38Th FGMR Discussion Meeting of the German Magnetic Resonance Society

38th FGMR Discussion Meeting of the German Magnetic Resonance Society

Düsseldorf, Germany September 12th to 15th, 2016

Impressum Heinrich-Heine-Universität Düsseldorf Universitätsstraße 1 40225 Düsseldorf www.hhu.de

Layout Andrew Dingley Vineet Panwalkar Marianne Schulte

Front Page Manuel Etzkorn

Printed by Heinrich-Heine-Universität Düsseldorf Stabsstelle Kommunikation Druckerei Gebäude 16.11 Raum 00.75 Universitätsstr. 1 40225 Düsseldorf

General Information

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Contents

1. Sponsors and Exhibitors ...... V 2. Organization ...... VI 3. General Information ...... VIII 4. Social Events ...... XI 5. Scientific Program ...... XII 5.1 Tutorials ...... XII 5.2 Felix-Bloch Lectureship 2016 ...... XII 5.3 Ernst Awards 2016 ...... XIII 6. Program Schedule ...... XV 7. Abstracts ...... 1 7.1 Tutorials ...... 1 7.2 Felix-Block-Lectureship ...... 6 7.3 Plenary Lectures 1 ...... 8 7.4 Parallel Session 1 ...... 11 7.5 Parallel Session 2 ...... 16 7.6 Plenary Lecture 2 ...... 21 7.7 Parallel Session 3 ...... 23 7.8 Parallel Session 4 ...... 28 7.9 Plenary Lectures 3 ...... 33 7.10 Plenary Lectures 4 ...... 36 7.11 Parallel Session 5 ...... 40 7.12 Parallel Session 6 ...... 45 7.13 Parallel Session 7 ...... 50 7.15 Parallel Session 8 ...... 55 7.16 Plenary Lectures 5 ...... 60 7.17 Parallel Session 9 ...... 63 7.18 Parallel Session 10 ...... 70 7.19 Plenary Lectures 6 ...... 77 8. Poster Presentations ...... 80 9. Index of Contributors ...... 168

General Information

1. Sponsors and Exhibitors

Financial support by sponsors and exhibitors (as of 16.8.2016) is gratefully acknowledged:

• ACD Labs, Frankfurt, Germany

• Barthel HF-Technik GmbH, Aachen, Germany

• Bruker BioSpin GmbH, Rheinstetten, Germany

• Cortec Net, Voisins-Le-Bretonneux, France

• Euriso-top, SAS, Saint-Aubin Cedex, France

• JEOL (Germany), Freising, Germany

• Magic Angle GmbH & Co., Offenbach, Germany

• Magritek GmbH, Aachen, Germany

• NMR-Bio SAS, Grenoble, France

• NMR Service GmbH Erfurt, Germany

• Rototec-Spintec GmbH, Griesheim, Germany

• Sigma-Aldrich/Merck GmbH, Taufkirchen, Germany

General Information

2. Organization

Local organization Henrike Heise (Chair) Andrew Dingley Manuel Etzkorn Philipp Neudecker Astrid Wies

Contact and conference homepage [email protected] http://www.hhu.de/fgmr2016

Scientific committee Henrike Heise (Chair) Jochen Balbach Marina Bennati Bernhard Blümich Eike Brunner Manuel Etzkorn Christophe Fares Steffen Glaser Josef Granwehr Wolfgang Jahnke Bernd König Wolfgang Lubitz Burkhard Luy Philipp Neudecker Kay Saalwächter Raphael Stoll Christina M. Thiele Dieter Willbold

General Information

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Ernst Award Committee Wolfgang Jahnke (Chair) Dariush Hinderberger Alexej Jerschow Herbert Kogler Till Maurer Alfred Ross Kay Saalwaechter Angelika Sebald Heinz-Jürgen Steinhoff

General Information

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3. General Information

Venue The conference is located in the main lecture hall building of the Heinrich Heine University, Düsseldorf. The conference building has the number 23.01 (see maps for more details). The campus is easily accessible by public transport (see map for respective lines/stops) as well as by car.

General Information

Registration/Conference Office Registration will open on Monday September 12th at 12:00 in lecture hall 3A.

Opening hours conference office (lecture hall 3A): Monday, Sept. 12th: 12:00 − 18:00 Tuesday, Sept. 13th: 8:00 − 18:00 Wednesday, Sept. 14th: 8:00 − 17:30 Thursday, Sept. 15th: 8:00 − 13:00

Wireless LAN is accessible either using your eduroam account or via the conference WiFi (fgmr2016, password: 78ah?xul)

General Information

Opening hours cafeterias: Mensa (lunch): 11:30 − 14:45 Cafeteria (in lecture hall complex): 8:00 − 15:00 Cafeteria Ex Libris (library): 8:00 − 17:00

Posters All posters shall be on display during the entire conference in the foyer of the lecture hall building. Posters can be mounted from Monday (Sept 12th) and need to be removed Thursday before 13:00. Poster session 1: Tue, 13.9. 15:45 − 17:30, odd numbers presenting Poster session 2: Wed, 14.9. 15:00 − 16:30, even numbers presenting

Coffee breaks and lunch Coffee, tea and snacks will be provided during the coffee breaks, i.e. after the first morning session and during the poster sessions. During lunch breaks you can get a variety of dishes at the central cafeteria (Mensa), which is located just across the lecture hall building. Alternatively, you can get sandwiches etc. at the different cafeterias around campus and fresh pizza and pasta in the Campus Vita, which is located in the Mensa building.

General Information

4. Social Events

Welcome mixer On the evening of Monday, September 12, a welcome mixer will take place at the conference site.

