experimental and theoretical data. 1Rodrigo Alexandre Ferreira, 1Mábio João Santana, The results of the theoretical calculations are directly linked 1 1 to the method chosen. Therefore, this study aims at verifying Luiz Henrique Keng, Luiz Henrique Keng Queiroz Júnior, 2Dhiogo Mendes da Silva, 2Emília Celma de Oliveira Lima, which method (GIAO or CSGT) provides the best correlation 1∗ with the experimental data of chemical shifts (δ) of 1H and Luciano Morais Lião 13C NMR for two synthetic Intermediates of guaianes and 1IQ-UFG, Laboratório de RMN, 2IQ-UFG, Laboratório de nor-guaianes. Materiais The structures of compounds were drawn with the The analysis of paramagnetic compounds by Nuclear Mag- GaussView4.1 program and a conformational search was per- netic Resonance (NMR) is commonly unfeasible due to the formed. The structures were optimized using Gaussian03 extremely broad signals observed. On the other hand this program at B3LYP/cc-pVDZ model. The calculations of δ analysis through High Resolution Magic Angle Spinning (HR- were performed at the B3LYP/cc-pVTZ, using GIAO and MAS) has proven to be an efficient strategy to study this CSGT methods. The solvent effect (chloroform) was in- kind of materials. HR-MAS NMR was applied to the study cluded. The study verified that for 13C the best results were of iron oxide nanoparticles (Fe2O3) functionalized with oleic achieved for calculations without solvent effect. acid providing spectra with excellent resolution. In these spectra multiplicity and coupling constant of olefin hydrogens The structures of compounds were drawn with the could be perfectly observed. The spectra were performed in GaussView4.1 program and a conformational search was per- a Bruker Avance III 500 NMR spectrometer, operating at formed. The structures were optimized using Gaussian03 11.75 Tesla, equipped with a 4-mm HRMAS probehead. The program at B3LYP/cc-pVDZ model. The calculations of δ experiments were carried out in five concentrations (6.25 x were performed at the B3LYP/cc-pVTZ, using GIAO and 10−5, 6.25 x 10−4, 6.25 x 10−3, 9.38 x 10−3, and 1.25 x CSGT methods. The solvent effect (chloroform) was in- 10−2 mol.L−1) and four spinning rates (1, 3, 5 and 8 kHz) cluded. to evaluate the importance of these parameters on the in- The study verified that for 13C the best results were achieved tensity and resolution of the NMR signals. The best result 1 was observed at 9.38 x 10−3 mol L−1 spinning at 8 kHz. for calculations without solvent effect. For H, the best re- 1 sults were achieved when solvent effect was taken into ac- The purification process monitored by H NMR shows that count. Taken these values as reference, the study compared five cycles of dispersion in n-hexane and precipitation with the GIAO and CSGT methods. ethanol is more efficient for remove 1-octadecene associated to the nanoparticles than five cycles of extraction with pure ethanol or butanol. We conclude the HR-MAS NMR tech- TH282: Transversal Relaxation Time Distribution nique is effective for improving the low resolution 1H NMR from Central Nervous System MRI Data: an Ill- spectrum of paramagnetic samples, showing better results in Conditioned Inverse Problem Analysis comparison with experiments in liquid phase. This improve- Bárbara Darós de Lelis Ferreira, João Pedro Braga, ment comes from the minimization of the effects of viscosity ∗Rita de Cássia Oliveira Sebastião and paramagnetic disturbances through the magic angle spin- UFMG - Universidade Federal de Minas Gerais ning, thus increasing both the resolution and the signal/noise ratio. The central nervous system (CNS), composed essentially of white and gray matter, is responsible to control the body Acknowledgements: CNPq, CAPES, FINEP and UFG. functions and the CNS functioning is very important for per- son monitoring. The common technique used for this purpose TU284: High Resolution Magic Angle NMR Profil- is magnetic resonance imaging, MRI, with physical charac- ing of Healthy and Pancreatic Ductal Adenocarci- teristics weighted images, for example, transversal relaxation nome Tissues time, T2 - weighted images. These properties are influenced 1∗Tatiana Onofre de Lira, 2Rafael Kemp, 1Tiago Venan- by chemical environment which perturb the resolution and 2 1 contrast of images, making difficult the tissue differentiation. cio, José Sebastião dos Santos, Antonio Gilberto Ferreira 1 This work intends to obtain the T2 distribution from CNS Departamento de Química de São Carlos, Universidade simulated MRI data. The process of obtaining the micro- Federal de São Carlos, 2Divisão de Cirurgia Digestiva, De- scopic property of proton relaxation time distribution, T2, partamento de Cirurgia e Anatomia, Faculdade de Medicina from the macroscopic spin echo MRI experiments is a prob- de Ribeirão Preto, Universidade de São Paulo lem classified as an ill-conditioned inverse problem. These Pancreatic cancer represents a challenge on diagnostic and problems arise by not satisfying one of the three conditions: therapy in modern medical science. The complex accessibil- existence, uniqueness and continuity of solution. Since the ity due to pancreas localization into abdominal cavity and model function is represented by a Fredholm integral equa- specific clinical symptoms developing time, lead a diagnostic tion, these three criteria do not work and robust techniques delay compromising prognosis for patients. Thus, medicine are required to solve this kind of problem. The Hopfield neu- has been seeking alternatives that can help with accuracy, T ral network was chosen to recover 2 distribution function specificity and early diagnosis of pancreatic cancer. Various and it was also analyzed considering errors in the simulated "omics" methodologies (genomics, transcriptomics and pro- data together with errors in the neurons initial condition. teomics) have been employed with this purpose. It is believed considering T2 distribution function instead an average parameter, i.e. considering the magnetic field hetero- In this work, a case of study was introduced to evaluate the geneities in each voxel, better contrast in the image and con- presence of different metabolites in healthy and pancreatic sequently improved diagnostic information can be achieved. ductal adenocarninome cancer (PDA) tissues with the aim The neural network approach was numerically stable and ro- to investigate cellular physiology alterations. All biopsy tis- bust with noises in the MRI simulated data and deviations sues samples were furnished by Digestive Surgical Division at

153 Poster Abstracts – HR-MAS

Hospital das Clínicas from University of São Paulo (Ribeirão Preto-SP). 1D and 2D HRMAS were carried out on Bruker Avance-III 400 spectrometer equipped with 9.4 T Oxford nar- row bore magnet and 4 mm with Z gradient HRMAS probe- head. All the experiments were conducted at 6◦C and the samples were spun at 4000Hz of spinning speed. As result several compounds could be characterized among sugars, amino acids, lipids and organic acids through J- Resolved, COSY 1H-1H, HSQC 1H-13C. Particularly, in PDA tissues L-lactate appeared and β-glucose significantly de- creases revealing preference in aerobic glycolysis, compromis- ing the normal cellular energy process. In addition, huge decreases of PUFA, MUFA and phospholipids levels were observed. These alterations affect signaling functions, intra and extracellular subtract transport, osmolises, synthesis and degradation of lipids resulting in a failure of amino acids stor- age as phospholipids such as glycerophosphocholine. At last, differences on amino acids levels such as choline, taurine, ala- nine and betaine could also be observed in PDA tissues. As conclusion, this case of study could demonstrate the poten- tial of HRMAS analysis for tissue physiology comprehension and clinical diagnosis. Acknowledgments: CAPES, FAPESP, CNPQ, CBIP

154 Poster Abstracts – Posters Upgraded to Oral Presentation

∗Viviane Silva de Paula, Robson Q. Monteiro, Fabio C. Posters Upgraded to Oral Presentation L. Almeida, Ana Paula Valente Universidade Federal do Rio de Janeiro TH285: Metallobiology of neurodegenerative dis- Human β-defensins (hBD) are believed to function as alarm eases: Structural and mechanistic basis behind the molecules that stimulate the adaptive immune system when acceleration of amyloid protein assembly a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of 1,2M.C. Miotto, 1,2A.A. Valiente Gabioud, 3Andres Bi- 4 5 1,2∗ a range of different cell types to the sites of inflammation. We nolfi, L. Quintanar, Christian Griesinger, Claudio O. have solved the structure of the human β-defensins 6 (hBD6) Fernández by NMR spectroscopy that contains a conserved β-defensin 1Instituto de Biología Molecular y Celular de Rosario (IBR- domain followed by an extended C-terminus. We also investi- CONICET), Universidad Nacional de Rosario, 2Max Planck gated the interaction of β-defensin 1 and 6 with microvesicles Laboratory of Structural Biology, Chemistry and Molecular shed by breast cancer cell lines using NMR. Chemical shift Biophysics of Rosario (MPLPbioR, 3Department of NMR- mapping of the interaction showed that both defensins inter- assisted Structural Biology, In-cell NMR, Leibniz Institute of act with microvesicles but in slightly different way, suggesting Molecular Pharmacology, 4Centro de Investigación y de Es- an inverse correlation with the aggressiveness potential of the tudios Avanzados (Cinvestav), 5Department of NMR-based cell. Furthermore, molecular docking using restraints derived Structural Biology, Max Planck Institute for Biophysical from the NMR chemical shift data produced a model of the Chemistry complex between hBD6 and a peptide derived from the extra- Alpha-synuclein (AS) aggregation is associated to neurode- cellular domain of CC chemokine receptor 2 (Nt-CCR2) that generation in Parkinson’s disease (PD). At the same time, reveals a contiguous binding surface on hBD6, which com- α β β alterations in metal ion homeostasis may play a pivotal role prises amino acid residues of the -helix, loop between 2- 3 in the progression of AS amyloid assembly and the onset of and C-terminal. The microvesicles binding surface partially PD. Elucidation of the structural basis directing AS-metal overlaps with the chemokine receptor interface. These data interactions and their effect on AS aggregation constitutes a offer new insights into the structure–function relation of the key step towards understanding the role of metal ions in AS hBD6–CCR2 interaction and may be helpful for the design amyloid formation and neurodegeneration. The structural of novel anti-cancer agents. properties of the AS-metal complexes were determined by the ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq combined application of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR), circular dichro- TU287: NMR Mapping of PCNA Interaction with ism (CD) spectroscopy, and matrix-assistedlaser desorption Translesion Synthesis DNA Polymerase Rev1 Medi- ionization mass spectrometry (MALDI MS). This work pro- ated by Rev1-BRCT Domain vides a comprehensive view of recent advances attained in the metallobiology of AS amyloid diseases. A hierarchy in Yulia Pustovalova, Mark W. Maciejewski, ∗Dmitry M. Ko- AS-metal ion interactions has been established: while diva- rzhnev lent metal ions interact at a non-specific, low-affinity binding University of Connecticut Health Center interface at the C-terminus of AS, copper binds with high affinity at the N-terminal region and it is the most effec- Rev1 is a Y-family translesion synthesis (TLS) DNA poly- tive metal ion in accelerating AS filament assembly.[1-4] The merase involved in bypass replication across sites of DNA strong link between metal binding specificity and its impact damage and postreplicational gap filling. In the process of on aggregation is discussed here on a mechanistic basis. A TLS high-fidelity replicative DNA polymerases stalled by detailed description of the structural features and coordina- DNA damage are replaced by error-prone TLS enzymes re- tion environments of copper to AS is presented and discussed sponsible for the majority of mutagenesis in eukaryotic cells. in the context of oxidative cellular events that might lead to The polymerase exchange that gains low-fidelity TLS poly- the development of PD. These new findings of the structural merases an access to DNA is mediated by their interactions and metallobiology of PD are discussed via a comparative with proliferating cell nuclear antigen (PCNA). Rev1 stands analysis with the binding and affinity features of metal ions alone from other Y-family TLS enzymes since it lacks the to the beta amyloid peptide of Alzheimer’s disease.[5] Over- consensus PCNA-interacting protein box (PIP-box) motif, all, the research findings presented here support the notion instead utilizing other modular domains for PCNA binding. that perturbations in the metabolism of metal ions may be a Here we report solution NMR structure of an 11 kDa BRCA1 common upstream event in the pathogenesis of neurodegen- C-terminus (BRCT) domain from S. cerevisiae Rev1, and erative processes.[6] demonstrate with the use of TROSY NMR methods that Rev1-BRCT domain directly interacts with an 87 kDa PCNA References in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2- [1] Rasia et al, Proc. Natl. Acad. Sci. U S A, 2005, 102, β4-α3-α4) typical for BRCT domain superfamily. PCNA- 4294-4299. binding interface of the Rev1-BRCT domain comprises con- served residues of the outer surface of the α1 helix, α1-β1, [2] Bertoncini et al, Proc Natl Acad Sci U S A, 2005, 102: β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT 1430-1435. binds to the inter-domain region of PCNA that overlaps with [3] Binolfi et al, J. Am. Chem. Soc., 2008, 130,11801-11812. the binding site for the PIP-box motif. Furthermore, Rev1- BRCT domain bound to PCNA can be displaced by increas- [4] Binolfi et al, J. Am. Chem. Soc., 2011, 133, 194-196. ing amounts of the PIP-box peptide from TLS DNA poly- [5] Valiente-Gabioud et al , J. Inorg Biochem. , 2012, 117:334- merase polη, suggesting that Rev1-BRCT and polη PIP-box 341. interactions with a given PCNA subunit are mutually exclu- sive. These results provide structural insights into PCNA [6] Binolfi et al, Coord. Chem Rev., 2012, 256:2188-2201. recognition by TLS DNA polymerases that help better un- Acknowledgements derstand TLS regulation in eukaryotes. Financial support from ANPCyT, CONICET, Laboratorios TH288: Detection of light induced intermediates of Pförtner, Fundación Bunge y Born, Max Planck Society and photoreceptor membrane proteins by in-situ photo- Alexander von Humboldt Foundation is acknowledged. irradiated solid-state NMR

MO286: Structural Basis for the Interaction of Hu- 1∗Akira Naito, 1Yuya Tomonaga, 1Tetsuro Hidaka, 1Hiroki man β-Defensins 1 and 6 and Its Putative Chemokine Yomoda, 1Teruki Makino, 1Izuru Kawamura, 2Yuki Sudo, Receptor CCR2 and Breast Cancer Microvesicles 3Akimori Wada, 3Takashi Okitsu, 4Naoki Kamo

155 Poster Abstracts – Posters Upgraded to Oral Presentation

1Yokohama National University, 2Nagoya University, 3Kobe channels) [2] with multiple receivers acquisition. Multi- de- Pharmaceutical University, 4Matsuyama University coupling enhances the resolution that reaches about five times Photoreceptor retinal proteins usually absorb photon to higher resolution power than the best synchrotron resolution generate photo-isomerization, and consequently change the power. structure and dynamics of proteins. To generate retinal iso- NMR Crystallography encompasses usage of prior data from merization, we have developed the photo-irradiation system XRD as cost function for the average structure determina- equipped to the solid-state NMR spectrometer [1]. tion as well as using new NMR hardware and software [3,4] Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a with additional pulse sequences [5] to solve all the structure negative phototaxis receptor of Natronomonus pharaonis and features of nanoporous crystals. forms a 2:2 complex with the cognate transducer (pHtrII), With this instrumental developments, one can devise for which transmits the photosignal into cytoplasm. Light ab- each structure resolution an original combination of pulse se- sorption of ppR initiates trans-cis photo-isomerization of the quence manipulating the resonances between spins in a com- retinal chromophore followed by cyclic chemical reaction con- plex choreography of spins, grouped in a variety of motives sisting of several intermediates (K, L, M and O). The M in- that would progressively address all the aspects of the crystal termediate is thought to be an active state for signal trans- complexity. duction. References We have successfully trapped and observed the M interme- diate by using newly developed photo-irradiated solid-state [1] B. Bouchevreau, C. Martineau, C. Mellot-Draznieks, A. NMR system. 13C NMR signal from [20-13C] retinal-ppR and Tuel, M. R. Suchomel, O. Lafon, J. Trébosc, J. P. Amoureux, ppR/pHtrII revealed that multiple M-intermediates (M1, M2 F.; Taulelle, Chem. Eur. J. in press. cis anti and M3) with 13- , 15- retinal configurations coexisted [2] C. Martineau, F. Engelke, F. Taulelle, J. Magn. Reson. under the continuously photo-irradiated condition [1]. Fur- 212 (2011) 311-319. ther, since the life time of M1 state was much longer than those of the other M states, this M1 state could be distin- [3] E. Kupce, R. Freeman, J. Magn. Reson. 206 (2010) 147- guished from the other M states and assigned as N interme- 153, J. Magn. Reson. 213 (2011) 1-13. diate. [4] E. Kupce, L. E. Kay, J. Biomol. NMR 54 (2012) 1-7. SrSRI (Salinibacter ruber sensory rhodopsin I) is a eubac- [5] Herbst, C.; Riedel, K.; Ihle, Y.; Leppert, J.; Ohlen- terium rhodopsin and acting multiple function as attractant schläger, O.; Görlach, M.; Ramachandran, R. MAS solid and repellent phototaxis. In the photocycle of SrSRI, M in- state NMR of RNAs with multiple receivers. J Biomol NMR termediate functions as attractant and P intermediate by ab- 2008, 41, 121–125. sorbing second blue light as a double photon process shows 13 as repellement. C NMR signal of M intermediate in SrSRI [6] C. Martineau, F. Engelke, F. Decker, F. Taulelle. J. Magn. was successfully trapped by illuminating 520 nm light, and Reson. Submitted. the configuration of retinal was revealed to be 13-cis, 15-anti. P intermediate was trapped by illuminating second 365 nm [7] V. Munch, F. Taulelle, T. Loiseau, G. Férey, A. Cheetham, light as a double photon process. S. Weigel, G.D. Stucky, Magn. Reson. Chem. 1999, 37, S100–S107. Photo-intermediate in photo-receptor membrane proteins is now possible to trap by using in-situ photo-irradiated solid- TU290: Optimizing Ionic Liquids for CO2 Capture: state NMR. an NMR Approach [1] Y. Tomonaga et al. Biophys. J. 2011, 101, L50-L52. 1Marta Corvo, 1João Sardinha, 2Sonia Maria Cabral de Menezes, 3Jairton Dupont, 3Graciane Marin, 4Sandra MO289: NMR Crystallography, from “Pas de deux” Einloft, 4Marcus Seferin, 1Teresa Casimiro, 1∗Eurico J. to Spin Choreography. Nanoporous Crystal Struc- Cabrita tures of Powders : Methods and Hardware. 1REQUIMTE,CQFB, Dept. Química, Fac. Ciên- 1∗Francis Taulelle, 2Frank Engelke, 2Frank Decker, 1Boris cias e Tecnologia, Univ. Nova Lisboa, Portugal, Bouchevreau, 1Charlotte Matineau 2PETROBRAS/CENPES, RJ Brazil, 3Dept. Química Orgânica, Inst. Química, Univ. Federal do Rio Grande do 1University of Versailles, 2Bruker-Biopspin GmbH Sul, Brazil, 4Fac. Química Pontificia Univ. Católica Rio Inorganic-organic hybrid nanoporous solids exhibits many Grande do Sul, Brazil challenges for their structure determination. They contain Global warming has prompted the scientific community to a framework, that is usually periodic or at least topologi- look for new strategies for CO2 capture and storage. Re- cally periodic. However, these materials are hard to form cent studies suggest that ionic liquids (ILs) can be alterna- single crystals, most of the time one has to resolve their tive materials for CO2 capture due to their high selectivity crystal structure from powder. Additionally, they contain for CO2 absorption. ILs physical and chemical properties can either additional templating agents, of the same nature than be enhanced and modified by both their cationic and anionic the organic linkers, making the activation (emptying the moieties and this is the reason for their broad range of appli- nanopores) of the materials difficult, if not impossible, and cations. Considering that ILs properties can be designed to by and large, partial. Therefore, at synthesis or later when satisfy specific application requirements, the optimization of exchanged with gases or molecules, these exchanged crystals ILs for CO2 capture is a matter of great interest.[1-3] exhibit a periodic network and a non-periodic sub-lattice. Herein we present a detailed NMR study focused on the eval- It will be shown on an alumina-phosphate lamellar uation of the interactions between ILs and CO2. Through nanoporous crystal [1] that X-ray diffraction has intrinsic lim- careful structural modifications we were able to identify CO2- its for structure computability from a powder and this limit philic features by the analysis of CO2 solvation in imida- can be overcome by using NMR data. With this combined zolium ILs. Our High Pressure NMR (HPNMR) methodol- usage of XRD and NMR the average periodic structure can ogy allows a direct measurement of CO2 solubility in ILs and be determined. At a second level of structure determina- an in-situ assessment of the relationship of all the species tion NMR can resolve the structural non-periodic part of the involved in the solvation process - cation/anion/CO2. HP- crystal. NMR experiments based on NOE and multinuclear diffusion In a second part, it will be shown that the large number of NMR experiments using the PGSE technique combined with NMR experiments that must be acquired to solve structures molecular dynamics simulations enabled a molecular based can be accelerated by using multiple channels probe (4 and 5 interpretation for the solvation mechanism. This systematic