Conference Dinner On the evening of Wednesday, September 14, the conference dinner will take place at the Theater der Träume, which is a completely renovated industrial hall that has maintained its historic flair, one of the nicest places in Düsseldorf. Bus shuttles will leave close to the lecture halls (see map) at 18:00 pm (please make sure that you are at the bus stop in time otherwise you will need to arrange traveling on your own). Return shuttles will depart at 23:00 and 0:00. In case you did not register for the conference dinner online, a limited amount of tickets are available at the conference office (first come first serve).

General Information

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5. Scientific Program

5.1 Tutorials These educational lectures on advanced magnetic resonance topics will take place on Monday, September 12th 2016, starting from 13:30 in the man lecture hall 3D.

Philipp Neudecker (Düsseldorf) “NMR relaxation dispersion spectroscopy for the investigation of conformational exchange in proteins”

Henrike Heise (Düsseldorf) “Solid-state NMR-Spectroscopy – Principles and Biological Applications”

Manuel Etzkorn (Düsseldorf) “Membrane systems“

Anton Savitsky (Mülheim a. d. Ruhr) “Introduction to modern EPR spectroscopy“

5.2 Felix-Bloch Lectureship 2016

Björn Corzilius

Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt am Main

“Advances in indirect and direct dynamic nuclear polarization of 13C and 15N”

General Information

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5.3 Ernst Awards 2016

Christian Hintze

Department of Chemistry, University of Konstanz

Christian Hintze, Dennis Bücker, Silvia Domingo Köhler, Gunnar Jeschke, and Malte Drescher “Laser-Induced Magnetic Dipole Spectroscopy“, J. Phys. Chem. Lett. 7 (2016) 2204−2209.

Katharina Märker

Univ. Grenoble Alpes, Grenoble, France

Katharina Märker, Morgane Pingret, Jean-Marie Mouesca, Didier Gasparutto, Sabine Hediger, and Gaël De Paëpe “A New Tool for NMR Crystallography: Complete 13C/15N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid- State NMR“, J. Am. Chem. Soc. 137 (2015) 13796−13799.

Johannes Wittmann

Physical Chemistry, ETH Zurich, Zurich, Switzerland

Johannes J. Wittmann, Valerie Mertens, Kazuyuki Takeda, Beat H. Meier, and Matthias Ernst, “Quantification and compensation of the influence of pulse transients on symmetry-based recoupling sequences”, J. Magn. Reson. 263 (2016) 7–18.

General Information

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5.4 Board & Members’ Meetings

FGMR Board Meeting

The board of the FGMR will meet on Monday, September 12th 2016 at 14:00 in room 23.21.02.21.

FGMR Members’ Meeting

The members of the FGMR will have a general assembly on Tuesday, September 13th 2016 at 19:00 in the main lecture hall 3D.

FGMR Small Molecules

People interested in joining the FG “Small Molecules” are welcome. The meeting takes place on Thursday, September 15th 2016 at 13:30 in the lecture hall 3B.

G-NMR Meeting

Members of the G-NMR consortium are also invited to their annual meeting on Thursday, September 15th at 14:30 in the lecture hall 3C.

General Information

6. Program Schedule

XVI

Monday, Sept. 12th, 2016

13:00 Registration 3A 14:00 FGMR Board Meeting Room 23.21.02.21 13:30 Tutorial Session 3D 13:30 Philipp Neudecker (Düsseldorf) NMR relaxation dispersion spectroscopy for the investigation of conformational exchange in proteins 14:10 Henrike Heise (Düsseldorf) Solid-state NMR-Spectroscopy – Principles and Biological Applications 15:00 Manuel Etzkorn (Düsseldorf) Membrane systems 15:40 Anton Savitsky (Mülheim a. d. Ruhr) Introduction to modern EPR spectroscopy 16:20 Short Break 17:00 Conference Opening 3D Henrike Heise Felix-Bloch-Lectureship Chair: Christina Thiele Björn Corzilius (Frankfurt am Main) Advances in indirect and direct dynamic nuclear polarization of 13C and 15N 18:30 Welcome Mixer Foyer

Program Schedule

XVII

Tuesday, Sept. 13th, 2016, morning

Plenary Lectures 1 3D 8:30 Ernst Awards Chair: Heinz-Jürgen Steinhoff Christian Hintze (Konstanz) Laser-Induced Magnetic Dipole Spectroscopy Katharina Märker (Grenoble) A New Tool for NMR Crystallography: Complete 13C/15N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid-State NMR Johannes Wittmann (Zürich/Frankfurt a. M.) Quantification and compensation of the influence of pulse transients on symmetry- based recoupling sequences 9:50 Arno Kentgens (Nijmegen) tba 10:30 Coffee Parallel Session 1 3D Parallel Session 2 3C Chair: Henrike Heise Chair: Burkhard Luy 11:00 Daniel Huster (Leipzig) Marcel Blommers (Novartis) NMR Investigations of the Structure and Sealed siRNAs: NMR guided design of Dynamics of Mutated Amyloid Protein novel therapeutic RNAs Fibrils 11:25 Martina Huber (Leiden) Kerstin Münnemann (Kaiserslautern) Amyloid and Intrinsically Disordered NMR hyperpolarization: Fighting the Proteins by Electron Paramagnetic lifetime issue Resonance Methods 11:50 Franziska Weirich (Düsseldorf) Klaus Woelk (Missouri) The beta-sheet Core of Recombinant High-Resolution NMR Relaxometry Fibrillar Human IAPP Includes the Central FGAILS Segment 12:10 Thomas Wiegand (Zürich) Wolfram Gronwald (Regensburg) Conformational switches of a DnaB Comprehensive metaboproteomics of helicase upon nucleotide and DNA Burkitt and diffuse large B-cell lymphoma binding studied by solid-state NMR 12:30 Lunch Break