156 Poster Abstracts – Posters Upgraded to Oral Presentation study allowed the identification of promising candidates for geometries of hydrogen bonds. Improved chemical shift pre- CO2 capture. dictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations [1] X Zhang, X Zhang, H Dong, Z Zhao, S Zhang and Y within conformational basins. Huang Energy Environ. Sci., 2012, 5, 6668- 6681. In the second approach, chemical shifts and anisotropic re- [2] M Ramdin, TW Loos and TJH Vlugt Energy Environ. straints obtained from relaxation dispersion experiments are Sci., 2012, 51, 8149-8177. used to determine the structure of a sparsely-populated, on- [3] JL Anderson, JK Dixon and JF Brennecke Acc. Chem. pathway folding intermediate that folds on the millisecond Res. 2007, 40, 1208-1216. timescale [2]. CPMG measurements are used to measure backbone chemical shifts, RDCs, and RCSAs of the 2% pop- The authors would like to thank Petróleo Brasileiro ulated folding intermediate of the A39V/N53P/V55L Fyn SA – PETROBRAS, Fundação para a Ciência e Tec- SH3 domain and the structure of the intermediate is cal- nologia (FCT) and Ministério da Educação (CQFB culated using a recently developed chemical shift restrained Strategic Project PEst-C/EQB/LA0006/2011 and Project molecular dynamics structure calculation protocol [3]. The PTDC/QUI-QUI/098892/2008) for financial support. The structure provides a detailed characterization of the non- NMR spectrometers are part of the National NMR Network native interactions stabilizing an aggregation-prone interme- (RNRMN) and are funded by FCT. diate under native conditions and insight into how such an intermediate can derail the folding pathway and initiate fib- TH291: Determining supramolecular organisation of rillation. ion channels by solid-state NMR and computational methods These results illustrate that NMR chemical shifts can be used to provide atomistic descriptions of protein motions on mul- 1∗ 1 Markus Weingarth, Elwin van der Cruijsen, tiple timescales. 1,2Alexander Prokofyev, 1Eline Koers, 1Alexandre M. J. J. Bonvin, 2Olaf Pongs, 1Marc Baldus [1] P Robustelli, KA Stafford, AG Palmer III, JACS, 134, 1Utrecht University, Bijvoet Center for Biomolecular Re- 6365-6374 (2012) 2 search, Utrecht, The Netherlands, Saarland University, [2] P Nuedecker, P Robustelli, A Cavalli, P Walsh, P Lund- Faculty of Medicine, Department of Physiology, Homburg, strom, A Zarrine-Afsar, S Sharpe, M Vendruscolo, LE Kay, Germany Science, 336, 362-366 (2012) Protein supramolecular structure invokes the conformation [3] P Robustelli, KJ Kohlhoff, A Cavalli, M Vendrusculo, beyond a single protein. While the protein is held together Structure, 18, 1-11 (2010) by intramolecular forces comprising strong interactions like covalent bonds, supramolecular structures are often com- posed of weaker intermolecular contacts. The latter ones TU293: High Resolution Para-Hydrogen Induced confer great diversity and sensitivity to their environment Polarization (PHIP) in Inhomogeneous Magnetic upon these multi-molecular arrangements. Fields 1,2∗ 1,2 3 Membrane protein are exposed to a partiuclarly complex Lisandro Buljubasich, Ignacio Prina, María Belén 3 3 habitat which crucially modulates protein structure and func- Franzoni, Kerstin Münnemann, Hans Wolfgang Spiess, 1,2 tions, however, the molecular basis of such modulations is Rodolfo Héctor Acosta hitherto poorly understood. What is more, its transient char- 1FAMAF – Universidad Nacional de Córdoba, 2IFEG acter renders the study of membrane protein supramolecular –CONICET, 3Max Plank Institut für Polymerforschung organization experimentally very challenging. The application of parahydrogen for the generation of hyper- Using the potassium channels KcsA and KcsA-Kv1.3 as ex- polarization has increased continuously during the last years. amples, we demonstrate how the combination of solid-state When the chemical reaction that deposits the parahydrogen NMR and computational approaches, assisted by electrophys- atoms into the target molecule is carried out at the same field iological measurements, allows dissecting various aspects of as the NMR experiment (PASADENA protocol [1]) an anti- membrane protein supramolecular structure, including, inter phase signal is obtained, with a separation of the resonance alia, annular and non-annular protein – lipid binding, the in- lines of only a few Hz. This imposes a stringent limit to the fluence of structural water or protein – protein interactions required homogeneity of the magnetic field in order to avoid in membranes. signal cancellation. In this work we show that the spectrum of the PHIP signal acquired with a Carr-Purcell-Meiboom- MO292: Atomistic Descriptions of Protein Dynamics Gil (CPMG) sequence, referred to as J-Spectroscopy [2-4], on Multiple Timescales From NMR Chemical Shifts not only presents an enhanced spectral resolution compared to standard the NMR-spectrum, but also avoids partial peak ∗ Paul Robustelli cancellation. Columbia University Experimental and numerical simulations concerning the hy- NMR chemical shifts are highly sensitive probes of molecular drogenation of Hexene with parahydrogen in PASADENA structure. This work illustrates two approaches for utilizing conditions are presented. Acquisition with a digital filter the structural information contained in chemical shifts to ob- is used to select a desired multiplet, namely a partial J- tain atomistic descriptions of the structural fluctuations of Spectrum acquisition [3]. The performance of the method proteins on the ns-µs and millisecond timescales. is tested on a thermally polarized sample, showing that the corresponding partial J-Spectra are unaffected by large inho- In the first approach, semi-empirical NMR chemical shift pre- mogeneities in the polarizing magnetic field. Finally, limita- diction methods are used to evaluate the dynamically aver- tions and applicability of the method to obtain either spectral aged values of backbone chemical shifts obtained from unbi- information of the sample or to monitor chemical reactions ased molecular dynamics (MD) simulations of proteins on the of very diluted samples will be discussed. ns-µs timescale [1]. MD-averaged chemical shift predictions generally improve agreement with experimental values when References compared to predictions made from static X-ray structures and a detailed analysis of the structural dynamics and confor- [1] Russell, C. and Weitekamp, D. P., J. Am. Chem. Soc. mational changes associated with the improvements provide 109, 5541-5542 (1987) support for specific motional processes in proteins. Chemical [2] Allerhand, A, J. of Chem. Phys. 44 (1966) shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, aromatic ring positions, and the [3] Freeman, R. and Hill H. D. W, J. Chem. Phys. 54 (1971)

157 Poster Abstracts – Posters Upgraded to Oral Presentation

[4] Vold, R. L. and Vold, R. R., J. Magn. Res. 13, 38-44 mammalian cells we were able to record highly reproducible (1974) In-cell heteronuclear NMR spectra. Moreover, residue spe- cific dynamic information from relaxation experiments was TH294: Human Macrophage Lectin (CLEC10A) obtained. By directly comparing these in-cell NMR results recognition of monosaccharides related to tumor with AS data from different in vitro environments mimicking marker Tn-antigen studied by 1H and 19F NMR. intracellular viscosity and macromolecular crowding, we are in the process of delineating physical and biological contribu- 1∗ 1 Francisco Javier Cañada, Anneloes Oude Vrielink, tions to AS’s different in vivo behaviours. Results emerging 1,2 1 1 1 Filipa Marcelo, Ana Manzano, Pilar Blasco, Jesús from this work contribute to the understanding of the native 3 3 Jiménez-Barbero, Sabine Andre, Hans-Joachim Gabius conformations of AS and lays the ground to further perform 1Centro de Investigaciones Biológicas, CIB-CSIC Chem- high-resolution in situ investigations under conditions that ical and Physical Biology Department. Ramiro de, lead to intracellular aggregation and neurodegeneration as 2REQUIMTE, CQFB, DQ, FCT-UNL, 2829-516 Caparica, observed in Parkinson’s disease. Portugal, 3Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, [1] Lashuel, H. A. et al (2012) Nat. Rev. Neurosci. 14, 38-48

In pathological tissues mucins, heavily O-glycosilated pro- [2] Ito, Y., and Selenko, P. (2010) Curr. Opin. Struct. Biol. teins of cellular surfaces, present perturbed glycosylation pat- 20, 640-648. terns that are being consider as tumor markers and are at- tracting a growing interest for the design and developing of antitumor vaccines. Interestingly, some of these carbohydrate TU296: Defining the Flexible and Fixed Sides of a related tumor markers are known carbohydrate antigens rec- Protein Channel by EPR ognized by C-type (calcium dependent) lectins expressed in ∗ cells of the immune system envisioning an important role Betty J. Gaffney for these interactions in both innate and adaptive immune Florida State University responses[1]. Human macrophages express in their surface Lipoxygenases are ubiquitous proteins that oxidize polyun- the so call Human Macrophage Lectin (HML-2, c-type lectin saturated chains at different positions, but do so with a highly from family 10, CLec10A) that recognizes carbohydrartes re- conserved structural motif of 20 helices. Because mechanistic lated to Tn antigen, (O-glicosides of N-Acetyl–Galactosamine details have been difficult to obtain by crystallography, EPR with serine or threonine). In the presented work the binding spectroscopy approaches have been implemented. Lipoxy- mode of Galactose and Galactosamine and fluorogalactoses genases are naturally paramagnetic due to an iron involved monosaccharides will be shown as it has been characterize by 1 19 in catalysis. We have made them additionally paramagnetic means of H and F NMR studies applying saturation trans- by adding site-directed spin labels at selected sites. Using fer difference (STD) and T2 perturbation strategies comple- several EPR approaches, we have found the entrance to the mented by molecular modeling and docking studies[2]. The active site and have examined an α-helix that must move to importance of all hydroxyl groups of the galactose-derived allow substrate to enter the deep cavity. The non-heme iron monosaccharide ligands and the dependency of calcium for is bound to five protein side chains and water and is located carbohydrate recognition by this c-type lectin has been de- centrally in the structure, 25-35 Å from the surfaces. The po- duced from those studies. lar end of a lipid substrate analog was found on the surface [1] "Sweet preferences of MGL: carbohydrate specificity and at one end the internal cavity in a lipoxygenase with pH- function", van Vliet SJ, Saeland E, van Kooyk Y. Trends gating of substrate access (Biophys J 103: 2134 (2012)). A Immunol. 2008 29(2):83-90. new bacterial lipoxygenase structure, with a lipid bound also seems to have a helical "lid" protecting a similar entrance to [2] "Carbohydrate-protein interactions: a 3D view by NMR", the active site. To examine how lipid binding "opens" the Roldós V, Cañada FJ, Jiménez-Barbero J., Chembiochem. way to catalytic iron, we have scanned the gating helix with 2011 12(7):990-1005. spin labels and examined, by power saturation, relaxation of Acknowledgments: This work has been carried out with the spin at many helix residues when a lysolipid is present. financial aid of the MINECO, Spain (Project number In comparison to substrate insensitive positions, measured CTQ2009-08536) in the reference above, the gating helix distances to iron in- crease in the presence of substrate analog, indicating a shift of the helix backbone. The EPR spectra of ferric lipoxyge- MO295: High resolution conformational description nases with and without lipids in the active site also confirm of Alpha-synuclein inside neurons using mammalian remarkable conservation of active site structure in lipoxyge- In-cell NMR nases from bacteria and eukaryotes. Andres Binolfi, Beata Bekei, Francois-Xavier Theillet, ∗ Honor M. Rose, Philipp Selenko TH297: Comparison of Structure of Cyanobacateria Leibniz Institut für Molekulare Pharmakologie (FMP-Berlin) and Spinach PSII Studied by PELDOR

The intrinsically disordered protein alpha-synuclein (AS) 1∗Asako Kawamori, 2Jiang-Ren Shen, 3Hiroyuki Mino has been involved in the onset of Parkinson’s disease (PD) 1AGAPE-Kabutoyama Institute of Medicine, 2Department through the conversion of its native monomeric state into of Biology, Okayama University, 3Department of Physics, beta-sheet rich amyloid fibrils in dopaminergic neurons [1]. Nagoya University So far, most of the structural studies aimed to understand this conversion were conducted on isolated protein samples, The Structure of Cyanobacterum has been determined re- under conditions that differ substantially from the crowded cently with resolution of 1.9 Å (Umena et al. NATURE in vivo environments of intact cells. The question remains (2011) 473 55) We have determined the distances between whether the features observed for AS in vitro correlate with electron transfer components of PSII in Spinach with PEL- its cellular behaviour. Until recently, there were no means of DOR( Pulsed Electron Double Resonance) with accuracy of looking into live cells with high enough resolution to address 1 Å, 27 Å for YD-YZ ,YD-Mn4 cluster by measurement of such important unresolved issues. The development of In-cell dipolar interactions between radicals. Usually Electron Spin NMR spectroscopy techniques changed this notion [2]. Here, is not localized and hyperfine constants reflecting spin distri- we present high-resolution In-cell NMR data on the structural bution are observed. Point dipolar approximation is not cor- and dynamic properties of AS in five different mammalian cell rect to determine radical distances. In this report we present lines that also include dopaminergic neurons of the Substan- the distance estimation by taking account of spin distribu- tia nigra. By using a novel approach to efficiently deliver iso- tion. Typically distances of chlorophylls and carotenoid from topically enriched protein samples into the cytosol of cultured YD are reported based on spin distributions, which were de-

158 Poster Abstracts – Posters Upgraded to Oral Presentation rived hyperfine constants of each radical, and crystal struc- In recent years, our group has demonstrated that high-field ture data. DNP in MAS solid-state NMR at low temperature ( 100 K) can be applied to investigate the structure of surfaces In a cynobacterium the distances obtained by PELDOR are of materials. In this approach, which we dubbed DNP sur- not coincident with those in spinachs but different values are face enhanced NMR spectroscopy (SENS), the polarizing obtained. Several radical pairs produced by light illumination agent (for example an organic di-nitroxide radicals such as will be compared for cyanobacteria and spinaches. bCTbK, etc.) is introduced into the sample by incipient wetting impregnation with a radical solution. Upon irra- MO298: Sensitivity enhancement in solution NMR diation with microwaves the DNP effect enhances polariza- 1 through dynamic nuclear polarization of encapsu- tion of the H nuclei of the solvent and the surface, and 1 lated proteins this enhanced H polarization can then be transferred to the surface hetero-nuclei (13C, 15N, 29Si or 27Al) by cross- 1 2 1 Kathleen G. Valentine, Guinevere Mathies, Nathaniel V. polarization (CP).[1,2] The SENS method can be extended 1 1 1 Nucci, Igor Dodevski, Sabrina Bédard, Matthew Stetz, to a large set of solvents, thus extending the nature of the 2 2 1∗ Thach V. Can, Robert G. Griffin, A. Joshua Wand accessible systems to include many modern materials, such 1University of Pennsylvania, 2Massachusetts Institute of as nanoparticulate silica, metal-organic-frameworks (MOF), Technology catalyst precursors or organometallic systems,... Here we will present the most recent developments in DNP SENS, and Solution NMR has contributed significantly to studies of the in particular show how this allows the acquisition of mul- structural and dynamic aspects of proteins and the informa- 1 29 1 13 tidimensional H- Si and or H- C correlation spectra of tion inherent in NMR phenomena offers much more. Yet, active surface species. The multi-dimensional spectra yield despite tremendous advances in technology, experimental de- detailed three-dimensional structural information of tethered sign and analytical strategies, solution NMR remains funda- surface species.[3] The structural information obtained by mentally restricted due to its inherent insensitivity. Thus, DNP SENS opens the way to a more rational design of sup- it seems important to improve the sensitivity of the solu- ported catalysis. tion NMR experiment in order to reduce experiment time, lower the absolute quantities of sample required and open a [1] A. Lesage, M. Lelli, D. Gajan, M. A. Caporini, V. lower concentration regime where proteins of limited solubil- Vitzthum, P. Mieville, J. Alauzun, A. Roussey, C. Thieuleux, ity can be accessed. With this in mind there has been a re- A. Mehdi, G. Bodenhausen, C. Copéret, L. Emsley, J. Am. vival of dynamic nuclear polarization (DNP). In solution, it is Chem. Soc. 2010, 132, 15459-15461; thought that such DNP transfer will occur primarily through the Overhauser effect. The basic strategy is to saturate the [2] M. Lelli, D. Gajan, A. Lesage, M. A. Caporini, V. electronic transition of a stable free radical and transfer this Vitzthum, P. Mieville, F. Heroguel, F. Rascon, A. Roussey, non-equilibrium polarization to the hydrogen spins of water, C. Thieuleux, M. Boualleg, L. Veyre, G. Bodenhausen, C. which will in turn transfer polarization to the hydrogens of Copéret, L. Emsley, J. Am. Chem. Soc. 2011, 133, 2104- the dissolved macromolecule. Unfortunately, technical as- 2107; pects of this approach seem to prove fatal to the idea in its current form. The primary reason is that the frequency of [3] M. Samantaray, J. Alauzun, D. Gajan, S. Kavitake, A. the electron transition of suitable radicals lies in the sub- Mehdi, L. Veyre, M. Lelli, A. Lesage, L. Emsley, C. Copéret, THz spectrum where water absorbs strongly and results in C. Thieuleux J. Am. Chem. Soc. 2013 in press catastrophic heating of the sample. In addition, the residence time of water on the surface of the protein is too short for TH300: In situ MR: Pore condensation at elevated efficient transfer of polarization. Here we take advantage of temperature and pressure the properties of solutions of encapsulated proteins dissolved in low viscosity solvents. Such samples are largely transpar- 1∗Matthew P. Renshaw, 1S. Tegan Roberts, 2Belinda S. ent to the subTHz frequencies required and thereby avoid Akpa, 1Mick D. Mantle, 1Andrew J. Sederman, 1Lynn F. significant heating during saturation of the electronic transi- Gladden tion. Nitroxide radical is introduced into the reverse micelle 1University of Cambridge, 2University of Illinois at Chicago system in three ways: covalently attached to the protein; co- valently attached to a surfactant embedded in the reverse Magnetic Resonance (MR) methods, being non-invasive and micelle shell; and free in the aqueous core. DNP experiments non-destructive, are ideal for probing heterogeneous catalytic at the X-band EPR frequency have yielded initial enhance- systems under realistic operating conditions [1,2]. However, ments from the nitroxide embedded in the surfactant to the until recently, the processes studied using MR have been water on the order of -35. In addition, we find that the hy- performed under mild conditions (typically < 5 atm and < ◦ dration properties of encapsulated proteins allow for efficient 200 C). We have recently commissioned a fixed-bed reactor, polarization transfer from water to the amide hydrogens in a compatible with operation inside a superconducting magnet, ◦ protein. Thus the merging of the reverse micelle technology which can be operated up to a temperature of 350 C and with DNP demonstrates the potential to provide a significant a pressure of 31 atm while simultaneously performing MR increase in the sensitivity of solution NMR spectroscopy of experiments. proteins and other bio-macromolecules.Supported by the NIH and the NSF. To demonstrate its capability we have studied the effect of confinement in mesopores on the vapour-liquid phase change of cyclohexane to elucidate pore filling and emptying mecha- TU299: DNP Surface Enhanced NMR (SENS): a nisms. The understanding of confinement effects, particularly Tool for Structural Investigation of Supported Catal- at realistic reaction conditions, is an important consideration ysis in heterogeneous catalysis. 1∗ 1 1 Moreno Lelli, Alexandre Zagdoun, David Gajan, Isothermal (150 ◦C and 188 ◦C) vapour-liquid phase change 1Aaron J. Rossini, 4Olivier Ouari, 4Paul Tordo, 3Chloé 2 1 1 cycles of cyclohexane in a bed of titania pellets were con- Thieuleux, Christophe Copéret, Anne Lesage, Lyndon ducted. 1H spin density images reveal changes in pore satu- Emsley ration of the pellets as the phase boundary is crossed. Pore 1Centre de RMN à Très Hauts Champs, Université de Lyon confinement effects are exhibited, as liquid remains within the (CNRS/ENS Lyon/UCB Lyon 1), 2Department of Chem- pores after bulk liquid has vaporised. Pore saturation dur- istry, ETH Zürich, Switzerland, 3Institut de Chimie de ing the evaporation-condensation cycles exhibits hysteresis, Lyon, Université de Lyon (CNRS-Université Lyon 1-ESCPE demonstrating the differences between the filling and emp- Lyon), 4Aix-Marseille Université, CNRS, Institut de Chimie tying mechanisms in these systems. The data are analogous Radicalaire (ICR), Marseille to N2 adsorption isotherms that characterise pore diameter

159 Poster Abstracts – Posters Upgraded to Oral Presentation and surface area, but are conducted for a relevant species at many hydraulic and mechanical properties of natural geoma- realistic conditions. terials such as hydraulic conductivity and shear resistance. 1 Pore size and sizes distribution of rocks depend on their Data from these MR images, in conjunction with H T1 and mineral composition and the geological processes involved T2 relaxation time measurements and pulsed field gradient in their formation, while for unconsolidated materials such diffusion measurements, are used to assess the mechansim of as soils, depend on grain sizes, grain sizes distribution and pore filling and emptying in addition to revealing information overburden pressure. The aim of this project is to corre- about the formation of liquid films. late transversal relaxation time (T2) with grain size of loose The use of the in situ MR reactor is currently being extended sandy soils in systems of single and double porosity. In this to study heterogenous catalytic reactions. research we applied Time Domain Nuclear Magnetic Reso- nance (TD-NMR) to measure the magnetization decaying [1] Gladden et al., Catalysis Today, 155 (2010) 157-163. curve of samples composed of selected sizes of sand grains, [2] Lysova and Koptyug, Chemical Society Reviews, 39 (2010) poor graded sands, silty sands, silt and clay. This was per- 4585-4601. formed by a CPMG pulse sequence using a low field (12 MHz) spectrometer (SLK-100). Obtained results show that MO301: Pore Sizes Distribution of Unconsolidated transversal relaxation time and its distribution are strongly Geomaterials correlated with grain sizes and their distribution. These results allow improving the knowledge about pore sizes dis- 2,3∗ 1,3 3 Marcos Montoro, Lucas C. Cerioni, Daniel José tribution when needed for modeling porous materials using Pusiol numerical techniques such as pore networks. 1Spinlock SRL, 2Universidad Nacional de Córdoba, 3CONICET ACKNOLEDGEMENTS: CONICET, SECyT-UNC, Pore size and pore sizes distribution of soils and rocks control FONTAR, FONCYT

160 Index of Authors

Index of Authors

Aakagi, Kenichi TU203 Alvarez-Martinez, Aronson, Matthew T. Banères, Jean-Louis Cristina OP148 OP009 UP027 Abboud, Khalil A. TU254 Alves, Nathalia S. TH048 Arshava, Boris TU002 Barberis, Gaston TH225 Abiko, Layara A. MO046 , Irene TU017, Arthanari, Haribabu Barbero, Jesús Jiménez Abreu, Valéria TU194 Amata UP054 TH036 TH009 Abriata, Luciano A. Arthur, Michel UP126 , Emeline TU008, OP075, TH216, Ambrosio, Andre Luis Barbet-Massin TH060 MO217 Berteli TH054, TU056 Asam, Claudia TH108 Achilles, Anja OP057 Amit, Eran MO073 Asami, Sam MO139, Barbosa, Layla Rosário OP070 TU281 Ackerbauer, Daniela Amo, Juan-Miguel Lopez TU047 del OP070 Ascona, Christian Rivera Barbosa, Lúcio Leonel TH087 TU266, MO268, TU269 Acosta, Rodolfo Héctor Amonette, James E. UP146, TH207, TU239, TU245 Ashbrook, Sharon E. Barcellos Jr., Ewerton de MO085 TH267, TU293 Amorim, Gisele Cardoso OP035 Adesso, Gerardo TU146 de TH033, MO034 Assunção, Bruno A. de Barreto, Jéssica Resende TH024 TU035 Afonso, Luciana Caminha Amorim, Mauro B. de TH180, MO208 TU176 Atala, Daniel I.P. TU164 Barros Jr., Wilson UP063 Agarval, Vipin UP135 Amoureux, Jean Paul Atanasov, Mihail OP020 Barros, Carlos Jonnatan UP036, MO139, TU140, Pimentel TH192, MO193 Aguiar, Daniel Lima TH141, TU143 Atchison, David A. Marques de TH084, TH090 MO205 Barsottini, Mario Ramos Anderson, Jason OP124 de Oliveira TU056 Aguiar, Paula Fernandes Atkinson, Kevin D. OP159 de MO115 Andrade, Fabiana Diuk Basso, Luis Guilherme M. Auccaise, Ruben TU146 TU092 TH051 Aguiar, Ramon Pinheiro Auger, Michele TU062 TH033 Andrade, Flavia Guedes Bastow, T. TU068 TH063 Azambuja, Ana Paula Ahn, Jinwoo TU038 Batista, Aline TH108 TU116 Andrade, R. H. S. MO187 Ahn, Sangdoo TU212 Baud, Matthias TU152 Azeredo, Rodrigo Ahola, Susanna UP098 Andre, Sabine UP017, Bagueira de Vasconcellos Bax, Ad OP132 TH294 MO247, TU263, MO277, Akoury, Elias TU005 Bazin, Laurent Terradot TH279 Andreeta, Mariane Barsi Alexandre OP137 Akpa, Belinda S. UP154, MO244 Azeredo, Sirlene O. F. TH300 Bechinger, Burkhard TH153, MO154 Andrews, A. Ballard TU050 Al-Hashimi, Hashim M. MO109 Azevedo, Eduardo N. de PL045 Becker, Stefan OP086, UP063 André, Sabine MO001 MO019, TU074, TH102, Alajarín, Mateo MO127 Angeli, Renata MO190 Azevedo, Mariangela B. OP133 Albernaz, Fabiana M. de MO076 Beedle, Christopher C. Pestana MO025 Angelis, Martin Hrabě de TH180, MO208 Azevêdo, Eduardo Ribeiro TU254 Alborghetti, Marcos de TU092, TH093, OP057, , Beata UP089, Rodrigo TU197 Anglister, Jacob TU002 TU146, TH276, MO280 Bekei MO295 Alcantara, Glaucia Braz AnoBom, Cristiane D. Baaden, Marc UP027 MO097, TU188, TH189 TH048 Benavente, E. MO229, Baaske, Philipp TU023 TU230 Alencastro, Ricardo Bicca Antzutkin, Oleg N. OP073 Bacchi, Pedro TU191, de TH084, TH090 Benedetti, Celso E. Apparecido, Rafael do TH195 TU053, TH054 Alkorta, Ibon MO064 Prado TU188 Bagryanskaya, Elena Benoit, Matthieu MO100 Allix, Mathieu OP038 Arachchige, Rajith J. TU218, TH219, OP019 OP134 Bento, Edson de Souza Almeida, Fabio C. L. Bajaj, Vikram S. OP068 TH081, TH168 MO004, TH015, UP162, Arake, Luisa Mayumi Baker, David OP133 , Wolfgang OP118 MO025, TU026, TH030, Ribeiro TU032 Bermel TH033, MO034, MO037, Balbach, Jochen MO199 Bermudez, Kalah TH018 TU041, TH042, TU047, Araki, Koiti MO211 Balcom, Bruce J. OP097 Bernadó, Pau TU017 MO049, TH108, TH114, Arantes, Guilherme OP005, MO190, TU194, Menegon TH039 Baldus, Marc UP136, Bernal, Andrés TH165 MO286 TU119, TH291 Araújo, Ana P.U. OP025 Bernard, Guy MO067 Almeida, Queli Aparecida Balieiro, Fabiano de Rodrigues de TU155 Araújo, Renata Mendonça Carvalho MO265, MO274 Bernardinelli, Oigres TU173 Daniel TU092, TH093 Almeida, Vinícius Mansur Ballesteros, Rafael R. Dose Lage de TU266 Arcos, Daniel MO151 TU242 Bernardo, Camilla do Nascimento MO115 Alphonse, Sébastien Ardavan, A. TH222, Baltisberger, Jay H. TH096 MO223 OP034 Berná, José MO127 Alvares, Rohan TH135 Ardá, Ana MO001 Ban, David OP086 Berry, Andrew J. OP035

161 Index of Authors

Berry, Robert E. UP076 Braga, Carolina Alvares Cabrita, Eurico J. UP121, Carvalho, Mário Geraldo TH063 TU290 de TH159, MO160 Berto, Maria I. TU164 Braga, João Pedro TH282 Cadars, Sylvian OP009, Casano, Gilles TH234 , C. W. MO013 Bertoncini OP038 Braga, Vanessa L. Azevedo Casanova, Federico TH207 Beszonova, Irina TH015 MO025 Cai, Shuhui TU137, Case, David A. TU020 Beuerman, Roger W. TH138, MO202 Bragatto, Juliano TH093 TH162 Casimiro, María MO103 Caldarelli, Stefano OP161, Braide, Otonye TU227 Bhuniya, Sankarprasad TU242 Casimiro, Teresa UP121, TH213 , Raimundo TU290 Braz Filho Calderon Filho, Cesar TU173 Bianconi, M. Lucia TH048 José TH225 Cassus, Eduardo P. Braz, Daniel César TH087 MO256 Bickelhaupt, F. M. Calvini, Ítalo A. OP025 Castanheira, Pedro MO052 Breiteneder, Heimo Camargo Jr., Sérgio S. TU047 TU023 Bieschke, Jan OP070 MO094 Brey, William W. Castañeda-Valencia, Billeter, Martin MO004 Camargo, A. S. S. de Gloria MO169 Brindle, Kevin M. TH099, MO082 , Andres UP072, Binolfi OP065 Castellen, Patricia MO055 UP089, TH285, MO295 Cambui, Erika Verena Castilla, Aracelys López Brito, Luciana M. TU278 Figueiredo TH168 Bjerring, Morten OP073, MO031 UP059, TH201 Brito, Rui M. TU023 Camilloni, Carlo UP131, TH129, MO130 Castillo, Andrés M. TH165 Blackburn, Mandy E. Britt, R. David OP008 Castro, Cecilia MO184 OP074 Campos, E. C. Menchaca Britto, Manuel OP074 TH255 Castro, Eustáquio Vinícius Blasco, Pilar MO001, Brocksom, Timothy John Ribeiro de TU125, TU266, UP017, TH294 Can, Thach V. UP111, TU281 MO298 MO268, TU269, TU281 Bloch Jr., Carlos TU032 Brouwer, Darren H. Canales, Ángeles TH009 Catoire, Laurent UP027 Bloise, Antonio C. TU191, OP009 Cavalcante, Gustavo TH195 Canalis, M.S. Blanco Brown, Leonid MO118 TH267 Prione MO172 Blundell, S. J. MO223 Cavalli, Andrea UP131, Brown, Steven P. MO079, Canaviri, Marcelo Torrez MO130 Blümich, Bernhard OP056 TH249 TH207, OP155, MO259, Cavini, Ítalo Augusto Bruix, Marta MO001, Candido, Anne Caroline TU260, MO262 TH111 TH009 MO034 Bode, Bela OP127 Cañada, Francisco Javier Bruschweiler-Li, Lei Canevarolo, Rafael MO001, UP017, TH294 Bodenhausen, Geoffrey MO046, OP129 Renatino TU116, MO196, OP108, UP125, UP036 Brutscher, Bernhard TH204 Cerioni, Lucas C. UP080, OP152, MO301 Boelens, Rolf UP031 OP055 Cantrelle, François-Xavier UP016 Cerioni, Lucas Matías Boisbouvier, Jerome Brüschweiler, Rafael MO148, MO241, TH258 MO100 MO046, OP129 Caporini, Marc UP036 Cervantes, Hernán Joel Bok, Robert OP040 Bublin, Merima TU047 Cappellaro, Paola OP143 MO211 Bolton, David OP127 Budker, Dmitry OP010 Cara, Andrés Espinoza Chammas, Roger TU191, MO217 Bonagamba, Tito J. Buldain, Graciela Yolanda TH195 PL066 TU158 Carlier, Marie-France Chang, Kiyuk MO214 UP016 Buljubasich, Lisandro Bonagamba, Tito José Chapman, Bogdan E. TH066, UP146, TU293 TH087, TU146, TH147, Carlos, Eduardo Fermino TH144 TH210, TH237, TH243, TU188, TH189 Bunce, Chris OP091, MO244, TU248, MO265, Charlton, Lisa M. TU038 TU182 MO274, MO280 Carneiro, Giovanna F. TH246, MO247 Chary, K. V. R. TU200 Burdisso, Paula MO100 Bonfim, Rodrigo de Paiva Carnevale, Diego UP036 Chattah, Ana Karina Floro TH117 Buriol, Lilian MO127 TH066, TH069, TU158, Carosio, Maria Gabriela Bonvin, Alexandre M. J. J. Burz, David TH198, TH252 Aparecida TU272, TH273 UP130, UP136, TU119, UP053 Chavarín, Jorge Uruchurtu TH291 Carpinella, M. TH267 Bustos, Silvina O. TU191, TH255 Boralle, Nivaldo MO175 TH195 Carrara, Caroline TU242 Chen, Chunying MO178 Borré, Leandro B. MO091, Byeon, Chang-Hyeock Carrigan, John TU182 Chen, Dan UP007 MO094 TU038 Carvalho, Alexandre Chen, Der-Yow OP043 Bouchard, Louis OP067 Byeon, In-Ja L. TU038 Plastino de MO277 Chen, Hao TU137 Bouchevreau, Boris Bédard, Sabrina UP111, Carvalho, André de Souza , Hsueh-Ying OP109 UP012, MO289 MO298 OP153, TH261, TH273 Chen Chen, Qun MO139, TU140 Bougault, Catherine Böckmann, Anja UP135, Carvalho, Erika Martins UP126 OP137 de MO088, TU089, TU110 Chen, Shizhen MO133 Boyd, Austin TH210, Cabeça, Luis Fernando Carvalho, João Teles de Chen, Zhong TU137, MO247, TU248 OP153, TH270, TH273 TH087 TH138, MO202

162 Index of Authors

Cheong, Cheajoon TU212 Correia, Vinícius Soares Diehl, Annette TH063 Ernst, Matthias UP135 da Paixão TU131 Chiaro, S.S.X. MO091 Diercks, Tammo OP114 Escobar, Melissa Quintero Corvo, Marta UP121, TH204 Chikayama, Eisuke Dietz, Carsten OP077 TU290 MO181 Espindola, Ana Paula Ding, Fa-Xiang TU002 Costa-Filho, Antonio José TH186 Chimon-Peszek, Sandra OP025, TH051, TU113, Diuk, Fabiana TH276 TH018 Espinoza-Cara, Andrés TH231 Dlubac, Katherine OP096 OP075 Chinelatto Júnior, L S. Costa, João M. MO256 MO091 Doan, Peter E. TU233 Estrada, Ruben Auccaise Coutinho, Bernardo TH147, MO265, MO274, Dodd, Stephen OP043 Chmelka, Bradley F. TH246 MO280 OP009 Dodevski, Igor UP111, Craik, David J OP160 Falson, Pierre OP137 Cho, Janggeun TU212 MO298 Cruickshank, Paul OP127 Fam, Maged OP078 Cho, Jee-Hyun TU212 Dolde, Florian OP157 Cruijsen, Elwin A. W. van Fan, Ying MO118 Domingues, Mariane N. Cho, Min-Kyu TH102 der TU119 TU053, TH054 Fanucci, Gail E OP074, Christou, George TU254 , Elwin van der TU227 Cruijsen Donoso, J. P. MO229, UP136, TH291 Cilli, Eduardo Maffud TU230 Farah, Chuck OP148 TH051 Crusca Júnior, Edson Donovan, Kevin J. MO016 Farah, Chuck S. MO043 Cilli, Eduardo Maffut TH051 Doty, F David MO151 Faria, Gregório C. OP057 TU113 Crusca, Edson TU113, Cipriano, Daniel F. TU086 TH231 Driehuys, Bastiaan OP039 Faria, Roberto de Barros TU155 Ciulli, Alessio TU023, Cruz, Joao Felipe Alves da Drozdyuk, Irina OP019 TU152 TU134 Duckett, Simon B OP159 Faria, Tiago TU023 Claramunt, Rosa M. Cunha, Alfredo G. MO085 Dugar, Sneha TH072 Farias, Fábio L. L. MO256 MO064 Cvitanic, Tonci MO073 Dupont, Jairton UP121, Fattori, Juliana MO076 Clausen, Katherine TH018 D’Andréa, Everton Dias TU290 Favaro, Denize C. MO046, Claytor, Kevin UP041 TU029 Dutta, Kaushik TH096 OP148 Clendinen, Chaevien D’Eurydice, Marcel N. Duyn, Jeff OP142 Favaro, Denize Cristina TH237, TU248 MO043 Cobas, Carlos TH132 Duzzioni, Eduardo Inácio D’Eurydice, Marcel MO280 Fayon, Franck OP038 Cobo, Marcio F. OP057 Nogueira MO244 Dyszy, Fábio H. OP025 Fedin, Matvey TU218, Cobra, Paulo Falco Danieli, Ernesto TH207, TH219, OP019 MO112, OP153 TU260, MO262 Eckert, Hellmut UP104, MO082, UP037 Fekete, Marianna OP159 Coelho, Felipe B. TU209 Daraku, T. MO052 Edison, Arthur S. Felli, Isabella C. OP051 Coen, Donald M. TH036 Darbello, Daniel de Edwards, John C. MO109, Feng, Yesu UP041 Coey, Aaron UP116 Menezes TH057, MO058 TU275 Fenwick, R. Bryn OP086 Cohen, Tal TU275 Dasari, Muralidhar OP070 Eiden, Michael MO184 Ferella, Lucio TU020 , Angie Lizeth Cole, Jonathan TU149 Davalos MO043 Einloft, Sandra UP121, Ferguson, Stuart J. OP004 Coletta Filho, Helvecio TU290 Davis, Ryan M. UP041 Fernandes, R. G. MO082 Della TH186 Elguero, José MO064 Decker, Frank UP012, Fernandes, Tácio Vinício Colnago, Luiz Alberto Ellis, Paul MO151 TU077, MO106, MO112, MO289 Amorim TH051 TU134, OP153, TH261, Demers, Jean-Philippe Emami, Sanaz MO118 Fernández, Claudio O. TH264, TH270, MO271, OP133 Emmerich, Francisco G. OP052, MO013, UP072, TU272, TH273 TH285 Deng, Feng MO070, MO085 Comellas, Gemma OP117 TU140, TH141 Emsley, Lyndon TH060, Fernández, Ignacio MO103 Conesa, Pablo TH183 Denkert, Carsten MO184 TH234, UP110, TU299 Ferreira Júnior, Eli da Conibear, Anne OP160 Dente, Axel D. TH066 Emwas, Abdul-Hamid TH078 Silva TU098 Conradi, Mark OP102 Denysenkov, Vasyl PL003 Endeward, Burkhard Ferreira, Antonio Gilberto Constable, R. Todd Deschamps, Michael PL003 MO136, OP092, TU284 OP061 OP038 Engelke, Frank UP012, Ferreira, Arthur Gustavo Copéret, Christophe Deshpande, Rashmi MO289 de Araujo MO244 TH234, UP110, TU299 UP104 Engelsberg, Mario UP063 Ferreira, Arthur Gustavo Cordeiro, Tiago OP029 Deville, Célia UP016 de Araújo TH147, MO280 Entzminger, George Cordeiro, Yraima MO007, Dias, David M. TU023, MO151 Ferreira, Bárbara Darós de TH126 TU152 Lelis TH282 Eon, Jean Guillaume Correa, Daniel H A TH063 Dias, Sandra Martha TH117 Ferreira, Daniel R. TU245 Gomes TU056, TH204 Correa, Maria Elvira Erlach, Markus Beck Ferreira, Fatima TU047, Pizzigatti TU197 Didry, Dominique UP016 TH045 TH108