Program Schedule

XVIII

Tuesday, Sept. 13th, 2016, afternoon

Plenary Lecture 2 3D Chair: Dieter Willbold 13:30 Stephan Grzesiek (Basel) Insights into GPCR function by solution NMR Parallel Session 3 3D Parallel Session 4 3C Chair: Dieter Willbold Chair: Kay Saalwächter 14:15 Sonja Dames (München) Jörn Schmedt auf der Günne (Siegen) Characterization of the regulation of M. Defects in inorganic solids tuberculosis protein kinase G by NMR and MD simulations 14:40 Jerôme Boisbouvier (Grenoble) Thomas Risse (Berlin) Structural and Functional Studies of EPR spectroscopy of spin labeled protein a 1 MDa Chaperonin in Action by NMR single crystals: insights into structure and dynamics 15:05 Robert Schneider (Lille) Ilya Shenderovich (Regensburg) Studying intrinsically disordered proteins Reconstructing the morphology of under true in vivo conditions by noncrystalline solids from NMR of combined cross polarization and carbonyl noncovalent interactions detection NMR 15:25 Wolfgang Hoyer (Düsseldorf) Frank Haarmann (Dresden) Beta-hairpin motifs of disease-related Local order in Intermetallics resolved by amyloidogenic intrinsically disordered NMR proteins 15:45 Poster Session 1 (+ Coffee) odd numbers presenting Plenary Lectures 3: Complementary Methods 3D Chair: Bernd König 17:30 Gunnar Schröder (Jülich) Protein Structure and Dynamics from Single-particle Cryo-EM Data 18:10 Claus Seidel (Düsseldorf) hybridFRET - deciphering biomolecular structure and dynamics 19:00 FGMR Members’ Meeting 3D

Program Schedule

XIX

Wednesday, Sept. 14th, 2016, morning

Plenary Lectures 4 3D Chair: Manuel Etzkorn 8:30 Guido Pintacuda (Lyon) Fully protonated proteins and proton-detected NMR at > 100 kHz magic-angle spinning 9:10 Enrica Bordignon (Bochum) Exploring conformational equilibria of heterodimeric ABC exporters by EPR 9:50 Volker Dötsch (Frankfurt am Main) Solution NMR studies of detergent sensitive integral membrane proteins 10:30 Coffee Parallel Session 5 3D Parallel Session 6 3C Chair: Johannes Liermann Chair: Josef Granwehr 11:00 Patrick Giraudeau (Nantes) Nicholas Cox (Mülheim a. d. Ruhr) Ultrafast 2D NMR: from reaction High-Field Pulse EPR: A New monitoring to quantitative Biophysical Toolbox for the Study of metabolomics the biological water splitting catalysis 11:25 Dariush Hinderberger (Halle) Olav Schiemann (Bonn) Elucidating the Nanoscale of Ionic EPR-based Metal Ion Trilateration Liquids with EPR spectroscopy 11:50 Christina Thiele (Darmstadt) Tufa Assafa (Bochum) New chiral alignment media based on Light-induced conformational changes polyglutamates and polyaspartates of the sensory module of phytochrome Cph2 12:10 Jens Haller (Karlsruhe) Jason W. Sidabras (Mülheim a.d. CLIP-COSY: A Clean In-Phase Ruhr) Experiment for the Rapid Acquisition Micro-Resonators for Electron of COSY-type Correlations Paramagnetic Resonance Spectroscopy of Size Limited Samples at 9.5 GHz 12:30 Lunch Break

Program Schedule

Wednesday, Sept. 14th, 2016, afternoon

Parallel Session 7 3D Parallel Session 8 3C Chair: Philipp Neudecker Chair: Bernhard Blümich 13:30 Paul Schanda (Grenoble) Peter Blümler (Mainz) Solid-state NMR studies of functional Ultra-Precise NMR-Magnetometers for dynamics in a half-megadalton enzyme High Fields complex 13:55 Katja Petzold (Stockholm) Siegfried Stapf (Ilmenau) Characterizing transient structures of Classical and dynamic phase transitions RNA using NMR of supercooled ionic liquids in bulk and in confinement 14:20 Petra Rovo (München) Jennifer Flohr (Aachen) Proton transverse relaxation as a Intra-aneurysmal flow and thrombosis sensitive reporter on microsecond motions in solid-state 14:40 Nils-Alexander Lakomek (Zürich) Ulrich Scheler (Dresden) Microsecond protein dynamics probed by Dynamics in thin polymer films and at solid-state NMR 15N R1ρ interfaces 15:00 Poster Session 2 (+ coffee) even numbers presenting Plenary Lectures 5 3D Chair: Wolfgang Lubitz 16:30 Heinz-Jürgen Steinhoff (Osnabrück) Conformational dynamics of the phototaxis membrane protein complex revealed by EPR spectroscopy 17:10 Song-I Han (Santa Barbara, CA) Dual DNP and EPR capabilities at 7 Tesla enable mechanistic studies to new applications 18:00 Conference Dinner

Program Schedule

XXI

Thursday, Sept. 15th, 2016

Parallel Session 9 3D Parallel Session 10 3C Chair: Raphael Stoll Chair: Anton Savitsky 8:30 Malene Ringkjoebing Jensen (Grenoble) Ümit Akbey (Aarhus) Protein intrinsic disorder in the MAPK cell High Field DNP enhanced NMR signalling pathways Spectroscopy: Higher efficiency, limiting factors and applications 8:55 Stefan Knauer (Bayreuth) Paul Shubhajit (Leipzig) Exploring RNA Polymerase Regulation by Cryptochrome model compound F10T: NMR Spectroscopy photo-CIDNP NMR studies on low and Earth’s magnetic field 9:15 Vineet Panwalkar (Düsseldorf) Monu Kaushik (Frankfurt a. M.) The Nedd4-1 WW domain recognizes the Dynamic nuclear polarization enhanced PY motif peptide through coupled folding solid state NMR using bis-Gd3+ polarizing and binding equilibria agents 9:35 Benesh Joseph (Frankfurt a. M.) Yury Kutin (Mülheim a. d. Ruhr) EPR Spectroscopy on Membrane Proteins EPR study of assembly mechanisms of the in Native Environments: New Mn/Fe cofactors in R2lox and R2c proteins Developments 9:55 Markus Schade (Aachen) Sigrun Rumpel (Mülheim a. d. Ruhr) NMR Fragment Screening for Challenging NMR spectroscopy as a novel tool in Targets hydrogenase research 10:15 Elisa Colas Debled (Grenoble) Müge Kasanmascheff (Göttingen/Bochum) Chaperonin stabilizes aggregation-prone Advanced EPR spectroscopy at high proteins and interferes with fibrillation of fields/frequencies sheds light on the radical amyloids transfer in E. coli ribonucleotide reductase 10:35 Coffee Plenary Lectures 6 3D Chair: Henrike Heise 11:05 Józef Lewandowski (Warwick) Structure and dynamics of protein complexes by solid-state NMR 11:45 Ruth Gschwind (Regensburg) Intermediates and Interactions in Photo- and Organocatalysis 12:25 Closing, Farewell 13:30 Meeting of the AG “Small Molecules” 3B 14:30 G-NMR Meeting 3C

Program Schedule

7. Abstracts 7.1 Tutorials

Tutorial

NMR relaxation dispersion spectroscopy for the investigation of conformational exchange in proteins P. Neudecker1,2 1Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany 2ICS-6, Forschungszentrum Jülich, 52425 Jülich, Germany E-mail: [email protected]

It is now widely accepted that the function of a protein is determined not only by its static three-dimensional structure but also by the sum of all its dynamic properties. NMR spectroscopy offers the capability of obtaining information about both, structure and dynamics on virtually all time-scales, at atomic resolution and under near-physiological solution conditions. Recent methodological advances now allow not only accurate quantification of the kinetics and thermodynamics of dynamic processes on the biochemically particularly relevant micro- to millisecond time-scale by NMR relaxation dispersion spectroscopy, but also high-resolution structure determination of the low and transiently populated states involved [1-4]. In this tutorial the principles of CPMG relaxation dispersion experiments and related techniques are introduced and the advantages as well as the experimental challenges of these techniques are illustrated based on example applications.

References [1] A. G. Palmer, Chem. Rev. 104, 3623-3640 (2004) [2] D. F. Hansen, P. Vallurupalli and L. E. Kay, J. Biomol. NMR 41, 113-120 (2008) [3] P. Neudecker, P. Lundström and L. E. Kay, Biophys. J. 96, 2045-2054 (2009) [4] P. Neudecker et al., Science 336, 362-366 (2012)

Tutorials & Awards

Tutorial

Solid-State NMR-Spectroscopy – Principles and Biological Applications H. Heise1,2 1Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany 2ICS-6, Forschungszentrum Jülich, 52425 Jülich, Germany E-mail: [email protected]

Solid-state NMR-spectroscopy is a powerful technique for obtaining structural information on the molecular level in systems which are not accessible by X-ray crystallography or solution-state NMR-spectroscopy, because they are neither crystalline nor undergoing rapid motional averaging. In the past 15 years, progress in instrumentation as well as methodology and isotope labelling strategies have led to substantial improvements, and today, solid-state- NMR-spectroscopy is readily applied to the structural investigation of complex biological systems, such as membrane proteins in lipid bilayers, large protein assemblies, amyloid fibrils, biominerals, and even full virus particles or full cells. A drastic improvement in the inherently low signal to noise ratio could be achieved by DNP, i.e. the hyperpolarization of nuclear spins by exploiting the high gyromagnetic ratio of the electron spin.

In this tutorial, I will first explain the fundamental principles of solid-state-NMR. Then I will give an overview over recent improvements and present some illustrative examples. Finally, I will give a short introduction to DNP-enhanced solid-state NMR-spectroscopy.

References [1] Müller, H.; Etzkorn, M.; Heise, H. In Modern NMR Methodology; Heise, H., Matthews, S., Eds.; Springer-Verlag Berlin: Berlin, Vol. 335, p 121. (2013).

Tutorials & Awards

Tutorial

Membrane systems M. Etzkorn1,2 1Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Germany E-mail: [email protected]

Membrane proteins belong to the most important and also most challenging systems currently studied in structural biology. Starting from the expression system used to produce larger amounts of (isotopically) labelled proteins, the choice of an adequate membrane- mimicking environment as well as the selection of the most suitable technique to obtain structural information, is of central importance. The tutorial will cover a selection of promising conventional and non-conventional approaches for NMR-based structural studies of membrane systems, including expression systems (e.g. cell-free and eukaryotic cells), membrane mimetics (e.g. nanodiscs, amphipols, whole cells), as well as suitable solution- and solid-state NMR techniques.