163 Index of Authors

Ferreira, Marcos Davi Fredriksson, Jonas MO004 Gardiennet, Carole Gonsalves, Elicardo MO271 OP137 MO190 Freed, Jack H. OP128 Ferreira, Rafaela Oliveira Garnero, Claudia TH069 Gonzalez, G. MO229, TH159 Freeman, Ray MO016, TU230 OP119 Garratt, Richard Charles Ferreira, Rodrigo TH111, TH231 Gonzalez, Mariano M. Alexandre MO283 Freire, Guilherme M. TU008 TU209 Garwood, Michael OP139 Ferreira, Vinicius Sousa González-Beltrán, Gataullin, Eduard M. TU080, TU104 Freitas-Fernandes, Liana Alejandra TH183 B. TU026, MO190, TU194 TU224, UP022 Ferretti, Giulia D. S. González-Pérez, Martha Gauvin, Régis TH141 TH126 Freitas, Deisy dos Santos TU098 TH189 Gava, Lisandra Marques Ferrone, Marcus OP040 Gonçalves, Eduardo Freitas, Jair C. C. MO085, TH063 Feuerstein, Sophie OP055 Corrêa MO277 TU086 Gehring, Kalle MO022 Fiat, Daniel TU206, UP141 Gonçalves, Gustavo R. Freitas, Monica Santos de Gelderen, Peter van MO085, TU086 Fidalgo, Tatiana Kelly S. TH063 OP142 TU026, MO190, TU194 Gonçalves, Kaliandra Fries, Pascal H. UP084 Gelves, Luis G. V. MO094 TH204 Fiehn, Oliver MO184 Fritzinger, Bernd MO238 Gennis, Robert B. OP117 Good, Jeremy A. TU149 Figueira, Elza C.S. TU257 Fritzsching, Keith J. Gentner, M. OP033 Gossert, Alvar TH003 Figueiredo, Luis de OP124 MO163 George, Julia M. OP117 Goulart, Henrique Fonseca Frizzo, Clarissa Piccinin TH081 Figueredo, Irineu MO250, MO127 Geraldes, Carlos F. G. C. TH279 TU023, TU152 Grandinetti, Philip J. Frost, Stefan TH012 OP034 Figueroa-Villar, José Geraldes, Carlos F.G.C. Daniel OP015, TH153, Frydman, Lucio MO016, OP062 Grangeasse, Christophe MO154, TU170 OP122, PL001 TH096 Ghose, Ranajeet TH096 Filho, Elenilson G. A. Fu, Riqiang TH072 Greco, Sandro José TU281 Giachetti, Andrea TU020 TH276 Fuchs, Helmut TH180, Green, Gary G. R. OP159 Giammatteo, Paul J. Filippov, Andrei OP073 MO208 TU275 Green, Richard A. OP159 Fillion, Matthieu TU062 Funk, Alexander M. UP084 Gil, Roberto R. OP085 Green, Steven J. TU149 Fink, Brian UP158 Fursova, Elena TH219 Gil, Rosane Aguiar da Silva Grey, Clare. P. OP101 Fink, Uwe OP070 Furtado, Joana TU023 San TH084, MO088, Griesinger, Christian TU089, TH090, MO091, Finkelstein-Shapiro, Furuita, K. MO052 OP086, MO013, UP072, MO094 Daniel TH228 TU074, TU122, OP133, Fändrich, Marcus OP070 Giller, Karin TH102, TH285 Firmino, Paulo Renato OP133 Alves TH192, MO193 G.N, Manjunatha Reddy Griffin, John OP101 TU242 Giotto, Marcus V. TU245 Flores, Douglas William Griffin, John M. OP035 Menezes MO271 Gabioud, A.A. Valiente Giraudeau, Patrick Griffin, Jules TH183, UP072, TH285 MO136 Florian, Pierre OP038 MO184 Gabius, Hans-Joachim Girolami, Davide TU146 Fogeron, Marie-Laure MO001, UP017, TH294 Griffin, Robert G. UP111, UP135 Giusti-Paiva, Alexandre OP013, MO298 Gabrienko, Anton TU218 MO172 Foguel, Débora MO007, Grigorjev, Igor TH219 TH063 Gaffney, Betty J. UP026, Gladden, Lynn F. OP079, Grombacher, Denys TU296 UP154, TH300 Foley, David A. TU275 OP096 Gailus-Durner, Valérie , Adriana Da Silva Gnida, Manuel UP031 Fontes TH180, MO208 Gromilov, Sergey TU218 TU221 Goddard, Alan D. OP004 Gajan, David UP110, Gronenborn, Angela M. Fontes, Sérgio Rodrigues TU299 Goddard, P.A. TH222 TU038, PL165 TH243 Galdeano, Carles TU152 Godoy, Michele Maria Groot, Berend L. de Forato, Lucimara Gonçalves de TH192, OP086 Aparecida TU077 Galiana, Gigi OP061 MO193 Grunewald, Elliot OP096 Forse, Alex OP101 Gambarra-Neto, Goldfarb, Daniella OP087 Francisco F. TH105, TU107 Grzesiek, S. OP033 Fortulan, Carlos Alberto Gollnick, Paul MO010 TH243 Gao, Min OP134 Gu, Yina OP129 Golotvin, Sergey TU161 Foster, Mark P. MO010, Garaga, Mounesha N. Gueiros-Filho, Frederico TU128 OP009 Gomes, Andre M. O. José MO055 TH048 Fraga, Luciana Elena M. S. Garbacz, Piotr MO067 Guerreiro Filho, Oliveiro TH030 Gomes, Bruna Ferreira TH057, MO058 García-Mayoral, María OP153, TH270 Franzoni, María Belén Flor TH009 Guimarães, Maria José C. UP146, TU293 Gomez, C.G. TU239 O. TU101 García, Jesús OP029 França, José Adonias Alves Gomez, Leonardo D. Guimarães, Vinícius de MO142, MO166 García, Julia Inés TH258 TH093 Ferreira MO172

164 Index of Authors

Guittet, Eric UP027, Hong, Mei PL069, OP124 Jaudzems, Kristaps Kettunen, Mikko I. OP065 UP016 TH060 Honorato, Hercílio D. A. Khanim, Farhat OP091, Guo, Qianni MO133 TU086 Jault, Jean-Michel OP137 TU182 Gupta, Rashmi OP133 Hooker, Jerris W. Javkhlantugs, Namsrai Khetrapal, C.L. TH177 TU059 Gupta, Shaan TH135 Hooper, Alex J. J. OP159 Khitrin, Anatoly UP145 Jayaseelan, Kalai Vanii Gustavsson, Martin Khramtsov, Valery V. Horev, Judith UP140 MO163 MO061 UP021 Horjales, Eduardo TU044 Jerschow, Alexej UP145 Günther, Ulrich L. OP091, Khrumkacheva, Olesya TU182, TU236 Hosztafi, Sándor MO028 Jeschke, Gunnar OP077, TH219 TH234 Hagens, Tona von OP077 Hou, Guangjin MO139 Kikuchi, Jun UP093, Jeziorna, Agata TU083 MO181 Hagn, Franz TH120 Hoyt, David W. UP099 Jiang, Peng MO232 Killner, Mario Henrique Hails, Guillermo MO100 Hritz, Jozef TU038 Montazzolli TU260, MO262 Jiang, Weiping MO133 Hallwass, Fernando Hsieh, Ming-Feng OP009 Kim, Hai-Young TH102 MO142, MO166, MO187 Hsu, Chaohsiung UP064 Jiménez-Barbero, Jesús MO001, UP017, TH294 Kim, Hyun Min TU215 Han, Oc Hee UP103 Hu, Bingwen MO139, Johnson, Robert L. OP124 Kim, Ji-Sun OP018 Han, Songi OP046 TU140 Jones, Catherine E. Kim, Jong Seung TH213 Han, Yi OP138 Hu, De-en OP065 MO205 Kim, Yongae OP018 Hanna, John V. TH075, Hu, Hong-Yu TU011 Jones, Oliver A.H. UP094 MO079 Hu, Jian Z. UP099 King, Scott P. MO079 Jung, Ji-Ho OP018 Hansen, Michael Ryan Huang, J.-r. OP033 Kira, Atsushi TU059 UP011 Junior, Luiz S. C. MO256 , Igor TH219 Huang, Jing TU179 Kirilyuk Harada, Yoshie TH150 Junker, Jochen MO115, Kleckner, Ian R. MO010, Huang, Susie Y. UP042 TU155, TH171 Harvey, Peter UP084 TU128 Huang, Xi OP074 Júnior, Antônio M. TU164 Harzstark, Andrea OP040 Knight, Michael J. TH060 Huber, Matthias UP135 Kabengi, Nadine J. TU245 Haug, Kenneth TH183 Knight, Rosemary OP096 Huber, Roland MO121 Kalbitzer, Hans Robert Hauk, Priscila MO046 Kock, Flávio Vinicius OP025, TH045, TH111 Hunkeler, Andreas OP137 Crizóstomo TU266, MO268 Hausnerova, Berenika Kaminski, D. TH222, MO259 Hunter, Robert OP127 Koczula, Katarzyna MO223 OP091 Hawkes, Geoffrey Ernest Hurlimann, Martin D. Kamo, Naoki UP088, TH081, TH168 TU248 Koehler, Joerg TH045 TH288 Hayes, Sophia E. MO145, , Clotilde UP016 Koers, Eline UP136, Husson Kang, Jongeun MO214 OP102 TH291 Hwang, Lian-Pin UP064 Kannan, Arvind MO130 He, Yao TU014 Kojima, C. MO052 Härd, Torleif OP073 Kaptein, Robert UP031 Hediger, Sabine UP126 Kolbe, Michael OP133 Hürlimann, Martin D. Karabanov, Alexander Heijenoort, Carine van Kolokolov, Daniil TU218 OP100 OP112 UP016 Kondo, Y. MO052 III, Arthur G. Palmer Kasthurirangan, Sanjeev Henriques, Sonia T OP160 MO124 MO205 Koretsky, Alan P. OP043 Hercík, Kamil TU038 III, George Cutsail TU233 Katahira, R. MO052 Korzhnev, Dmitry M. Heredia, Gabriela Pinheiro TH015, UP049, MO037, Igarashi, Ruji TH150 Kawamori, Asako UP150, TU029 TU287 Igea, Ana TU017, UP054 TH297 Herlein, Judith UP158 Kostadinova, Maria Kawamura, Izuru UP088, Ihms, Elihu MO010 TU047 Hidaka, Tetsuro UP088, TU059, MO118, TH288 TH288 Inomata, Kohsuke TU203 Kovacs, Helena TH003 Kay, Lewis E. MO016, Highton, Louise A. R. Iqbal, Anwar MO049 OP070 Kozlov, Guennadi MO022 OP159 Ivanshin, Vladimir A. Kear, Jamie L. OP074 Krebs, Manuel TH210 Hill, Stephen TU254 TU224, UP022 Keeble, David OP127 Kremer, Werner TH045 Hilty, Christian OP109 Izadi-Pruneyre, Nadia UP048 Keeler, Eric OP034 Kreutz, Christoph MO121, Hinderberger, Dariush TH123 Kemp, Rafael TU284 OP023 Jackowski, Karol UP120 Kritsiligkou, Paraskevi Keng, Luiz Henrique Ho, Oanh TH012 Jain, Sheetal Kumar OP004 MO283 UP059 Hoffman, Brian M. TU233 Kryukov, Eugeny TU149 Kennedy, Brett W.C. Jaremko, Lukasz MO019, Hogle, James M. TH036 OP065 Krzystek, J. MO232 TH102 Holland, D. J. OP079 Kenwright, Alan M. Kuchel, Philip W. OP095, Jaremko, Mariusz MO019, UP084 TH144 Holte, Laura MO151 TH102 Keren, Amit MO073 Kukic, Predrag TH129 Hong, Kwan Soo TU212, Jaroniec, Christopher P. TH213, MO214, TU215 OP134 Kessler, Naama TU002 Kuktaite, Ramune TU092

165 Index of Authors

Kulminskaya, Natalia Leite, Érika de Almeida LoSan, Talita TH114 Mahendraker, Tejasvi TH201 MO193 TH183 Login, Frederic TH012 Kumagaia, Patricia S. Leitão, Alexandre Amaral Mainz, Andi OP070 TH231 TH084, TH090 Loibl, Sibylle MO184 Makino, Teruki UP088, Kunert, Britta OP137 Lelli, Moreno UP110, Loiola, Rodrigo Azevedo TH288 TU299 MO211 Kupče, Eriks¯ MO016, Maldonado, Andres OP119 Lendel, Christofer OP073 Long, Dong OP129 TH198 Kurbatska, Victoria Leonciks, Ainars TU002 Long, Joanna TU227 Maltsev, Alex OP132 TU002 Leonov, Andrei TU122 Longhi, Marcela TH069 Manson, J. L. TH222 Kurhanewicz, John Lesage, Anne TH060, Lopes, Edmundo Pessoa de OP040 Mantle, Mick D. OP079, TH234, UP110, TU299 Almeida TH192, MO193 UP154, TH300 Kurizki, Gershon OP122 Leskes, Michal OP101 Lopes, Thiago I. B. TU164 Mantsyzov, Alexey OP132 Kuster, Ricardo M MO034 Levin, Judith G. TU038 Lopes, Thierry R. MO085 Manzano, Ana UP017, Kwiatkowski, Grzegorz Levitt, Malcolm H. PL044 Lopez, Claire TU242 TH294 OP112 Levstein, Patricia R. Loquet, Antoine OP133 Marana, Sandro Roberto Köckenberger, Walter TH066, OP144 TH039 OP112 Lorin, Aurelien TU062 León, A.E. TH267 Marassi, Francesca M. Kędziorek, Mariusz Loring, John S. UP099 OP024 MO067 Li, Dawei OP129 Loth, Karine UP031 Marcelo, Filipa UP017, Labate, Carlos Alberto Li, Dong TH294 TH093 Lourenço, Gustavo V. Li, Haidong MO133 TU209 Marco-Rius, Irene TH099 Lacerda Júnior, Valdemar Li, Yixuan TU140 TU125, TU266, MO268, Luchinat, Claudio OP156 Marconcini, Lucinéia TU269, TU281 Li, Zhao MO133, UP042, Ludwig, Christian OP091, Vizzotto MO271 UP064 Lacour, Jérôme TU242 TU182, TU236 Marin, Graciane UP121, Liedl, Klaus MO121 TU290 Ladizhansky, Vladimir Luján, Juan Fernando MO118, OP149 Lim, Sing Mei UP007 Collados TU185 Marques, Mayra de A. MO007 Lafon, Olivier UP036, Lim, Yong Taik TU215 Lungwitz, Bernhard MO139, TU140, TH141 TH210 Marquez, Brian L. TU275 Lima, Emília Celma de Lamberto, G. R. MO013 Oliveira MO283 Luo, Qing MO133 Marsaioli, Anita J. TU164 Lanfredi, Alexandre J. C. Lima, J. F. MO229, TU230 Luy, Burkhard OP082 Martin, Gary E OP118 MO226 Lima, Jerson MO025 Lye, Ming TH036 Martins, Carla TU194 Lange, Adam TU074, Lima, José Fernando de López-Prados, Javier OP133 Martins, Felipe Terra TH051 TH009 TU080, TU104 Laraoui, Abdelghani Lima, Maria E. de TH024 , Concepción MO064 OP157 López Martins, Gabriel R. TU176 Lima, Marisa Aparecida Larion, Mioara OP129 López, Jesús García TU185 TH093, TH237 Martins, Marcos A. P. Macdonald, Peter M. Larkin, Timothy J. TH099 MO127 Lima, Nerilson Marques TH135 Lavayen, V. TU230 MO175 Martins, Mateus TU209 Machado, Luciana Elena Lavorente, Gabriela Lin, Yanqin MO202 S. F. TH015, MO037, Martínez, Juan Manuel Bassetti TH093 Lin, Yulan TH138 MO049 Lázaro TU158, TH252 Leal, Ana Paula F. TU110 Lin, Yung-Ya UP042, Machado, Sérgio de Paula Maryunina, Ksenia OP019 TU155 Leal, Katia Z. TH090 UP064 Mascarenhas, Artur José Lecoq, Lauriane UP126 Lin, Zhongjie TH075 Machado, Vinicius de Santos TH081 França MO247, MO250, Maso, Lianna Di TH018 Lee, Brittany Linck, Yamila Garro TH279 TU260 Massiot, Dominique Lee, Chulhyun TU212 Maciejewski, Mark W. Lindahl, Martin TH201 OP038 Lee, Donghan OP086 UP049, TU287 Linden, Elisabeth G. van Mateo, Carlos TU251 Lee, Hyunseung TH213, Madhu, P. K. OP071 der MO094 Mathies, Guinevere TU215 Maffei, Mariano TU017, Linser, Rasmus TH120 UP111, MO298 Lee, Jae-Seung UP145 UP054 Matineau, Charlotte Lira, Tatiana Onofre de Mafra, Luís MO064, Lee, Sumin TH213 UP012, MO289 TU284 OP060, TH156 Lee, Youngbok OP109 Liu, J. TH222 Matsuoka, Hideto OP019 Magalhães, Mariana T. Q. Leftin, Avi PL001 Lião, Luciano Morais MO037 Mattea, Carlos TH240 Lehmann-Horn, J. A. TU080, TU104, TH105, Magon, Cláudio José Mauro, Eduardo Di TU068 TU107, TU188, MO283 TU071, TU134, MO229, MO220, TU221 Lehtivarjo, Juuso MO130 Llases, M. E. MO013 TU230 Mavridou, Despoina A.I. Leigh, Kendra TH036 Lloyd, Lyrelle S. OP159 Maguire, Eamonn TH183 OP004