Tutorials & Awards

Tutorial

Introduction to modern EPR Spectroscopy

Anton Savitsky Max-Planck-Institut für Chemische Energiekonversion Stiftstr. 34-36, Mülheim and der Ruhr [email protected]

During the last two decades, the chemistry, biology and physics communities apparently witness a boost of new EPR (electron paramagnetic resonance) applications. This is largely due to technological breakthroughs in the development of pulsed microwave sources and components, fast data-acquisition instrumentation. They enable the EPR spectroscopists to introduce multiple-pulse microwave irradiation schemes, very much in analogy to what is common practice in modern NMR (nuclear magnetic resonance), and to apply advanced multifrequency EPR techniques as powerful spectroscopic tools with unique potential for the elucidation of structure and dynamics of complex systems, for example membrane proteins in biological action. In this lecture the introduction to modern EPR spectroscopy is given. Starting from the basic EPR principles the multiresonance pulsed EPR techniques are explained. The hyperfine EPR methods (Electron-Nuclear DOuble Resonance (ENDOR), ELectron-DOuble Resonance (ELDOR)-detected NMR) and electron-electron dipolar EPR techniques (Double Electron- Electron Resonance (DEER), Relaxation-Induced Dipolar Modulation Enhancement (RIDME)) are discussed in details.

Tutorials & Awards

7.2 Felix-Block-Lectureship

Tutorials & Awards

Felix-Block-Lectureship

Advances in indirect and direct dynamic nuclear polarization of 13C and 15N B. Corzilius1 1Institute of Physical and Theoretical Chemistry, Institute of Biophysical Chemistry, and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue- Str. 7-9, 60438 Frankfurt am Main, Germany. E-mail: [email protected]

Generally, paramagnetic species such as nitroxides or other persistent organic radicals provide the large electron spin polarization to be transferred to the nuclei of interest by dynamic nuclear polarization. Recently, we have introduced paramagnetic metal complexes with high-spin states, such as Gd(III) and Mn(II).[1] The narrow central electron paramagnetic resonance (EPR) transition of these half-integer spin systems allows for well-resolved solid effect DNP at high magnetic field. Here, we present that it is possible to utilize paramagnetic bis-metal complexes as polarizing agents for cross effect (CE) DNP. We provide clear evidence of CE of 13C and 15N, with NMR signal enhancement factors exceeding 100 using compounds of the type Gd-spacer-Gd with well-defined Gd-Gd distances.[2] By variation of the spacer length we investigated the Gd-Gd distance dependence of DNP enhancements. These results will allow us to design improved bis-metal compounds for efficient CE DNP and might lead to DNP applications utilizing biomolecules doubly labeled with tags based on metal complexes. Additionally, we show that selective inversion and enhancement of methyl 13C resonances occurs by heteronuclear cross-relaxation when 1H are dynamically hyperpolarized using bis- nitroxides. Potential applications for selective NMR of functional groups are discussed in regards to multi-dimensional correlation spectroscopy of large biomolecules or one- dimensional spectroscopy of complex mixtures.

References [1] B. Corzilius, A. A. Smith, A. B. Barnes, C. Luchinat, I. Bertini and R. G. Griffin, J. Am. Chem. Soc. 133, 5648 (2011) [2] M. Qi, M. Hülsmann and A. Godt, J. Org. Chem. 81, 2549 (2016)

Plenary Lectures & Parallel Sessions

7.3 Plenary Lectures 1 Ernst Awards

Plenary Lectures & Parallel Sessions

Ernst Awards

1. Christian Hintze Department of Chemistry, University of Konstanz, 78464 Konstanz, Germany E-mail: [email protected]

Christian Hintze, Dennis Bücker, Silvia Domingo Köhler, Gunnar Jeschke, and Malte Drescher „Laser-Induced Magnetic Dipole Spectroscopy“, J. Phys. Chem. Lett. 7 (2016) 2204−2209.

2. Katharina Märker Univ. Grenoble Alpes, INAC, F-38000 Grenoble, France

Katharina Märker, Morgane Pingret, Jean-Marie Mouesca, Didier Gasparutto, Sabine Hediger, and Gaël De Paëpe „A New Tool for NMR Crystallography: Complete 13C/15N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid- State NMR“, J. Am. Chem. Soc. 137 (2015) 13796−13799.

3. Johannes Wittmann Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland Current address: Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Str. 7-9, 60438 Frankfurt am Main, Germany E-mail: [email protected]

Plenary Lectures & Parallel Sessions

Plenary Lectures 1

No title available A. Kentgens1 1Radboud University Nijmegen, Institute for Molecules and Materials, Heyendaalsweg 135, 6525 AJ Nijmegen, The Netherlands. E-mail: [email protected]

No abstract available.

Plenary Lectures & Parallel Sessions

7.4 Parallel Session 1

Plenary Lectures & Parallel Sessions

Parallel Session 1

NMR Investigations of the Structure and Dynamics of Mutated Amyloid Protein Fibrils

Juliane Adler,1 Alexander Korn,1 Holger A. Scheidt,1 Anand Kant Das,2 Anoop Rawat,2 Debanjan Bhowmik,2 Sudipta Maiti,2 Perunthiruti K. Madhu,2 Daniel Huster1,2 1Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany 2Tata Institute of Fundamental Research, Department of Chemical Sciences, Homi Bhabha Road, Colaba, Mumbai 400 005, India E-mail: [email protected]

A series of peptide mutants was studied to understand the influence of local physical interactions on the fibril formation mechanism of amyloid β (Aβ) (1-40). In the peptide variants, the well-known hydrophobic contact between residues phenylalanine 19 and leucine 34 was rationally modified. In single site mutations, residue 19 was replaced by amino acids that introduce higher structural flexibility or restrict the backbone flexibility. Next, the aromatic phenylalanine was replaced by aromatic residues to probe the influence of hydrogen bond forming capacity in the fibril interior. Furthermore, charged residues were introduced to probe the influence of electrostatics. While the fibrillation kinetics and the local structure and dynamics of the peptide variants were influenced by the introduction of these local fields, the overall morphology and cross-β structure of the fibrils remained very robust against all the probed interactions. However, characteristic local structural and dynamical changes indicate that amyloid fibrils show an astonishing ability to respond to local perturbations but overall show a very homogenous mesoscopic organization. Interestingly, we find that even conservative mutations perturbing an early folding contact can drastically reduce the membrane-affinity and toxicity of Alzheimer’s Amyloid-β oligomers, without substantially affecting the end-state structure. Given such a significant biological importance of putatively minor sequence alterations, we currently focus on finding the minimally tolerated variation of the F19-L34 hydrophobic contact in Aβ(1-40).

References [1] J. Adler et al., Phys. Chem. Chem. Phys. 16, (2014) 7461 [2] A. K. Das et al., ACS Chem. Neurosci. 6, (2015) 1290

Plenary Lectures & Parallel Sessions

Parallel Session 1

Amyloid and Intrinsically Disordered Proteins by Electron Paramagnetic Resonance Methods M. Huber1 1Department of Physics, Huygens-Kammerlingh-Onnes Laboratory, Leiden University, Leiden, The Netherlands E-mail: [email protected]

Intrinsically Disordered Proteins, IDP’s, gained notoriety by being involved in diseases, mostly of the neurodegenerative kind. These proteins are highly flexible and, in contrast to regular proteins, lack a folded ground-state structure. Functional IDP's are biologically active, and operate using special mechanisms. Generally, weak interactions and perturbations can have large effects on the protein itself, and the flexibility enables these proteins to change conformation in response to changes in environment and available partners, e.g. proteins or biological membranes of different types. Understanding IDP’s is a challenge, particularly since traditional structural methods do not work well to study them. Human α-Synuclein, αS, combines both functional and pathological aspects of IDP’s, as it is a natural protein in the brain, where it associates with neuronal junctions (synapses), however, with so far unexplained function. It is also the major component of the plaques (Lewy bodies) in the brains affected by Parkinson’s disease. We show that Electron Paramagnetic Resonance can address membrane interaction and relevant fibrillar states of this protein [1,2].

References [1] Three long-range distance constraints and an approach towards a model for the α- Synuclein-fibril fold M. Hashemi Shabestari, P. Kumar, I. M.J. Segers-Nolten, M. M.A.E. Claessens, Bart D. van Rooijen, V. Subramaniam, and M. Huber Applied Magnetic Resonance 46, 369-388 (2015) [2] Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics P.Kumar, I. M.J.Segers-Nolten, N. Schilderink, V. Subramaniam, M. Huber PLOS One (2015) DOI: 10.1371/journal.pone.0142795

Plenary Lectures & Parallel Sessions

Parallel Session 1

The β-sheet Core of Recombinant Fibrillar Human Islet Amyloid Polypeptide Includes the Central FGAILS Segment Franziska Weirich[1,2], Lothar Gremer[1,2], Ewa A. Mirecka[2], Elke Reinartz[2], Wolfgang Hoyer[1,2], Henrike Heise[1,2]

1Institute of Structural Biochemistry (ICS-6), Forschungszentrum Jülich, 52425 Jülich, Germany 2Institut für Physikalische Biologie, Heinrich Heine Universität Düsseldorf, 40225 Düsseldorf, Germany E-mail: [email protected], [email protected]

Amyloid forming peptides and proteins are under intense research due to their causal relationship with some severe disorders. The 37-residue human Islet Amyloid Polypeptide (IAPP or Amylin) has a high propensity to form fibrillar aggregates with cytotoxic properties. Amyloid deposits formed from IAPP are a hallmark of type II diabetes mellitus and are known to be cytotoxic to pancreatic beta-cells [1]. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist [2-6]. We present results of solid-state NMR experiments on recombinantly expressed and uniformly 13 15 C, N-labeled human IAPPCOOH after fibrillation [7]. Complete sequential resonance assignments and resulting constraints on secondary structure are obtained. We find that the central FGAILS segment is part of the core region and forms a β-strand in our fibril preparation of IAPPCOOH. The eight N-terminal amino acid residues of IAPP, building a ring- like structure due to a disulfide bridge between cysteine residues at positions 2 and 7, appear to be well defined but with an increased degree of flexibility as compared to the β- strands.

References [1] P. Westermark, A. Andersson, G.T. Westermark, Physiol. Rev. 91 (2011) 795-826. [2] S. Bedrood, Y. Li, J.M. Isas, B.G. Hegde, U. Baxa, I.S. Haworth, R. Langen, J. Biol. Chem. 287 (2012) 5235-5241. [3] A.V. Kajava, U. Aebi, A.C. Steven, J. Mol. Biol. 348 (2005) 247-252. [4] A.T. Alexandrescu, PloS one 8 (2013) e56467. [5] J.J. Wiltzius, S.A. Sievers, M.R. Sawaya, D. Cascio, D. Popov, C. Riekel, D. Eisenberg, Protein Sci. 17 (2008) 1467-1474. [6] S. Luca, W.M. Yau, R. Leapman, R. Tycko, Biochemistry (Mosc). 46 (2007) 13505-13522. [7] E.A. Mirecka, L. Gremer, S. Schiefer, F. Oesterhelt, M. Stoldt, D. Willbold, W. Hoyer, J. Biotechnol. 191 (2014) 221-227.