166 Index of Authors

McDonough, Kathleen Monteiro, Robson Q. Nadal, Jurandir MO190 Nordlund, Pär UP007 UP053 UP162, MO286 Nadaud, Philippe S. Norman, David OP127 McElroy, Craig A. MO010 Monti, Gustavo Alberto OP134 , Béla TH027, TU158, TU239, TH252 Noszál McInnes, E. J. L. MO223 Naider, Fred TU002 MO028 Montoro, Marcos MO241, McQueen-Mason, Simon UP080, MO301 Naito, Akira UP088, Nour, Zalfa OP009 J. TH093 TU059, TH288 Montrazi, Elton Tadeu Nourikyan, Julian TH096 Meier, Beat H. UP135, TH243 Nand, Deepak TU119 OP137 Novotny, Etelvino Moon, Hyeyoung TH213, Nantes, Iseli L. MO226 Henrique MO265, MO274 Meira, Douglas Adamoski MO214 TU056 Nardini, Viviani TH174 Nucci, Nathaniel V. Moore, Eric PL002 UP111, MO298 Melo, Carolina Pereira de Nascimento, Claudia Souza MO196 Moore, Jeremy OP102 Jorge do TU170 Nunes, Luiza Maria da Silva OP153, TH270 Melquiond, Adrien UP130 Moraes, Adolfo G. TH108 Nascimento, L. S. MO187 Nunes, Wilian da Silva Nascimento, Otaciro R. Mendes, Ygara S. TH048 Moraes, Adolfo H. MO037, MO097 TH042, TU047 MO226, MO229 Menegola, Julia TU116 Nyman, Tomas UP007 Moraes, Tiago Bueno de Nascimento, Patrícia Menezes, Rachel Santos de MO106, TU134 Coelho TU131 Ohashi, William TH186 MO040 Moreira, Raphaell MO082 Nast, Robert E. Ohno, Ayako TU203 Menezes, Sonia Maria Cabral de MO091, UP121, Moreno, M. MO229 Naughton, Shannon Okitsu, Takashi UP088, TU125, MO256, TU290 TH018 TH288 Moreth, Kristin MO208 Meriles, Carlos A. OP157 Naumann, Christoph Oliveira Junior, Marcos Morgada, Marcos N. TH144 de TU071 Merkle, Hellmut OP142 OP075, TH216 Navas, D. TU230 Oliveira Júnior, Ivan dos Messinger, Robert J. Morgan, Vinícius Gomes Santos TH147 OP009, OP038 TU269 Navon, Gil UP140 Oliveira-Silva, Rodrigo de , Ryan E. OP159 , S. OP033 Nebreda, Angel R. TU017, Mewis Morin TH237, MO274 UP054 Meyer, Jacques TU233 Morozov, Denis TH219 Oliveira, Andre Marco de Neese, Frank OP020 Miljak, D. G. TU068 Moré, Cristian Sebastián MO025 TH258 Nelson, Sarah OP040 Miller, Brian OP129 Oliveira, Andrea Cheble Moura, Luiza Cristina de Neschen, S TH180 de MO025, TH048 Millet, Oscar OP029, TH117 OP114 Neto, Alvaro Cunha Oliveira, Caroline Silva de Moussa, Miled Hassan TU125, TU281 TU188 Min, Hlaing OP109 Youssef MO280 , Denise Cristian Oliveira, Eduardo Barreto Mino, Hiroyuki UP150, Neto Mozley, Erin C. OP004 TH249 TH297 Ferreira TH153 Muhammad, Asif TU263, , Emanuele Miotto, M.C. UP072, Neto, Francisco Gomes Oliveira TH279 TH285 TH030 Catarina da Silva TU125 Mujica, Vladimiro TH228 Miranda Filho, Fernando Neto, Roberto P. Cucinelli Oliveira, Everton Lucas de Luis TU095 Mullen, Dan MO061 MO094 MO244 Miranda, Fabio da Silva Munhoz, Victor H. O. Netto, Antonio Marchi Oliveira, Guilherme A. P. TU263 TU050 MO259 de MO007, TH126 Miranda, Manoel Messias Muniz, Adriane M. S. Neugebauer, Petr PL003 Oliveira, Ivan dos Santos TU146, MO280 P. TU077 MO190 Nicholson, Jeremy K. Mirzahosseini, Arash Munro, Rachel MO118 PL106 Oliveira, Jessica TU041 MO028 Munte, Claudia Elisabeth Nielsen, Jakob TH201 Oliveira, Juliana Ferreira de TU056 Mitchell, J. OP079 OP025, TU044, TH045, Nielsen, Niels Christian TH051, TH111, TU113, Mitra, Mithun TU038 OP073, UP059, OP123, Oliveira, Kleber Thiago de TH231 TH201 MO136, TU281 Miyamoto, Catarina A. Murakami, Mario T. MO049 Nieto, Pedro N. TH009 Oliveira, Livia Roberta P. TH054 de TU194 Mkami, Hassane El OP127 Nieuwkoop, Andrew J. Murayama, Syuhei TU203 OP117 Oliveira, Luciana Moeginger, Bernhard Murphy-Boesch, Joseph Coutinho de MO043, MO259 Nishchenko, Alena TU218 OP043 MO046, OP148 Mohorič, Aleš MO238, Nishimura, Katsuyuki Muthu, Dhanasekaran Oliveira, Paulo Sérgio TU251 TU059 UP076 Lopes de TU056 Molle, Inge Van TU152 Nisius, L. OP033 Muñoz, Roberto Rodriguez Oliveira, Thais MO034 , Marcos Roberto TH255 Noel, Mathieu TU062 Monteiro Ono, A. MO052 TH105, TU107, TH276 Münnemann, Kerstin Nogueira, Marcela Opella, Stanley OP151 Monteiro, Mavis L. UP146, TU293 Christina Oliveira TU155, MO076 TH171 Orcellet, M. L. MO013 N.Garaga, Mounesha Monteiro, Paulo TH078 OP038 Noh, Young-Woock TU215 Oresic, Matej MO184

167 Index of Authors

Orgován, Gábor TH027, Pereira, Wagner Luiz Potrzebowski, Marek J. Reinhard, Friedemann MO028 TU170 TU083, TU143 OP157 Ortega, Gabriel OP114 Peres, Augusto C. C. Pourpoint, Frédérique Religa, Tomasz OP070 MO256 TH141 Ortiz, Fernando López Ren, Jinjun MO082, MO103, TU185 Perez, Javier UP016 Požek, Miroslav MO073, UP037 MO253 Oschkinat, Hartmut Perez, Yolanda TU017 Ren, Pingping TU179 TH063, OP028 Perlo, Josefina TH207 Prado, Paula Favoretti Vital do TU056 Renault, Louis UP016 Ota, Shoko TU098 Pervushin, Konstantin Prates, Maura Viana Renshaw, Matthew P. Ott, Maria UP058 UP007, TH162 TU032 UP154, TH300 Ouari, Olivier TH234, Peters, Dana C. OP061 , Ignacio UP146, Resende, Jackson A. L. C. UP110, TU299 Prina Petronilho, Elaine da C. TU293 TH090 Ovcharenko, Victor TH153 Prisner, Thomas F. PL003 Resende, Jarbas M. OP019 Piló-Veloso, Dorila TH024, TU050 , Alexander Ozarowski, Andrew TH024, TU050 Prokofyev UP136, TU119, TH291 Reverdatto, Sergey MO232 Pimentel, H.R.X. MO091 TH198, UP053 Prosser, Scott TH135 Pagès, Guilhem OP095 Pinheiro, Natália G. S Rezende, Camila Alves Paim, A. P. S. MO187 TH024 Provencher, Marie-Eve TH093, TH237 TU062 Paiva, Fernando Fernandes Pintacuda, Guido TH060 Rezende, Carlos A. TH090 Puckeridge, Max OP095, TH210 Pinto, Andressa P. A. TH144 Ribeiro, Tatiana Santana Paluch, Piotr TU083, OP025 TU077 TU143 Puglisi, Joseph D. UP116 Pinto, Diogo O. Soares Ribotta, P.D. TH267 Parella, Teodor OP083 TH087, TU146 Purusottam, Rudra Narayan UP125 Riedel, Dietmar OP133 Park, Hyo Eun MO214 Pinto, Thiago Rodrigues MO025 Pusiol, Daniel José Rienstra, Chad M. OP117 Park, Junwon TH213 MO148, MO241, UP080, Pinto, Vinícius S. TH105, Rio, Edmilson TH246 Parker, David UP084 TU107 TH258, OP152, MO301 Rios, Edmilson Helton Pascutti, Pedro Geraldo Pires, Diego Arantes Pustovalova, Yulia MO247, MO250, MO256, TH051 Teixeira TU032, TU170 TH015, UP049, TU287 TH279 Pastawski, Horacio M. Pires, José Ricardo Murari Pérez-Aparicio, Roberto Rivlin, Michal UP140 TH066, OP144 TU029, MO031, MO040 UP058 Roberts, S. Tegan UP154, Patiny, Luc TH165 Pizetta, Daniel C. TU209 Qian, Chunqi OP043 TH300 , Luiz Paula, Viviane Silva de Placial, Jean-Pierre UP016 Queiroz Júnior Robertson, Duncan MO004, TH015, UP162, Henrique Keng TU080, Plevin, Michael OP055 OP127 TU041, MO286 TU104, MO136, MO283 Robles, Ivan Erick Plivelic, Tomás S. TU092 , L. UP072, Paulsen, Harald OP077 Quintanar Castañeda TH255 Pol, Rostislav TU161 TH285 Pawlak, Tomasz TU083, Robustelli, Paul UP115, Rabbani, Said R. TU191, TU143 Polenova, Tatyana MO139 MO124, MO292 TH195, MO211 Paëpe, Gaël De UP126 Polienko, Yulija TH219 Rocca-Serra, Philippe Ragavan, Mukundan TH183 Pelc, Damjan MO253 Polikarpov, Igor TH093, OP109 TH237 Rocha, Cristiane B. Pell, Andrew J. TH060 Ramaswamy, Vijaykumar Polli, Roberson Saraiva MO007 Pellecchia, Maurizio TH087, TH210, TH243 Ramos, Bruna M. C. Rocha, João TH156 OP014 TH054 Polyhach, Yevhen OP077 Rochaa, Cláudia Quintino Penzel, Susanne UP135, Ramos, Carlos Henrique Pomarico, Luciana TU194 da MO172 OP137 TH063 Pomin, Vitor Hugo TU041 Roditi, Itzhak TH147, Peobody, David MO025 Ramos, Ingrid Graça MO280 Pepin, Alexandre TH210 Pongs, Olaf UP136, TH081 TU119, TH291 Rodrigues Filho, Rangel, Luciana Pereira Pereira, Carla Karine Ubirajara P. TU071 Barbosa TH168 Pons, Miquel OP029, MO025 TU017, UP054 Rodrigues, Diego Alves Pereira, Elen G. TH126 Raschle, Thomas TH120 Ponte, Santiago L. TH132 TU080, TU104 Pereira, Fabiola Manhas Rasia, Rodolfo Rodrigues, Letícia V. Verbi TH264 Poole, Katye M. TU254 Maximiliano MO100 MO127 , James M. MO205 Pereira, G. E. MO187 Pope Ravotti, Francesco UP135 Rodrigues, Raquel Popot, Jean-Luc UP027 Pereira, Gonçalo Redfield, Christina OP004 Pantoja Barreiros MO115, Amarante Guimarães TU155 Porto, Christiane Feijó de Reibarkh, Mikhail OP118 TU056 Castro TU281 Rodrigues, Stephen Reich, Daniel OP142 Pereira, Luciana TU194 Postigo, Matheus Pereira Fernandes de Paula MO211 MO112 Reichert, Detlef OP057 Pereira, Natália Corrêa Rodrigues, Tiago B. TH030 Potapov, Alexey PL002 Reif, Bernd MO139, OP070 OP065

168 Index of Authors

Rohwedder, Jarbas José Santos, Carlos Henrique Schwarten, Melanie Silva, Afonso OP142 Rodrigues TU260, MO262 Corrêa dos MO160 OP055 Silva, André L. MO226 Rojas, Leonardo MO076 Santos, Carolina G. dos Schwartz, Lawrence Silva, Antonio Jorge MO226 TH246 Romanuka, Julija UP031 Ribeiro da TU176 Santos, Daniel M. TH024 Schwiegk, Claudia OP086 Romeiro, Luiz Antônio Silva, Daniel F. OP025 Soares TU131 Santos, Fabiane Aparecida Schwieters, Charles D. Silva, Daniel Varón MO001 Batalha dos MO136 TH102, OP134 Roos, Annette UP007 Silva, Danilo M.D. Delfino Santos, José Sebastião dos Schütz, Anne OP137 Rosato, Antonio TU020, da TU209 TU284 TH021 Scramin, Juliana TU077 Silva, Dhiogo Mendes da Santos, Marcelo Henrique Rose, Honor M. UP089, Sears, Jesse A. UP099 MO283 MO295 dos MO172 Sebastião, Pedro José Silva, Dulce Helena Santos, Raquel MO190 Rosengren, K Johan TU278 Siqueira MO175 OP160 Santos, Reginaldo Bezerra Sebastião, Rita de Cássia Silva, Emerson Oliveira Da dos TU281 Rossini, Aaron J. TH234, Oliveira TH282 TU095 UP110, TU299 Santos, Sérgio M. MO064, Sederman, Andrew J. Silva, Fernanda B. da OP060, TH156 Rosso, Kevin M. UP099 OP079, UP154, TH300 TU101 , Tereza Cristina dos Ruiz, Naira Machado da Santos Seferin, Marcus UP121, Silva, Gil V. J. da TH174 Silva MO256 TU089 TU290 Silva, Glauciane R.D. Römelt, Michael OP020 Sant’Anna, Raphaela Seidl, Peter R. TU101 TU164 Teresa TU155 SZE, Kong Hung TU011 Selenko, Philipp UP089, Silva, Hana Karina Pereira Sant’Anna, Ricardo MO295 da TH093 Saalwächter, Kay UP058, Oliveira TH063 OP057 Semaan, Felipe Silva Silva, I. D. A. MO229 Sardinha, João UP121, TU263 Sabo, Michael OP086 TU290 Silva, Isabela Almeida Sengupta, Ishita OP134 TU146 Sagdeev, Renad OP019 Sardo, Mariana MO064 Separovic, Frances OP163 Silva, Jerson L. MO007, Saha, Dipta MO145 Saridakis, Emmanuel Serial, M.R. TH267 TH048, TH126 Sahakyan, Aleksandr OP004 MO130 , Eva M. OP065 Silva, Lorena M. A. TH276 Sarthour Júnior, Roberto Serrao Salager, Elodie OP101 Silva TU146, TH147, Sesti, Erika MO145 Silva, Milton Neto da MO280 TU110, TU257 Salek, Reza TH183, Seyedlar, Amin Ordikhani MO184 Sarzedas, Carolina Galvão TH240 Silva, Pablo Nascimento da TU035 MO277 Salinas, Roberto Kopke Sforça, Mauricio Luis TH039, MO043, MO046 Sass, H.-.J. OP033 TU053, TH054, MO055, Silva, Ricardo Oliveira da TU056, TU197 TH192, MO193 Salinas, Roberto OP129, Sati, Pascal OP142 OP148 Sgourakis, Nikolaos Silva, Rodrigo de Oliveira Sattler, Michael OP090 OP133 TH087, MO265 Salvatella, Xavier OP086 Sayegh, Raphael Santa Shanks, Brent OP124 Silva, Romeu Ricardo da Sam, My D. TH036 Rosa TH039 TU194 Sharma, Mayuri TH036 Samoson, Ago UP135 Scheler, Ulrich MO238 Silva, Ronaldo Dionísio da Shekhtman, Alexander Samouilov, Alexandre TH192 Scherf, Tali TU002 TH198, UP053 MO235 Silva, Simone P. MO094 Schettino Jr., Miguel A. Shemesh, Noam OP122 Sanders, Kevin OP034 MO085, TU086 Silva, Tania Maria S. da Shen, Jiang-Ren UP150, Sano, Walter TU221 TH159 Schiemann, Olav OP127 TH297 Sansone, Susanna-Assunta Silva, Virginia Cláudia da Schmidt-Rohr, Klaus Shen, Yang OP132 TH183 OP124 MO160 Shevelkov, Veniamin Sant’Ana, Antônio Silveira, Camila P. Schmidt, Rita PL001 OP133 Euzébio Goulart TH081, MO076, TU101 Schneider, Horst UP058 , Chaowei TH168 Shi Silveira, Edilberto Rocha Sant’Anna, Quézia da Schneider, José Fabian Shi, Lichi MO118 TU131, TU173 Silva TH171 TU065, UP104, TU071 Shi, Pan Silveira, Ivna A. F. B. Santana, Juliana Santos Schnur, Einat TU002 TU110 Shi, Yunyu OP050 TH063 Schomburg, Benjamin Simorre, Jean-Pierre Shirakawa, Masahiro Santana, Mábio João TU074 UP126 TH150, TU203 MO283 Schreier, Shirley TH114 Simões, Clara MO154 Shundrina, Inna TU218 Santos Jr., Emanuel Schubert, Volker MO001 Singh, Himanshu TU200 MO094 Siepel, Florian TU122 Schug, Alexander UP006 Sivalingam, Kantharuban Santos-Filho, Osvaldo Silletta, Emilia V. TH207, OP020 Andrade MO088 Schulthes, Cristian P. TU239 TU245 Sivitz, William UP158 Santos, Bernardo Coutinho Silva Filho, Eloi Alves da Camilo dos MO247, MO250 Schwalbe, Harald UP032 MO268 Skora, L. OP033

169 Index of Authors

Smith, Colin OP086 Stelzl, Lukas OP004 Teo, Hsiangling UP007 Tuna, F. MO223 Smith, Graham OP127 Stepanov, Alexander Terskikh, Victor MO067 Turcu, Flaviu R.V. UP099 TU218 Smith, Pieter ES OP122 Theillet, Francois-Xavier Tuttle, Marcus D. OP117 Stepišnik, Janez MO238, UP089, MO295 Soares, Vera L. P. MO094 , Robert PL002 TU251 Tycko Theis, Thomas OP138, Solomon, Eddy PL001 , Galia UP140 Stetz, Matthew UP111, UP041 Tzarfaty Solyom, Zsofia OP055 MO298 Thiemann, Otavio Tzarfaty, Ilan UP140 Song, Yi-Qiao OP081, Stevens, Julie M. OP004 Henrique MO112 Täubert, Sebastian TU122 TH246 Strizhakov, Rodion TH219 Thieuleux, Chloé UP110, Uchiyama, Mayara Soprano, Adriana S. TU299 Klimuk MO211 TU053 Strumia, M.C. TU239 Thompson, Karen TH201 Ueda, Kazuyoshi TU059 Sorenson, Martha M. Stupp, Gregory S. Thurber, Kent PL002 MO007 Suarez, Irina Paula MO100 Ugurbil, Kamil PL164 Tian, Changlin TU014, Sotoma, Shingo TH150 Suarez, Marisa C. MO007 Ulzega, Simone UP036 Tiezzi, Henrique Oliveira Sotta, Paul UP058 Umeyama, Masako TU059 Subramanian, M. A. TU056 Sousa Júnior, Paulo MO232 Unden, Gottfried TU074 Timco, G. A. MO223 Teixeira de MO160 Sudo, Yuki UP088, TH288 Unverzagt, Carlo MO001 Tinoco, Luzineide Sousa, Eduardo Gomes Surface, J. Andrew OP102 Wanderley TU035, MO169 , Viviane da Silva Rodrigues de MO088, Vaiss TH084, TH090 TU089 Suter, Dieter OP147 Tischer, I. TU167 Souza, Andre TH210, Sychrovsky, Vladimir Tiziano, Grazielle. V. Vajpai, N. OP033 MO247, TU248 MO052 MO256 Vaknin, David TH225 Souza, André Alves de Takahashi, Hiroki UP126 Tochio, Hidehito TH150, Valdés-Solis, Maria José TH279 TU203 Takegoshi, K. PL107 Iglesias TU185 Souza, Claudia M. G. de Toma, Sergio Hiroshi Tam, Leo OP061 Valente, Ana Paula TU191, TH195 MO211 MO004, TH015, UP162, Tamaki, Fabio Kendi Souza, Diorge Paulo de Tomatis, Pablo E. TU008 OP047, TU026, TH030, TH039 MO043 TH033, MO037, TU041, Tomonaga, Yuya UP088, Tanaka, Yoshiyuki MO052 TH042, TU047, TH048, Souza, Diorge OP148 TH288 MO049, TH108, TH114, Tang, Huiru MO157, Souza, Eugênio Furtado de Toms, Harold TH081 TH126, MO190, TU194, MO178, TU179 TH084 MO286 Tordo, Paul TH234, Tang, Joel A. TH072 Souza, Isis Torres TH168 UP110, TU299 Valentine, Kathleen G. , Ming OP117 UP111, MO298 Souza, Ivete P. R. de Tang Tozoni, José Roberto MO190, TU194 Tannús, Alberto TU209, TH087 Valezi, Daniel Farinha MO220 Souza, Ivete R. P. de TH210 Traaseth, Nathaniel J TU026 Tars, Kaspars TH060 MO061 Varani, Luca TH042 Souza, Tatiana A.C.B. Tasic, Ljubica MO076, Trbovic, Nikola MO124 Vargas, Geovana TH042 TU053 TU101, TH186 Trease, Nicole M. OP101 Veber, Sergey OP019 Souza, Theo L. F. MO025, Taulelle, Francis UP012, Trebosc, Julien TU140, Vega, Daniel TU158 TH048 MO289 TH141 Veglia, Gianluigi OP030, Souza, Tiago A. TU053 Tavares, Maria Inês Bruno Trellet, Mikael UP130 MO061 Sperling, Lindsay J. MO094, TU278 Tretyakov, Evgeny , A.K. OP117 Velez-Jurado Tayler, A. B. OP079 TH219, OP019 TU167 Spielberg, E. T. MO223 Tayler, Michael C. D. Trevizan, Willian Veloro, Angelo M. OP074 Spiess, Hans Wolfgang TH099 Andrighetto TH246, Venancio, Tiago TU284 UP146, TU293 Taylor, Rae TH078 MO247, MO250 Vendruscolo, Michele Spindler, Phillip PL003 Teixeira, Francesco Trzeciak-Karlikowska, Katarzyna TU083 UP131, TH129, MO130 Spitzer, Romana MO121 Brugnera TU044 Trébosc, Julien UP036, Venâncio, Tiago TH276 Spitzmesser, JB MO151 Teixeira, Paulo José Pereira Lima TU056 MO139 Vera, I.M.S. de OP074 Staab, John MO151 Teixeira, Róbson Ricardo Trésaugues, Lionel UP007 Verardi, Raffaello MO061 Stanton, Christopher J. TU170 Tse, Ho Sum TU011 MO145 Verly, Rodrigo M. TH024, Tekely, Piotr UP125 Tsimanis, Alexander TU050 Stapf, Siegfried TH240, TU002 TU251 Teles, Rubens Rodrigues Verma, Abhinav UP006 MO142, MO166 Tsuchida, Jefferson Staudacher, Tobias OP157 Viani, Elisabetta UP116 Esquina TU065, TH237 Telkki, Ville-Veikko UP098 Steinbeck, Christoph Vicente, Eduardo Festozo Tsuchida, Jefferson UP104 MO163, TH183 Telser, Joshua MO232, TH051, TU113 TU233 Tsutsumi, Atsushi TU059 Steinhaus, Johannes Vicentin, Bruno Luiz MO259 Temel, Deniz B. TH096 Tugarinov, Vitali OP129 Santana TU221