Plenary Lectures & Parallel Sessions

Parallel Session 1

Conformational switches of a DnaB helicase upon nucleotide and DNA binding studied by solid-state NMR Thomas Wiegand1, Katharina Keller1, Riccardo Cadalbert1, Carole Gardiennet3, Denis Lacabanne2, Maxim Yulikov1, Laurent Terradot2, Gunnar Jeschke1, Anja Böckmann2 and Beat H. Meier1 1Laboratorium für Physikalische Chemie, ETH Zürich, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland 2IBCP BMSSI, 7 passage du Vercors, 69007 Lyon, France 3CRM2, Université de Lorraine, BP 70239, Boulevard des Aiguillettes, 54506 Vandoeuvre-lès- Nancy, France E-mail: [email protected]

DnaB helicases are ATP-driven enzymes that separate double-stranded DNA into their single- stranded analogues as a key step during the DNA replication process [1]. In this contribution, we study the helicase-nucleotide and helicase-DNA binding of the dodecameric DnaB helicase from Helicobacter pylori [2]. In a first step, the complex between DnaB, MgCl2 and the non-hydrolysable ATP-analogue Adenylyl-imidophosphate (AMP-PNP) is characterized by 2D and 3D solid-state NMR experiments. Chemical-shift perturbations (CSPs) were used to monitor the AMP-PNP binding and to identify the binding interaction interfaces, as well as to highlight a conformational switch of the helicase upon AMP-PNP binding. The substitution of diamagnetic Mg2+ ions complexed with AMP-PNP by paramagnetic ions allows to use paramagnetic NMR and EPR spectroscopy as further tools for investigating the helicase- AMP-PNP interactions. In a second step, a polythymine stretch consisting of 20 nucleic acids is added to the helicase-AMP-PNP-MgCl2 complex to mimic the binding of single-stranded DNA (ssDNA). The formed complex is studied by solid-state NMR experiments. 31P NMR experiments are performed for distinguishing between bound and unbound AMP-PNP and ssDNA.

References [1] J. H. LeBowitz, R. McMacken, J. Biol. Chem. 1986, 261, 4738-4748 [2] A. Bazin, M. V. Cherrier, I. Gutsche, J. Timmins, L. Terradot, Nucleic Acids Res. 2015, 43, 8564-8576

Plenary Lectures & Parallel Sessions

7.5 Parallel Session 2

Plenary Lectures & Parallel Sessions

Parallel Session 2

Sealed siRNAs: NMR guided design of novel therapeutic RNAs M. Blommers Novartis Institutes for BioMedical Research, Switzerland E-mail: [email protected] siRNAs are 21-nucleotide duplex oligonucleotides that down regulate complementary target mRNA. For their potential use as therapeutics, the pharmacological properties of siRNAs need to be improved, both by chemical modification of the siRNAs themselves and by proper formulation. siRNAs lead to target mRNA cleavage by association of one RNA strand with the Argonaute-2 protein (Ago-2) in the silencing complex (RISC). The ends of the guide strand are recognized by distinct domains of Ago-2: the two 3’-terminal nucleotides are associated in a pocket of the PAZ domain. In order to improve siRNA stability as well as to prevent immune stimulation, we explored the possibility of substituting the 3’-terminal dinucleotide by a mimic that still interacts with the PAZ domain. A fragment library was designed using the crystal structure of the PAZ domain, and the fragments were screened by NMR for binding to the PAZ domain. Novel PAZ domain ligands could be identified this way. From the best ligands, building blocks for RNA synthesis were derived and the corresponding conjugates were prepared. The most potent RNA-conjugates display enhanced chemical stability, without compromising PAZ domain binding. The structure and dynamics of the oligonucleotide-conjugates were characterized by NMR. These activities resulted in novel therapeutic molecules, sealed siRNAs, that have significantly enhanced potency and duration of action as demonstrated by in vitro and in vivo functional assays.

Plenary Lectures & Parallel Sessions

Parallel Session 2

NMR hyperpolarization: Fighting the lifetime issue K. Münnemann1 1Laboratory of Engineering Thermodynamics (LTD), University of Kaiserslautern, Germany E-mail: [email protected]

Despite its wide applicability in natural sciences and medicine, Nuclear Magnetic Resonance (NMR) still suffers from its inherently low sensitivity. This problem can be addressed by hyperpolarization techniques, such as Dynamic Nuclear Polarization (DNP) and Parahydrogen Induced Polarization (PHIP). Exploiting the large signal enhancements associated with these techniques, however, NMR or MRI qualify for monitoring dynamic processes in real time. Each hyperpolarization technique has important applications, but several general problems remain. One severe limitation is the limited lifetime of the hyperpolarized state caused by T1 relaxation. In liquids efficient relaxation processes restrict the hyperpolarization to last from typically seconds to at best a few minutes. This drawback can be partially overcome by storing the fast decaying hyperpolarization in slowly relaxing singlet states. Another shortcoming inherent to all hyperpolarization techniques is the partial destruction of the hyperpolarization by the application of RF-pulses which renders the usage of complex pulse sequences for multi-dimensional NMR experiments difficult. An adequate concept to avoid these severe limitations is to use the hyperpolarization techniques in a continuous flow mode providing a continuous supply of hyperpolarized molecules. In this contribution the continuous production of hyperpolarized liquids using two different polarization methods (PHIP and Overhauser DNP) will be demonstrated.