170 Index of Authors

Vidal, Thaís Jerônimo Wand, A. Joshua UP111, Williamson, R. Thomas Yunes, José Andrés MO196 TU035 MO298 OP118 Zaballa, María-Eugenia Vidoto, Edson Luiz Gea Wang, Conan OP160 Wimperis, Stephen OP035 OP075 TU209, TH210, TH243 Wang, Hao OP101 Winpenny, R. E. P. Zagdoun, Alexandre Vieira, Fernando Henrique MO223 TH234, UP110, TU299 TH261 Wang, Qiang TU140, TH141 Wist, Julien TH165, Zanotto, Edgar Dutra Vieira, Ygor W. TU281 TU167 TU065 Wang, Qiu UP041 Vigneron, Daniel B. Withers, Richard S. Zell, Mark T. TU275 OP040 Wang, Shenlin MO118 Wolf-Watz, Hans TH012 Zeng, Haifeng OP109 Vila, Alejandro J. TU008, Wang, Yulan MO157, Wolf-Watz, Magnus Zeri, Ana Carolina de OP075, TH216, MO217 MO178 UP006, TH012 Mattos TU053, TH054, Wanker, Erich E. OP070 Vilegas, Wagner MO172 Wrachtrup, Jörg OP157, MO055, TU056, MO058, TU116, MO196, TU197, Vilela, Fabiana Cardoso Warren, Warren S. OP138, OP105 UP041 TH204 MO172, MO172 Wu, Fangming TU014, Zeri, Carolina de Mattos Vinaik, Roohi MO022 Washington, Jacqueline Wunderlich, Christoph TH057 UP053 Vizthum, Veronika UP036 MO121, TH123 Zhang, Chenwei TU014 Wasielewski, Emeric Voyer, Normand TU062 Wylde, Richard OP127 TU023 Zhang, Hongjun UP076 Vrielink, Anneloes Oude Xu, Jun TU140 Wasylishen, Roderick E. Zhang, Ji MO133 UP017, TH294 MO067 Yamanaka, D. MO052 Zhang, Limin MO178, Véron, Emmanuel OP038 Webber, A. L. TH222, Yamauchi, Seigo OP019 TU179 Wada, Akimori UP088, MO223 Yan, Si MO139 Zhang, Zhiyong TU137, TH288 Weber, David J. TH006 Yang, Bai TH162 TH138 Wagner, Gerhard TH036, Zhong, Guiming TH072 TH120, OP113 Wedge, C. J. MO223 Yang, Yong TH072 Zhou, Lina OP101 Walbrecker, Jan O. Weingarth, Markus Yau, Wai-Ming PL002 UP136, TU119, TH291 OP096 Ye, Chaohui MO133 Zhou, Pei UP041 , Christoph TH012 Walker, F. Ann UP076 Weise Ying, Jinfa OP132 Zhou, Xin MO133 Wallner, Michael TH108 Wheeler, Dustin MO145 Yokota, Hiroaki TH150 Zhou, Zijian OP138 Walls, Jamie D. UP042 Wheeler, Patrick TU161 Yomoda, Hiroki UP088, Zwart, Jacco de OP142 TH288 Walsh, David OP096 Whittaker, Sara B-M. Zweckstetter, Markus TH054 Yoshinari, Yosuke TH150 TU005, MO019, TH102 Walter, Eric D. TU245 Wiktor, M. OP033 Yu, Liping UP158 Álvarez, Gonzalo A OP122 Walter, Glenn A. Willbold, Dieter OP055 Yu, Xin OP043 Åden, Jörgen UP006 Walter, Korvin F. A. OP086 Williams, Mark TH183 Yulikov, Maxim TH234 šebera, J. MO052

171 Directory of Participants

Directory of Participants

Afonso de Andre, Carlos Alborghetti, Marcos R Anglister, Jacob SPWLA Brazilian Biosciences National Labo- Weizmann Institute MACAE, Brazil ratory - LNBio/CNPEM Rehovot, Israel [email protected] Campinas, Brazil [email protected] [email protected] Abbots, Frances T Arake, Luisa M BG E&P Brasil Ltda Alcantara, Glaucia B. Embrapa Cenargen Rio de Janeiro, Brazil Universidade Federal de Mato Grosso Brasilia, Brazil [email protected] do Sul [email protected] Campo Grande, Brazil [email protected] Abiko, Layara Akemi Araujo, Renata Mendonça University of São Paulo Universidade Federal do Ceará São Paulo, Brazil Almeida, Fabio C.L. Natal, Brazil [email protected] Federal University of Rio de Janeiro [email protected] Rio de Janeiro, Brazil [email protected] Abrantes, Luiza T Ardá, Ana Petroleo Brasileiro S.A Centro de Investigaciones Biológicas- Rio de Janeiro, Brazil Alvares, Rohan CSIC [email protected] University of Toronto Madrid, Spain Mississauga, Canada [email protected] [email protected] Acosta, Rodolfo H. FAMAF-Universidad Nacional de Arthanari, Haribabu Cordoba Alves, Nathalia S Harvad Medical School CORDOBA, Argentina Universidade Federal do Rio de Boston, United States [email protected] Janeiro [email protected] Rio de Janeiro, Brazil [email protected] Afonso, Luciana Caminha Ascona, Christian Rivera Helmholtz Center Munich Instituto de Física de São Carlos - Neuherberg, Germany Alves da Cruz, Joao Felipe Universidade de São Paulo luciana.afonso@helmholtz- Universidade Federal de São Carlos São Carlos, Brazil muenchen.de São Carlos, Brazil [email protected] [email protected] Aguiar, Daniel L.M. Ashbrook, Sharon Universidade Federal do Rio de Alves Rodrigues, Diego A. R. University of St Andrews Janeiro Universidade Federal de Goiás St Andrews, United Kingdom Rio de Janeiro, Brazil APARECIDA DE GOIÂNIA, Brazil [email protected] [email protected] [email protected] Auccaise, Ruben Ahola, Susanna Amata, Irene Empresa Brasileira de Pesquisa NMR Research Group, University of Insitute for Research in Biomedicine Agropecuária Oulu (IRB) of Barcelona, and University Rio de Janeiro, Brazil Oulu, Finland of Barcelona [email protected] susanna.ahola@oulu.fi Barcelona , Spain [email protected] Auger, Michele Ailion, David C Universite Laval University of Utah Amorim, Gisele C Quebec, Canada Salt Lake City, United States Centro Nacional de Ressonância [email protected] [email protected] Magnética Nuclear - Universidade Federal do Rio de Janeiro Rio de Janeiro, Brazil Azeredo, Rodrigo Akoury, Elias [email protected] Universidade Federal Fluminense Max-Planck-Institute for Biophysical Niteroi, Brazil Chemistry [email protected]ff.br Göttingen, Germany Amoureux, Jean Paul [email protected] Lille University Villeneuve d’Ascq, France Azevedo, Clerio F [email protected] D’Or Institute for Research and Ed- AL, Susmitha ucation (IDOR) Tata Institute of Fundamental Re- Rio de Janeiro, Brazil Andrade, Fabiana D search [email protected] Mumbai, India Institute of Physics of São Car- [email protected] los/USP São Carlos , Brazil Azevedo, Graziella V T [email protected] Petróleo Brasileiro S.A Al-Hashimi, Hashim rio de Janeiro, Brazil University of Michigan [email protected] Ann Arbor, Brazil Andrade, Raphael H.S. [email protected] Universidade Federal de Pernambuco / Instituto Federal de Educação, Bagryanskaya, Elena G. Ciência e Tecnologia de Perna Novosibirsk Institute of Organic Albernaz, Fabiana P. Recife, Brazil Chemistry SB RAS Unversidade Federal do Rio de [email protected] Novosibirsk, Russia 166 Rwanda Janeiro [email protected] Rio de janeiro, Brazil [email protected]

172 Directory of Participants

Bai, Yang Bax, Ad Bonvin, Alexandre M.J.J. Nanyang Technological University, NIDDK, National Institutes of Utrecht University, Faculty of Sci- School of Biological Sciences Health ence, Bijvoet Center singapore, Singapore Bethesda, United States Utrecht, Netherlands [email protected] [email protected] [email protected]

Baines, Victoria Belisle, Katherine Borovskaya, Irina Schlumberger Cambridge Isotope Laboratories, Schlumberger Rio de Janeiro, Brazil Inc. Rio de Janeiro, Brazil [email protected] Andover, United States [email protected] [email protected] Bajaj, Vikram Bouchard, Louis-Serge University of California, Berkeley Bento, Edson de Souza University of California, Los Angeles and Lawrence Berkeley National Universidade Federal de Alagoas Los Angeles, United States Laboratory Maceio, Brazil [email protected] Berkeley, United States [email protected] [email protected] Boyd, Austin Bernardinelli, Oigres Daniel Schlumberger Balbach, Jochen USP IFSC Rio de Janeiro, Brazil University of Halle São Carlos, Brazil [email protected] Halle, Germany [email protected] [email protected] Braide, Otonye Bernardo, Camilla Nascimento University of Florida Balcom, Bruce J Fundação e Instituto Oswaldo Cruz Gainesville, United States University of New Brunswick Rio de Janeiro, Brazil [email protected] Fredericton, Canada [email protected] [email protected] Brito, Luciana M. Billeter, Martin IMA-UFRJ Barberis, Gaston E University of Gothenburg Rio de Janeiro, Brazil Instituto de Fisica UNICAMP Gothenburg, Sweden [email protected] Campinas, Brazil [email protected] barberis@ifi.unicamp.br Britt, R David Binolfi, Andres University of California, Davis Barbet-Massin, Emeline Leibniz-Institut für Molekulare Davis, United States Centre de RMN à Très Hauts Pharmakologie (FMP) [email protected] Champs Berlin, Germany Villeurbanne, France binolfi@fmp-berlin.de Brown, Steven P. [email protected] University of Warwick Bize-Forest, Nadege Coventry, United Kingdom Barbosa, Lúcio Leonel Schlumberger [email protected] Federal University of Espírito Santo Rio de Janeiro, Brazil Vitória, Brazil [email protected] Bruix, Marta [email protected] IQFR-CSIC Blümich, Bernhard Madrid, Spain Barison, Andersson RWTH Aachen University [email protected] Paraná Federal University Aachen, Germany Curitiba, Brazil [email protected] Bruschweiler, Rafael [email protected] Florida State University and Böckmann, Anja NHMFL Barros, Carlos J. P. Institut de Biologie et Chimie des Tallahassee, United States Universidade Federal de Pernambuco Protéines, CNRS-Université de Lyon [email protected] Recife, Brazil Lyon, France [email protected] [email protected] Brutscher, Bernhard Institut de Biologie Structurale Barros Junior, Wilson Bodenhausen, Geoffrey Grenoble, France Federal University of Pernambuco EPFL SB ISIC LRMB [email protected] Recife, Brazil LAUSANNE, Switzerland [email protected] geoffrey.bodenhausen@epfl.ch Buarque Muller, Camilla Djenne Pontifícia Universidade Católica do Basso, Luis GM Boelens, Rolf Rio de Janeiro University of Sao Paulo Utrecht University Rio de Janeiro, Brazil Sao Carlos, Brazil Utrecht, Netherlands [email protected] [email protected] [email protected] Budker, Dmitry Batista, Aline L. Bonagamba, Tito José University of California at Berkeley Universidade Federal do Rio de Instituto de Física de São Carlos - Berkeley, United States Janeiro USP [email protected] Rio do Janeiro, Brazil São Carlos, Brazil [email protected] [email protected] Buljubasich, Lisandro Fa.M.A.F.-IFEG, Córdoba- Bonfim, Rodrigo P F Argentina UFRJ Córdoba, Argentina Rio de Janeiro, Brazil [email protected] rodrigobonfi[email protected]

173 Directory of Participants

Burai, Laszlo Carpinella, Mariela Chmelka, Bradley F Servier Research Institute of Medici- FaMAF_CONICET - Universidad University of California, Santa Bar- nal Chemistry Nacional de Córdoba, Argentina bara Budapest, Hungary Córdoba, Argentina Santa Barbara, United States [email protected] [email protected] [email protected]

Burdisso, Paula Carvalho, André S Cho, Jee-Hyun Instituto de Biología Molecular y Universidade Federal de São Carlos Korea Basic Science Institute Celular de Rosario São Carlos, Brazil Ochang-eup, South Korea Rosario, Argentina [email protected] [email protected] [email protected] Cassús, Eduardo P Cobas Gomez, Carlos Byeon, Chang-Hyeock Petróleo Brasileiro S.A. MESTRELAB RESEARCH, S.L. University of Pittsburgh Rio de Janeiro, Brazil Santiago de Compostela, Spain PIttsburgh, United States [email protected] [email protected] [email protected] Castañeda, Ivan Erick Ivan E. Cole, Jonathan Byeon, In-Ja L Universidad Autónoma del Estado de Cryogenic Ltd. University of Pittsburgh Morelos London, United Kingdom Pittsburgh, United States Cuernavaca, Mexico [email protected] [email protected] [email protected] Colnago, Luiz Alberto Cabrita, Eurico J Castañeda Valencia, Gloria Embrapa REQUIMTE, Faculdade de Ciências Maria São Carlos, Brazil e Tecnologia - UNL Iris Valencia Rivera [email protected] Caparica, Portugal Rio de Janeiro, Brazil [email protected] [email protected] Correia, Maury D Petrobras Caldarelli, Stefano Castellen, Patricia Rio de Janeiro, Brazil ICSN CNRS UPR 2301 LNBio [email protected] PALAISEAU, France Campinas, Brazil [email protected] [email protected] Corvo, Marta REQUIMTE, CQFB Calderon, Cesar J Catoire, Laurent J Caparica, Portugal UNICAMP CNRS - IBPC [email protected] Campinas, Brazil Paris, France [email protected] [email protected] Costa-Filho, Antonio J University of São Paulo Camilloni, Carlo Cavini, Ítalo A Ribeirao Preto, Brazil University of Cambridge Instituto de Física de São Carlos - ajcosta@ffclrp.usp.br Cambridge, United Kingdom USP [email protected] São Carlos, Brazil Cowsik, Sudha M [email protected] Jawaharlal Nehru University, New Cañada, Francisco Javier Delhi Centro Investigaciones Biológicas, Cerioni, Lucas M C New Delhi, India CIB CSIC Spinlock SRL - CONICET [email protected] Madrid, Spain Cordoba, Argentina [email protected] [email protected] Coy, Andrew Magritek Canevarolo, Rafael R Chattah, Ana K Wellington, New Zealand Laboratório Nacional de Biociências FaMAF and CONICET, Cordoba, [email protected] Campinas, Brazil Argentina [email protected] Córdoba, Argentina Craik, David J [email protected] The University of Queensland Cappellaro, Paola Brisbane, Australia Massachusetts Institute of Technol- Chaykovsky, Mark [email protected] ogy Bruker do Brasil Cambride, United States Atibaia, Brazil Crossley, Nigel [email protected] Mark.Chaykovsky@bruker- Oxford Instruments biospin.com Abingdon, United Kingdom Carneiro, Giovanna [email protected] Schlumberger Chinelatto-Júnior, Luiz S Rio de Janeiro, Brazil Petroleo Brasileiro S.A. Crusca, Edson [email protected] Rio de Janeiro, Brazil University of São Paulo [email protected] São Carlos, Brazil Carneiro, Sandra R R [email protected] Petrobras Chira, Pedro O Rio de Janeeiro, Brazil Halliburton Cvitanic, Tonci [email protected] Rio de Janeiro, Brazil University of Zagreb [email protected] Zagreb, Croatia Carosio, Maria G. A. [email protected] Institute of Chemistry of São Carlos São Carlos, Brazil [email protected]

174 Directory of Participants

D’Andréa, Éverton Dias de Souza Filho, Jose D El-Bacha, Tatiana Universidade Federal do Rio de Universidade Federal de Minas Universidade Federal do Rio de Janeiro Gerais Janeiro Rio de Janeiro, Brazil Belo Horizonte, Brazil Rio de Janeiro, Brazil [email protected] [email protected] [email protected] da Silva, Gil V. J. Della Vecchia, Miguel A Emwas, Abdul-Hamid M FFCLRP / University of São Paulo Instituto Nacional de Tecnología In- King Abdullah University of Science Ribeirão Preto, Brazil dustrial and Technology [email protected] San Martín, Argentina Thuwal, Saudi Arabia [email protected] [email protected] da Silva, Milton N. Instituto de Tecnologia em Imunobi- Deng, Feng Engelsberg, Mario ológicos Wuhan Institute of Physics and Universidade Federal de Pernambuco RIO DE JANEIRO, Brazil Mathematics, Chinese Academy of Recife, Brazil milton@bio.fiocruz.br Sciences [email protected] Wuhan, People’s Republic of China [email protected] Darbello, Daniel de Menezes Escobar, Rodolfo Laboratório Nacional de Biociências Halliburton Campinas, Brazil Deslauriers, Roxanne Rio de Janeiro, Brazil [email protected] iRDs [email protected] Headingley, Canada [email protected] de Araujo Ferreira, Arthur G Espindola , Ana Paula Instituto de Física de São Carlos Ana Paula Espindola São Carlos, Brazil Di Mauro, Eduardo Barueri , Brazil [email protected] Universidade Estadual de Londrina [email protected] LONDRINA, Brazil [email protected] de Azevedo, Eduardo R Espinoza Cara, Andrés Universidade de São Paulo Instituto de Biología Molecular y São Carlos - SP, Brazil Dias, David M Celular de Rosario [email protected] University of Cambridge Rosario, Argentina Cambridge, United Kingdom [email protected] [email protected] de Carvalho, Erika Martins Instituto de Tecnologia em Farmacos Falco Cobra, Paulo Rio de Janeiro, Brazil Dinse, Klaus-Peter Instituto de Química de São Carlos, erikamc@far.fiocruz.br Physics Dept. FU Berlin Germany Universidade de São Paulo Berlin, Germany São Carlos, Brazil [email protected] [email protected] de Carvalho, Mário Geraldo Universidade Federal Rural do Rio de Janeiro Doty, Francis David Fam, Maged Seropédica, Brazil Doty Scientific, Inc. Halliburton [email protected] Columbia, United States Bogota, Colombia [email protected] [email protected] De Goitia, Adriana Mendes PUC-RIO Doty, Judy Fanucci, Gail E Rio De Janeiro, Brazil Doty Scientific, Inc. University of Florida [email protected] Columbia, United States Gainesville, United States [email protected] [email protected]fl.edu de Oliveira, Guilherme A. P. Federal University of Rio de Janeiro Driehuys, Bastiaan Farias, Fábio LL Rio de Janeiro, Brazil Duke University Petróleo Brasileiro SA [email protected] Durham, United States Rio De Janeiro, Brazil [email protected] [email protected] de Oliveira, Livia R. P. Universidade Federal do Rio de Duckett, Simon B Favaro, Denize C. Janeiro - Faculdade de Odontologia University of York University of Sao Paulo Rio de Janeiro, Brazil York, United Kingdom Sao Paulo, Brazil [email protected] [email protected] [email protected] de Paula, Viviane S Duyn, Jeff H Fedin, Matvey V. Universidade Federal do Rio de National Institutes of Health, USA International Tomography Center SB Janeiro Bethesda, United States RAS Rio de Janeiro, Brazil [email protected] Novosibirsk, Russia 166 Rwanda [email protected] [email protected] Eckert, Hellmut de Souza , João Carlos IFSC, University of Sao Paulo Felli, Isabella C. Agilent Technologies Sao Carlos, Brazil CERM, University of Florence, Italy Barueri , Brazil [email protected] Sesto Fiorentino (Florence), Italy [email protected] [email protected]fi.it Edwards, John C Process NMR Associates Feng, Yesu Danbury, United States Duke University [email protected] Durham, United States [email protected]

175 Directory of Participants

Fernandez, Cesar Figueroa-Villar, José D Frey, Michael Novartis Pharma AG Military Institute of Engineering JEOL USA, Inc Basel, Switzerland Rio de Janeiro, Brazil Peabody, United States [email protected] [email protected] [email protected]

Fernandez, Claudio Oscar Finkelstein-Shapiro, Daniel Frizzo, Clarissa P. Universidad Nacional de Rosario, In- Arizona State University Universidade Federal de Santa Maria stituto de Biologia Molecular y Celu- Evanston, United States Santa Maria , Brazil lar de Rosario d-finkelstein- [email protected] Rosario, Argentina [email protected] [email protected] Frydman, Lucio Florian, Pierre Weizmann Institute Ferraris, Paolo CEMHTI-CNRS Rehovot, Israel Schlumberger Orléans, France [email protected] Rio de Janeiro, Brazil pierre.fl[email protected] [email protected] Fu, Riqiang Foster, Mark P Natl High Magnetic Field Lab Ferraz, Erica K P The Ohio State University Tallahassee, United States Petrobras S.A. Columbus, United States [email protected] Macae, Brazil [email protected] [email protected] Funk, Alexander M França, Camila Durham University Ferreira, Antonio Gilberto BG Group Durham, United Kingdom Federal University of São Carlos Rio de Janeiro, Brazil [email protected] São Carlos, Brazil [email protected] [email protected] Gaffney, Betty J. França, José Adonias Florida State University Ferreira, Bárbara D Universidade Federal de Pernambuco Tallahassee, United States UFMG - Universidade Federal de Mi- Recife, Brazil gaff[email protected] nas Gerais [email protected] Belo Horizonte, Brazil Galiana, Gigi [email protected] Fredriksson, Jonas MB Yale University Gothenburg University Sweden New Haven, United States Ferreira, Fernanda G Meireles Gothenburg, Sweden [email protected] Centro Nacional de Bioimagem [email protected] Rio de Janeiro, Brazil Gambarra-Neto, Francisco F. [email protected] Freed, Jack H Universidade Federal de Goiás Cornell University Gioania, Brazil Ferreira, Matheus E Ithaca, United States [email protected] Instituto Nacional de Ciência e Tec- [email protected] nologia de Biologia Estrutural e Bioimagem Garbacz, Piotr Rio de Janeiro, Brazil Freire, Ronaldo C University of Warsaw [email protected] Petrobras Warsaw, Poland Rio de Janeiro, Brazil [email protected] [email protected] Ferreira, Rodrigo Alexandre Ferreira Alexandre García Mayoral, María Flor Universidade Federal de Goiás Freitas, Daniel Instituto de Química Física Roca- Goiânia , Brazil Merck Millipore solano, Consejo Superior de Investi- gaciones Científicas [email protected] Rio de Janeiro, Brazil [email protected] Madrid, Spain [email protected] Ferreira Neto, Denise C. Instituto Militar de Engenharia Freitas, Deisy S. Garro Linck, Yamila Rio de Janeiro, Brazil Universidade Federal de Mato Grosso do Sul RWTH Aachen University [email protected] Campo Grande, Brazil Aachen, Germany [email protected] [email protected] Fiat, Daniel University of Illinois at Chicago Freitas, Jair C C Garwood, Michael Chicago, United States Department of Physics, Federal Uni- University of Minnesota [email protected] versity of Espirito Santo (UFES) Minneapolis, United States Vitoria, Brazil [email protected] Fidalgo, Tatiana KS [email protected] Federal University of Rio de Janeiro Gataullin, Eduard M. Niterói, Brazil Freitas, Mônica Santos de Kazan (Volga Region) Federal Uni- tatianaksfi[email protected] Universidade Federal do Rio De versity Janeiro Kazan, Russia 166 Rwanda Figueira, Elza Cristina S Rio de Janeiro, Brazil [email protected] Instituto de Tecnologia em Imunobi- [email protected] ológicos Geraldes, Carlos F. Rio de Janeiro, Brazil Freitas-Fernandes, Liana B University of Coimbra marcela.soares@bio.fiocruz.br Federal University of Rio de Janeiro Coimbra, Portugal Rio de Janeiro, Brazil [email protected] [email protected]

176 Directory of Participants

Ghiviriga, Ion Gore, John C Gusmao, Karla A G University of Florida Vanderbilt University Universidade Federal de Minas Gainesville , United States Nashville, United States Gerais [email protected]fl.edu [email protected] Belo Horizonte, Brazil [email protected] Ghose, Ranajeet Grandinetti, Philip City University of New York Ohio State University Hallwass, Fernando New York, United States Columbus, United States Universidade Federal de Pernambuco [email protected] [email protected] Recife, Brazil [email protected] Gil, Ana M Graterol Parodi, Jusmell J University of Aveiro Han, Oc Hee Aveiro, Portugal Rio de Janeiro, Brazil Korea Basic Science Institute [email protected] [email protected] Daegu, South Korea [email protected] Gil, Roberto R. Green, Derrick Carnegie Mellon University Green Imaging Technologies Han, Songi Pittsburgh, United States Fredericton, Canada Department of Chemistry and Bio- [email protected] [email protected] chemistry, University of California Santa Barbara Santa Barbara, United States Gimbaud, Sylvain Grey, Clare P [email protected] Bruker do Brasil University of Cambridge Atibaia, Brazil Cambridge, United Kingdom Hansen, Michael Ryan [email protected] [email protected] Max-Planck-Institute for Polymer Research Giotto, Marcus Griesinger, Christian Mainz, Germany University of Connecticut Max-Planck-Institute for Biophysical [email protected] STORRS, United States Chemistry [email protected] Goettingen, Germany Härd, Torleif [email protected] Swedish University of Agricultural Gladden, Lynn F Sciences University of Cambridge Griffin, Robert G. Uppsala, Sweden Cambridge, United Kingdom Massachusetts Institute of Technol- [email protected] [email protected] ogy Cambridge, United States Hayes, Sophia E [email protected] Goldfarb, Daniella Washington University Weizmann Institute of Science Saint Louis, United States Gromov, Igor Rehovot, Israel [email protected] [email protected] Bruker do Brasil Atibaia, Brazil He, Yao [email protected] Gomes, Bruna F University of Science and Technology Universidade de São Paulo of China SÃO CARLOS, Brazil Gronenborn, Angela M. Hefei, People’s Republic of China [email protected] University of Pittsburgh School of [email protected] Medicine Pittsburgh, United States Gomes, Rosana Garrido Herrera , Ricardo [email protected] Pontifica Universidade Católica doe Halliburton Rio de Janeiro Rio de Janeiro, Brazil Grunewald, Elliot Rio de Janeiro, Brazil [email protected] [email protected] Vista Clara, Inc. Mukilteo, United States Hilty, Christian [email protected] Goncalves, Eduardo Correa Texas A&M University Universidade Federal Fluminense College Station, United States Grunin, Leonid Niteroi, Brazil [email protected] [email protected] CEO of Resonance Systems Ltd. Yoshkar-Ola, Russia 166 Rwanda Hinderberger, Dariush [email protected] González, Carlos Max Planck Institute for Polymer Instituto de Química Física Roca- Research solano - CSIC Grzesiek, Stephan Mainz, Germany Madrid, Spain University of Basel dariush.hinderberger@mpip- [email protected] Basel, Switzerland mainz.mpg.de [email protected] Gonzalez, Mariano M. HONG, Kwan Soo IBR - CONICET Guenther, Ulrich L Korea Basic Science Institute Rosario, Argentina University of Birmingham Cheongwon, South Korea [email protected] Birmingham, United Kingdom [email protected] [email protected] González-Pérez, Martha Hong, Mei Instituto de Pesquisas Tecnológicas Guittet, Eric Iowa State University São Paulo, Brazil CNRS-ICSN Ames, United States [email protected] Gif sur Yvette, France [email protected] [email protected]

177 Directory of Participants

Honorato, Hercilio D. A. Jaroniec, Christopher P Kawamori, Asako Petrobras Ohio State University AGAPE-Kabutoyama Institute of Rio de Janeiro, Brazil Columbus, United States Medicine [email protected] [email protected] Nishinomiya, Japan [email protected] Hosur, Ramakrishna V Jayaseelan, Kalai Tata Institute of Fundamental Re- EMBL-EBI Kerssebaum, Rainer search Cambridge, United Kingdom Bruker do Brasil Mumbai, India [email protected] Atibaia, Brazil [email protected] Rainer.Kerssebaum@bruker- biospin.de Jegede, Olamide M Hurlimann, Martin BG E&P Brasil Ltda Schlumberger - Doll Research Rio de Janeiro, Brazil Khetrapal, C L Cambridge, United States [email protected] Centre of Biomedical Magnetic Res- [email protected] onance Lucknow, India Jerschow, Alexej [email protected] Iglesias, María José New York University Universidad de Almería New York, United States Khramtsov, Valery V Almería, Spain [email protected] [email protected] The Ohio State University Columbus, United States Jeschke, Gunnar [email protected] Iqbal, Anwar ETH Zurich Federal University of Rio de Janeiro Zurich, Switzerland Kikuchi, Jun Rio de Janeiro, Brazil [email protected] [email protected] RIKEN Yokohama, Japan Jimenez-Barbero, Jesus [email protected] Iulianelli, Gisele Cristina Valle Centro de Investigaciones Biologicas, Universidade Federal do Rio de CSIC Janeiro MADRID, Spain Kikuchi, Marcelo M Rio de Janeiro, Brazil [email protected] Petrobras [email protected] Santos, Brazil [email protected] Jonaitis, Ivan Ivanshin, Vladimir A. Agilent Technologies Kazan (Volga Region) Federal Uni- Barueri , Brazil Killner, Mario H. M. versity [email protected] Instituto de Química Unicamp Kazan, Russia 166 Rwanda Campinas, Brazil [email protected] [email protected] Jones, Oliver A.H. RMIT University Izadi-Pruneyre, Nadia Melbourne, Australia Kim, Hyun Min Institut Pasteur [email protected] Korea Basic Science Institute Paris, France Cheongwon-Gun, Chungbook, South [email protected] Korea Júnior, Gilson S [email protected] Petrobras Jackowski, Karol Rio de Janeiro, Brazil Kim, Yongae Faculty of Chemistry, University of [email protected] Warsaw, Poland Hankuk University of Foreign Stud- Warszawa, Poland ies Junker, Jochen [email protected] Yong-In, South Korea Fundação Oswaldo Cruz [email protected] Rio de Janeiro, Brazil Jaen, Damaris Quesada junker@cdts.fiocruz.br Bruker do Brasil King, Scott P Atibaia, Brazil University of Warwick Kaminski, Danielle [email protected] Coventry, United Kingdom University of Oxford [email protected] Oxford, United Kingdom Jain, Sheetal Kumar [email protected] Aarhus University Kock, Flávio Vinicius Crizós- Aarhus, Denmark tomo Kannan, Arvind [email protected] Universidade Federal do Espirito Cambridge University Santo Cambridge, United Kingdom Conçeição da Barra, Brazil Jaremko, Lukasz [email protected] kock.fl[email protected] Max Planck Institute for Biophysical Chemistry Goettingen, Germany Kaptein, Robert Kockenberger, Walter [email protected] Utrecht University University of Nottingham Culemborg, Netherlands Nottingham, United Kingdom [email protected] Jaremko, Mariusz [email protected] Max Planck Institute for Biophysical Chemistry Karipidis, Taisiya K Koretsky, Alan P. Goettingen, Germany OAO Gazprom do Brasil NIH, NINDS, LFMI [email protected] Rio de Janeiro, Brazil Bethesda, United States [email protected] [email protected]

178 Directory of Participants

Korzhnev, Dmitry M. Lee, Hyunseung Lopes, Thierry R University of Connecticut Health Korea Basic Science Institute Universidade Federal do Espírito Center Chungcheungbuk-Do, South Korea Santo Farmington, United States [email protected] Serra, Brazil [email protected] [email protected] Lehmann-Horn, Jochen A Kovacs, Helena CSIRO López Castilla, Aracelys Bruker BioSpin Lucas Heights, Australia Universidade Federal do Rio de Fällanden, Switzerland [email protected] Janeiro [email protected] Rio de Janeiro, Brazil [email protected] Lelli, Moreno Kozlov, Guennadi CNRS/ENS Lyon/UNiversité Lyon-1 McGill University Villeurbanne, France López Ortiz, Fernando Montreal, Canada [email protected] Universidad de Almería [email protected] Almería, Spain fl[email protected] Levitt, Malcolm H Kuchel, Philip W University of Southampton, UK University of Sydney Southampton, United Kingdom Luchinat, Claudio Sydney, Australia [email protected] CERM University of Florence - Italy [email protected] Sesto Fiorentino, Italy [email protected]fi.it Li, Zhao Kukic, Predrag University of California, Los Angeles University of Cambridge, UK Los Angeles, United States Luy, Burkhard Cambridge, United Kingdom [email protected] Karlsruhe Institute of Technology [email protected] (KIT) Karlsruhe, Germany Liao, Luciano M [email protected] Kulminskaya, Natalia V Universidade Federal de Goiás Aarhus University Goiânia, Brazil Machado, Luciana ESF Aarhus, Denmark [email protected] [email protected] Universidade Federal do Rio de Janeiro Lima, Nerilson Marques Rio de Janeiro, Brazil Kupce, Eriks Núcleo de Bioensaios, Biossíntese e [email protected] Bruker Ltd. Ecofisiologia de Produtos Naturais - Coventry, United Kingdom Instituto de Química Machado, Vinicius [email protected] Araraquara, Brazil [email protected] Petrobras S.A. Rio de Janeiro, Brazil Lacerda Jr., Valdemar [email protected] Departamento de Química, Centro Lin, Yung-Ya de Ciências Exatas, Universidade University of California, Los Angeles Federal do Espírito Santo Los Angeles, United States Madhu, Perunthiruthy Vitória, Brazil [email protected] TIFR, Mumbai, India [email protected] Colaba, Mumbai, India [email protected] Lin, Zhongjie Ladizhansky, Vlad University of Warwick University of Guelph Coventry, United Kingdom Mafra, Luís Guelph, Canada [email protected] University of Aveiro (CICECO labo- [email protected] ratory) Aveiro, Portugal Lins, Fernando [email protected] Laker, Timothy J SIGMA-ALDRICH Sigma-Aldrich Sao Paulo, Brazil Magalhaes, Mariana T.Q. Miamisburg, United States [email protected] [email protected] Universidade Federal do Rio de Janeiro Linser, Rasmus J Rio de Janeiro, Brazil Lange, Adam UNSW Sydney & Harvard Medical [email protected] Max Planck Institute for Biophysical School Boston Chemistry, Goettingen Sydney, Australia Magon, Claudio J Goettingen, Germany [email protected] [email protected] Instituto de Física de São Carlos - Universidade de São Paulo Llases, María Eugenia São Carlos, Brazil Latorre, Leandro Institute of Molecular and Cell Biol- [email protected] Advanced Chemistry Development, ogy of Rosario; Max-Planck Labora- Inc. (ACD/Labs) tory of Rosario Toronto, Canada Rosario, Argentina Manrao, Suraj P [email protected] [email protected] Isotec Stable Isotope Group Old Bridge, United States [email protected] Lázaro Martínez, Juan M. Lopes, Thiago I.B CONICET & Universidad de Buenos Universidade Estadual de Campinas Aires Campinas, Brazil Marassi, Francesca M Ciudad Autónoma de Buenos Aires, [email protected] Sanford-Burnham Medical Research Argentina Institute [email protected] La Jolla, United States [email protected]

179 Directory of Participants

Marathias , Bill Millis, Kevin Morgan, Vinícius G. Agilent Technologies Cambridge Isotope Laboratories, Universidade Federal do Espírito Barueri , Brazil Inc. Santo [email protected] Andover, United States Serra, Brazil [email protected] [email protected] Marchi Netto, Antonio Rheinisch-Westfaelische Technische Miotto, Marco C Mossinger, Alan Hochschule Aachen Institute of Molecular and Cell Biol- Petrobras Aachen, Germany ogy of Rosario; Max-Planck Labora- Rio de Janeiro, Brazil [email protected] tory of Rosario [email protected] Rosario, Argentina [email protected] Marco-Rius, Irene Muhammad, Asif University of Cambridge and Cancer Universidade Federal Fluminense Research UK Miranda Filho, Fernando Luis Niterói , Brazil Cambridge, United Kingdom Instituto de Macromoléculas Profes- [email protected] [email protected] sora Eloisa Mano Rio de Janeiro, Brazil Munhoz, Victor H. O. [email protected] Marsaioli, Anita J Universidade Federal de MInas Chemistry Institute - University of Gerais Campinas Moll, Jorge Belo Horizonte, Brazil Campinas, Brazil D’Or Institute for Research and Ed- [email protected] [email protected] ucation (IDOR) Rio de Janeiro, Brazil Munte, Claudia E [email protected] Martin, Gary E. Instituto de Física de São Carlos Merck Research Laboratories São Carlos, Brazil Montenegro, André Summit, United States [email protected] [email protected] Sigma-Aldrich São Paulo, Brazil Naides, Claudio H [email protected] Martins, Gabriel R Petrobras S.A. Universidade Federal Rio de Janeiro Rio de Janeiro, Brazil Monti, Gustavo A Rio de Janeiro, Brazil [email protected] [email protected] FaMAF-UNC and IFEG-CONICET Cordoba, Argentina Naito, Akira [email protected] Medeiros, Murilo T Yokohama National University BG E&P Brasil Ltda Yokohama, Japan Montoro, Marcos A Rio de Janeiro, Brazil [email protected] [email protected] Spinlock SRL - CONICET - Univer- sidad Nacional de Córdoba Córdoba, Argentina Nantes, Iseli L. Meguellatni, Sylvain [email protected] Universidade Federal do ABC Bruker do Brasil Santo André, Brazil Atibaia, Brazil [email protected] Montrazi, Elton T Sylvain.Meguellatni@bruker- biospin.de São Carlos Institute of Physics - Uni- versity of São Paulo Nascimento, Claudia J Piracicaba, Brazil Universidade Federal do Estado Meier, Beat [email protected] do Rio de Janeiro/Universidade de ETH Zurich Brasília Zurich, Switzerland Rio de Janeiro, Brazil Moraes, Adolfo H [email protected] [email protected] Universidade Federal do Rio de Janeiro Menezes, Rachel S Rio de Janeiro, Brazil Nascimento, Karina RPR Universidade Federal do Rio de adolfofi[email protected] Petróleo Brasileiro S.A Janeiro Rio de Janeiro, Brazil Rio de Janeiro, Brazil [email protected] Moraes, Tiago Bueno [email protected] University of São Paulo São Carlos, Brazil Nascimento, Marco Antonio Menezes, Sonia Cabral de [email protected] Chaer Petrobras S.A. Universidade Federal do Rio de Rio de Janeiro, Brazil Janeiro Morcomber , Corey [email protected] Rio de Janeiro, Brazil Agilent Technologies [email protected] Barueri , Brazil Meriles, Carlos A [email protected] City College of New York - CUNY Navon, Gil New York, United States Tel Aviv University Moreira Silva, Raphaell Junnio [email protected] Tel Aviv, Israel São Carlos Institute of Physics [email protected] São Carlos, Brazil Millet, Oscar [email protected] CIC bioGUNE Neese, Frank Derio, Spain Max Planck Institute for Chemical Morgada, Marcos N [email protected] Energy Conversion Instituto de Biología Molecular y Muelheim an der Ruhr, Germany Celular de Rosario [email protected] Rosario, Argentina [email protected]

180 Directory of Participants

Netto, Paulo Oliveira, Juliana F. Pawlak, Tomasz Petrobras Laboratorio Nacional de Biocien- Centre of Molecular and Macro- Rio de Janeiro, Brazil cias/LNBio molecular Studies Polish Academy of [email protected] Campinas, Brazil Science [email protected] 90363, Poland [email protected] Nicholson, Jeremy K Imperial College London Oliveira, Luciana C London, United Kingdom Instituto de Química- Universidade Pelc, Damjan [email protected] de São Paulo University of Zagreb, Faculty of Sci- São Paulo, Brazil ence [email protected] Zagreb, Croatia Nicot, Benjamin [email protected] Schlumberger Rio de janeiro, Brazil Oliveira, Rodolfo Pellecchia, Maurizio [email protected] Schlumberger Rio de Janeiro, Brazil Sanford-Burnham Medical Research [email protected] Institute Nielsen, Niels Chr. La Jolla, United States Aarhus University, Denmark [email protected] Aarhus , Denmark Oliveira Junior, Marcos [email protected] Universidade de São Paulo São Carlos, Brazil Pereira, Fabíola M. V. [email protected] Embrapa Instrumentação Noszál, Béla São Carlos, Brazil Semmelweis Universíty [email protected] Budapest, Hungary Onofre de Lira, Tatiana [email protected] Universidade Federak de São Carlos Pereira, Natália C. univ.hu Sao Carlos, Brazil [email protected] Universidade Federal do Rio de Janeiro Nunes, Wilian S Rio de Janeiro, Brazil Opella, Stanley Mato Grosso do Sul University [email protected] Campo Grande, Brazil University of California, San Diego [email protected] La Jolla, United States Pereira, Rosana C. L. [email protected] Petrobras/CENPES Oliveira, Aline L Rio de Janeiro, Brazil Orlandi, Aristides N Universidade de Brasilia [email protected] Brasilia, Brazil Schlumberger [email protected] Rio de Janeiro, Brazil Perona Jiménez, Ricardo [email protected] Repsol Sinopec Oliveira, Caroline S Rio de Janeiro, Brazil Oschkinat, Hartmut Universidade Federal de Mato Grosso [email protected] do Sul Leibniz-Institut für Molekulare Londrina, Brazil Pharmakologie Pervushin, Konstantin [email protected] Berlin, Germany [email protected] Nanyang Technological University Singapore , Singapore Oliveira, Eduardo B [email protected] Petrobras Ott, Maria Rio de Janeiro, Brazil Martin-Luther University Halle- Pilo-Veloso, Dorila [email protected] Wittenberg Halle, Germany Universidade Federal de Minas [email protected] Gerais Oliveira, Emanuele Silva Belo Horizonte, Brazil Universidade Federal do Espírito [email protected] Santo Ozaki, Elisa Serra, Brazil Sigma-Aldrich Pinheiro-Aguiar, Ramon [email protected] São Paulo, Brazil [email protected] Federal University of Rio de Janeiro Rio de Janeiro, Brazil Oliveira, Everton L [email protected] São Carlos Institute of Physics - Uni- Paluch, Piotr versity of São Paulo Center of Molecular and Macro- Araraquara, Brazil molecular Study Polish Academy of Piovani, Mônica R [email protected] Science Shimadzu do Brasil Comercio LTDA Lodz, Poland São Paulo, Brazil [email protected] [email protected] Oliveira, Jessica M Universidade Federal do Rio de Janeiro Parella, Teodor Pires, Diego A. T. Rio de Janeiro, Brazil Universitat Autònoma de Barcelona Universidade de Brasília [email protected] Bellaterra, Spain Brasilia, Brazil [email protected] [email protected] Oliveira, Juliana Faculdades católicas Pastawski, Horacio M. Pires, Jose R Rio de Janeiro, Brazil Instituto de Física Enrique Gaviola- Universidade Federal do Rio de [email protected] Universidad Nacional de Córdota Janeiro Córdoba, Argentina Rio de Janeiro, Brazil [email protected] [email protected]

181 Directory of Participants

Pizetta, Daniel C Ramos, Bruna MC Rios, Edmilson H Instituto de Fisica de São Carlos - Laboratório Nacional de Biociências National Observatory USP Campinas, Brazil Niterói, Brazil sao carlos, Brazil [email protected] [email protected] [email protected] Ramos, Paulo Frederico de Robustelli, Paul Polinski, Ralf Oliveira Columbia University Schlumberger Baker Hughes Incorporated New York, United States Rio de Janeiro, Brazil Rio de Janeiro, Brazil [email protected] [email protected] [email protected] Rocha, Cláudia Q. Polli, Roberson S Rasia, Rodolfo M. Instituto de Quimica-Unesp- Universidade de São Paulo - Instituto Instituto de Biología Molecular y Araraquara de Física de São Carlos Celular de Rosario, Argentina Arararquara, Brazil São Carlos, Brazil Rosario, Argentina [email protected] [email protected] [email protected] Rodrigues, Tiago Brandão Pons, Miquel Redfield, Christina Cancer Reserch UK Cambridge Insti- University of Barcelona University of Oxford tute Barcelona, Spain Oxford, United Kingdom Cambridge, United Kingdom [email protected] christina.redfi[email protected] [email protected]

Poole, Katherine M Reif, Bernd Romero, Pedro A. University of Florida TU Muenchen Halliburton Gainesville, United States Garching, Germany Rio de Janeiro, Brazil [email protected]fl.edu [email protected] [email protected]

Pope, James M Reis, Alvaro Campassi Rosato, Antonio Queensland University of Technol- Petrobras University of Florence ogy, Brisbane, Australia Natal, Brazil Sesto Fiorentino, Italy Brisbane, Australia [email protected] [email protected]fi.it [email protected] Renshaw, Matthew P Ruiz, Naira M.S. Porto, Christiane F. C. University of Cambridge PUC-Rio Universidade Federal do Espírito Cambridge, United Kingdom Rio de Janeiro, Brazil Santo [email protected] [email protected] vitoria, Brazil [email protected] Resende, Jarbas M Salek, Reza Universidade Federal de Minas EMBL-EBI Prisner, Thomas Ferdinand Gerais Cambridge, United Kingdom J. W. Goethe University Belo Horizonte, Brazil [email protected] Frankfurt, Germany [email protected] [email protected] Sales, Caroline R Rey, Nicolás A. Petrobras Puglisi, Joseph D Pontifícia Universidade Católica do Rio de Janeiro, Brazil Stanford University Rio de Janeiro [email protected] Stanford, United States Rio de Janeiro, Brazil [email protected] [email protected] Sales, Edijane Matos Instituto Militar de Engenharia Pusiol, Daniel Rezende, Carlos A Rio de Janeiro, Brazil IFEG-CONICET-SPINLOCK Universidade Federal Fluminense [email protected] Córdoba, Argentina Rio de Janeiro, Brazil [email protected] [email protected] Salinas, Roberto K. Universidade de São Paulo Queiroz, Ricardo Ribeiro, Tatiana Santana Sao Paulo, Brazil BG E&P Brasil Ltda Universidade Federal de São Carlos [email protected] Rio de Janeiro, Brazil São Carlos, Brazil [email protected] [email protected] Salomoni, Roberto R Petrobras Queiroz Junior, Luiz H K Rienstra, Chad M Rio de Janeiro, Brazil Universidade Federal de Goias University of Illinois at Urbana- [email protected] Goiânia, Brazil Champaign [email protected] Urbana, United States Samouilov, Alexandre V. [email protected] The Ohio State University Wexner Quintero Escobar, Melissa Medical Center LNBIO Rigotti, Italo Columbus, United States campinas, Brazil Petrobras S.A. [email protected] [email protected] Rio de Janeiro, Brazil [email protected] San Gil, Rosane A.S. Rabbani, Said R. Universidade Federal do Rio de São Paulo University Riley, Scott A Janeiro, Instituto de Quimica São Paulo, Brazil Tecmag, Inc. Rio de Janeiro, Brazil [email protected] Houston, United States [email protected] [email protected]

182 Directory of Participants

Sanderson, Mark R Schwalbe, Harald Silva, Monica T King’s College London Goethe-University Frankfurt Petrobras London, United Kingdom Frankfurt, Germany RIO DE JANEIRO, Brazil [email protected] [email protected] [email protected]

Sant’Anna, Quézia Separovic, Frances Silva, Rodrigo de Oliveira Fundação Oswaldo Cruz University of Melbourne São Carlos Institute of Physics - Uni- Rio de Janeiro, Brazil Melbourne, Australia versity of São Paulo [email protected] [email protected] São Carlos, Brazil [email protected] Santana, Juliana S Serra, Anna Carolina Instituto de Bioquímica Médica Tedia Brazil Silva Pinto, Vinícius Cidade Universitária - Rio de Rio de Janeiro, Brazil Universidade Federal de Goiás Janeiro, Brazil [email protected] Goiânia, Brazil [email protected] [email protected] Sforça, Maurício L Santos, Bernardo C C Centro Nacional de Pesquisa em En- Silveira, Camila P Petrobras ergia e Materiais UFRJ - UNICAMP Rio de Janeiro, Brazil Campinas, Brazil Valinhos, Brazil [email protected] [email protected] [email protected]

Santos, Daniel Shekhtman, Alexander Silveira, Edilberto R. Merck Millipore State University of New York at Al- Universidade Federal do Ceará Rio de Janeiro, Brazil bany Fortaleza, Brazil [email protected] Albany, United States [email protected] [email protected] Santos, Daniel Ribas Simões, Clara Bruker do Brasil Shi, Yunyu Instituto Militar de Engenharia Atibaia, Brazil University of science and technology Rio de Janeiro, Brazil [email protected] of China [email protected] Hefei, People’s Republic of China [email protected] Santos, Talita Simões, Marcelo Luiz UFRJ (CNRMN) EMPRESA BRASILEIRA DE Rio de Janeiro, Brazil Shimada, Ichio PESQUISA AGROPECUÁRIA [email protected] The University of Tokyo São Carlos, Brazil Tokyo, Japan [email protected] [email protected] Sarzedas, Carolina G Universidade Federal do Rio de Simões, Vanessa S Janeiro Silletta, Emilia V. Schlumberger Rio de Janeiro, Brazil FAMAF – Universidad Nacional de Rio de Janeiro, Brazil [email protected] Córdoba, IFEG –CONICET [email protected] Córdoba, Argentina [email protected] Sattler, Michael Simorre, Jean-Pierre Helmholtz Zentrum München & CNRS Technische Universität München Silva, Afonso C. GRENOBLE , France Neuherberg, Germany National Institutes of Health [email protected] [email protected] Bethesda, United States [email protected] Smith, Graham M Sayegh, Raphael S R University of St Andrews University of São Paulo Silva, Geovana Vargas St Andrews, United Kingdom São Paulo, Brazil Universidade Federal do Rio de [email protected] [email protected] Janeiro Rio de Janeiro, Brazil Soares, Viviane Faria [email protected] Schefer, Alexandre Bezerra Empresa Brasileira de Pesquisa Bruker do Brasil Agropecuária Atibaia, Brazil Silva, Isabela A São Carlos, Brazil [email protected] Universidade de São Paulo - Instituto [email protected] de Física de São Carlos São Carlos - SP, Brazil Schmidt-Rohr, Klaus Soeiro, Paulo Antero Sure [email protected] Iowa State University Petrobras Ames, United States Rio de Janeiro, Brazil Silva, Marcio S. [email protected] [email protected] om.br University of São Paulo São Paulo, Brazil Schneider, Jose F. Song, Yiqiao [email protected] Universidade de São Paulo Schlumberger São Carlos, Brazil Cambridge, United States Silva, Maria Isabel Pais da [email protected] [email protected] Faculdades Católicas Rio de Janeiro, Brazil Schomburg, Benjamin Sotoma, Shingo [email protected] Max-Planck-Institute for Biophysical Kyoto university Chemistry kyoto, Japan Goettingen, Germany [email protected] [email protected]

183 Directory of Participants

Sousa, Eduardo G. R. de Sze, Kong Hung Trébosc, Julien Farmanguinhos/FIOCRUZ University of Hong Kong Unité de Catalyse et Chimie du Rio de Janeiro, Brazil Hong Kong SAR, People’s Republic Solide - Université de Lille 1 esousa@far.fiocruz.br of China Villeneuve d’Ascq, France [email protected] [email protected] Sousa Ferreira, Vinicius Universidade Federal de Goiás Takegoshi, Kiyonori Trevizan, Willian A Goiânia, Brazil Kyoto University Petrobras [email protected] Kyoto, Japan Rio de Janeiro, Brazil [email protected] [email protected] Souza, Andre A. Schlumberger Brazil Research and Tang, Huiru Tsuchida, Jefferson E Geoengineering Center Wuhan Institute of Physics and Universidade de São Rio de Janeiro, Brazil Mathematics Paulo/Universidade Federal de [email protected] Wuhan, People’s Republic of China São Carlos [email protected] São Carlos, Brazil [email protected] Souza, Anelise L Petrobras Tasic, Ljubica Rio de Janeiro, Brazil University of Campinas Turcu, Flaviu [email protected] Campinas, Brazil "Babes-Bolyai" University [email protected] Cluj-Napoca, Romania [email protected] Souza, Claudia M G de Instituto de Pesquisas Tecnológicas Täubert, Sebastian do Estado de SP Max-Planck-Institute for Biophysical Tycko, Robert São Paulo, Brazil Chemistry National Institutes of Health [email protected] Göttingen, Germany Bethesda, United States [email protected] [email protected] Souza, Isis T Universidade Federal de Alagoas Taulelle, Francis Ugurbil, Kamil Maceió, Brazil University of Versailles Saint University of Minnesota [email protected] Quentin en Yvelines Minneapolis, United States Versailles, France [email protected] [email protected] Spiess, Hans Wolfgang Max-Planck-Institute Polymer Re- Valente, Ana Paula search Mainz Tavares, Maria Ines Bruno Federal University of Rio de Janeiro Mainz, Germany IMA-UFRJ Rio de Janeiro, Brazil spiess@mpip-mainz. mpg.de Rio de Janeiro, Brazil [email protected] [email protected] Spitzer, Romana Valezi, Daniel F University of Innsbruck Teixeira, Francesco B. Universidade Estadual de Londrina Innsbruck, Brazil Instituto de Física de São Carlos - Londrina, Brazil [email protected] USP [email protected] São Carlos, Brazil [email protected] Stael, Giovanni C Veglia, Gianluigi Observatório Nacional University of Minnesota Rio de Janeiro, Brazil Tekely, Piotr Minneapolis, United States [email protected] Ecole Normale Superieure/CNRS [email protected] Paris, France [email protected] Steinbeck, Christoph Velez Jurado, Ana K EMBL-EBI Universidad del Valle Cambridge, United Kingdom Teles, Rubens Rodrigues Cali, Colombia [email protected] Universidade Federal de Pernambuco [email protected] Recife, Brazil [email protected] Stepišnik, Janez Venâncio, Tiago University of Ljubljana, FMF and In- Universidade Federal de São Carlos stitute "Jozef Stefan" Telser, Joshua São Carlos, Brazil Ljubljana, Slovenia Roosevelt University [email protected] [email protected] Chicago, United States [email protected] Viani Puglisi, Elisabetta Suter, Dieter Stanford University TU Dortmund Tochio, Hidehito Stanford, United States Dortmund, Germany Kyoto University [email protected] [email protected] Kyoto, Japan [email protected] Vicentin, Bruno L S Sychrovsky, Vladimir Universidade Estadual de Londrina Institute of Organic Chemistry and Tovar-Moll, Fernanda Freire Londrina, Brazil Biochemistry, Academy of Sciences IDOR/UFRJ [email protected] of the Czech Republic Rio de Janeiro, Brazil Prague, Czech Republic [email protected] Vieira de Luca, Pedro H [email protected] Repsol Sinopec Trail , John Rio de Janeiro, Brazil Magritek [email protected] Wellington, New Zealand [email protected]

184 Directory of Participants

Vigneron, Daniel Wheeler, Patrick University of California San Fran- Advanced Chemistry Development, cisco Inc. (ACD/Labs) San Francisco, United States Toronto, Canada [email protected] [email protected]

Vila, Alejandro J Whittaker, Sara Instituto de Biologia Molecular y The Henry Wellcome Building for Celular de Rosario Biomolecular NMR Spectroscopy, Rosario, Argentina University of Birmingham, UK [email protected] Birmingham, United Kingdom [email protected] Vilcachagua, Janaina D Instituto de Quimica - Universidade Wist, Julien de São Paulo - USP/SP NMR Lab, Universidad del Valle Sao Paulo, Brazil Cali, Colombia [email protected] [email protected]

Vivero, Santiago Domingez Wolf-Watz, Magnus MESTRELAB RESEARCH, S.L. Umeå University Santiago de Compostela, Spain Umeå, Sweden [email protected] [email protected]

Wagner, Gerhard Wrachtrup, Joerg Harvard Medical School 3rd Physical Institute, University of Boston, United States Stuttgart [email protected] Stuttgart, Germany [email protected] Walker, Frances Ann University of Arizona Wunderlich, Christoph H Tucson, United States Universität Innsbruck [email protected] Innsbruck, Austria [email protected] Wand, Joshua University of Pennsylvania Yu, Liping Philadelphia, United States University of Iowa [email protected] Iowa City, United States [email protected] Wang, Yulan Wuhan Institute of Physics and Zagdoun, Alexandre Mathematics CRMN ENS Lyon Wuhan, People’s Republic of China Villeurbanne, France [email protected] [email protected]

Warren, Warren S Zeri, Ana Carolina M Duke University Brazilian Biosciences National Labo- Durham, United States ratory [email protected] campinas, Brazil [email protected] Weber, David J. University of Maryland School of Zhou, Xin Medicine Wuhan Institute of Physics and Baltimore, United States Mathematics [email protected] Wuhan, People’s Republic of China [email protected] Wegner, Sebastian Bruker do Brasil Zinn, Fabiano Kauer Atibaia, Brazil Shimadzu do Brasil Comercio LTDA [email protected] São Paulo, Brazil [email protected] Weingarth, Markus Utrecht University De Bilt, Netherlands [email protected]

Werdan, Ciro Cordeiro Tedia Brazil Rio de Janeiro, Brazil [email protected]

185 Late Participants

Late Participants

Apparecido, Rafael do P Ivanshin, Vladimir A. UFMS Kazan Federal (Volga region) Univer- Campo Grande, Brazil sity [email protected] Kazan, Russia 166 Rwanda [email protected] Barbosa, Rodrigo Jorge V Universidade Federal do Rio de Martel, Laura B.C. Janeiro Halliburton Rio de Janeiro, Brazil Rio de Janeiro, Brazil [email protected] [email protected]

Bharatam, Jagadeesh Paiva, Fernando F Indian Institute of Chemical Tech- Universidade de São Paulo nology, Hyderabad São Carlos, Brazil Hyderabad, India [email protected] [email protected] Pestana, Fernanda Bramati, Ivanei UFRJ D’OR Institute for Research and Ed- Rio de Janeiro, Brazil ucation [email protected] Rio de Janeiro, Brazil [email protected] Quinting, Gregory R Anasazi Instruments Buckley, Michael Dean Indianapolis, United States Cryogenic - Insight Técnica [email protected] São Paulo, Brazil [email protected] Ribeiro, Abel dpUNION Corbett, Patrick WM São Paulo, Brazil Heriot-Watt University [email protected] Edinburgh, United Kingdom [email protected] Salazar , Jesus P Baker Hughes do Brasil de Moraes, Fernanda H P Rio de Janeiro, Brazil UFRJ / IDOR [email protected] Rio de Janeiro, Brazil [email protected] Teixeira, Camila CENABIO-UFRJ de Souza, Andrea S Rio de Janeiro, Brazil IDOR / UFRJ / REDE D’OR [email protected] Rio de Janeiro, Brazil [email protected] van Daalen, Rob Elsevier Gauthier, Alan Amsterdam, Netherlands BRUKER [email protected] ATIBAIA, Brazil [email protected] Vezin, Hervé CNRS LASIR UMR 8516 Université Gelev, vladimir Lille 1 FB Reagents DBA Villeneuve d’Ascq, France cambridge, United States [email protected] [email protected]

Goncalves, Eleno Paes SUPERLAB Instrumentação Analítica LTDA São Paulo, Brazil [email protected]

Hawkins, Elizabeth Springer-Verlag GmbH Heidelberg, Germany [email protected]

Holmes, Elaine Imperial College London, United Kingdom [email protected]

186