Plenary Lectures & Parallel Sessions

Parallel Session 2

High-Resolution NMR Relaxometry K. Woelk1 and R. J. Klingler2 1 Missouri S&T, Department of Chemistry, 400 West 11th Street, Rolla, Missouri 65409-0010, USA 2 Argonne National Laboratory, Chemistry Division, 9700 South Cass Avenue, Argonne, Illinois 60439, USA E-mail: [email protected]

NMR relaxometry is commonly used to probe into mobility and diffusivity of and around NMR-active nuclei, and to gain information about porosity and pore size distribution of permeable materials such as clay and shale samples. When time-dependent relaxation data are recorded from samples with distributions of relaxation time constants, the mathematical reconstruction of the distribution is understood as an ill-posed discrete inverse Laplace transformation. However, standard computations utilize numerical non-negative least- squares (NNLS) procedures with stabilizing functions or regularizing operators.1 Depending on the specific problem, regularizing functions may rank from zeroth to fourth order in the coefficient distribution. Because regularizations prevent the detection of sharp features in a coefficient distribution, a revised computational approach was developed that doesn’t rely on stabilizing functions or regularizing operators but faithfully reproduces even delta-function features from synthetic data (with or without random noise added).

Pressure-dependent T1 data of methane in the bulk gas phase and in pores of clay materials were recorded using a high-pressure toroid cavity probe.2 Because the revised computation approach is most successful when data are collected decaying toward zero, a two-scan pulse experiment with phase-cycled split inversion pulse was utilized. The sequence also suppresses residual probe ring-down of the toroid-cavity metal pressure vessel probe.

References [1] J. Mitchell, T.C. Chandrasekera, L.F. Gladen, Prog. Nucl. Magn. Res. 62, 34-50 (2012). [2] H. G. Niessen, P. Trautner, R. Backhausen, K. Woelk, Concepts Magn. Reson. 16B, 15-21 (2003)

Plenary Lectures & Parallel Sessions

Parallel Session 2

Comprehensive metaboproteomics of Burkitt and diffuse large B- cell lymphoma

Philipp Schwarzfischer1, Jörg Reinders1, Katja Dettmer1, Karsten Kleo2, Lora Dimitrova2, Michael Hummel2, Maren Feist3, Dieter Kube3, Monika Szczepanowski4, Wolfram Klapper4, Franziska Taruttis1, Julia C. Engelmann1, Rainer Spang1, Peter J. Oefner1, Wolfram Gronwald1,*

1University of Regensburg, Institute of Functional Genomics, Regensburg, Germany 2Charité-Universitätsmedizin Berlin, Institute of Pathology, Berlin, Germany 3University Medical Center Göttingen, Department of Hematology and Oncology, Göttingen, Germany 4Institute of Hematopathology, University Hospital Schleswig-Holstein Campus Kiel/Christian- Albrechts University Kiel, Germany *[email protected]

Burkitt’s lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are biologically, clinically and pathologically distinct subtypes of aggressive B-cell lymphoma. Combining NMR and mass spectrometry based metabolomics, proteomics and transcriptome analysis we investigated these lymphoma types using cell lines, as well as both cryopreserved and formalin-fixed paraffin-embedded (FFPE) human tissue sections and give a comprehensive overview of their differences [1]. Strikingly, metabolomics analyses revealed that DLBCL cells and cryopreserved tissues produced and secreted significantly more pyruvic acid than BLs. Subsequent proteome analysis using nanoLC-SWATH-MS allowed the quantitative determination of 3041, 2939, and 1442 proteins in cell culture, in cryopreserved and FFPE tissue sections, respectively. Key metabolic pathways like glycolysis and one carbon metabolism showed the same consistent regulation in both cell lines and primary tumor specimens. In conclusion, differences in metabolism and protein expression between BL and DLBCL were highly preserved in cultured cells and tissue sections. These results clearly demonstrate the advantage of combining an array of experimental techniques and sources of specimens to achieve a comprehensive overview of the distinct subtypes of aggressive B- cell lymphoma.

References [1] Schwarzfischer et al., (2016), submitted

Plenary Lectures & Parallel Sessions

7.6 Plenary Lecture 2

Plenary Lectures & Parallel Sessions

Plenary Lecture 2

Insights into GPCR function by solution NMR 1 1 1 1 2 2 2 S. Grzesiek , S. Isogai , C. Opitz , A. Grahl , X. Deupi , G. Schertler , D. Veprintsev 1Biozentrum, University of Basel, CH-4056 Basel, Switzerland 2Paul Scherrer Institute, CH-5232 Villigen, Switzerland E-mail: [email protected]

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signaling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood due to missing dynamical information. We have recently shown [1] that receptor motions can be followed at virtually any backbone site in a 15 thermostabilized mutant of the turkey 1-adrenergic receptor ( 1AR). Labeling with N- valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor including the extracellular ligand entrance pocket. Time permitting we will also discuss recently developed strategies for economic isotope labeling in insect cells based on isotope-labeled yeast extracts [2].

References [1] Isogai, S.; Deupi, X.; Opitz, C.; Heydenreich, F. M.; Tsai, C.-J.; Brueckner, F.; Schertler, G. F. X.; Veprintsev, D. B.; Grzesiek, S. Nature 530, 237 (2016) [2] Opitz, C.; Isogai, S.; Grzesiek, S. J. Biomol. NMR 62, 373 (2015)

Plenary Lectures & Parallel Sessions

7.7 Parallel Session 3

Plenary Lectures & Parallel Sessions

Parallel Session 3

Characterization of the regulation of M. tuberculosis protein kinase G by redox changes, phoshphorylation, and membrane binding by NMR and MD simulations M. Wittwer1a Q. Luo1b V. R. I. Kaila1b and S. A. Dames1a,2 1Technische Universität München, Department of Chemistry, aBiomolecular NMR b Spectroscopy/ Computational Biocatalysis, Lichtenbergstr. 4, 85747 Garching, Germany 2Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany E-mail: [email protected]

Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). The N-terminal ∼75 residues were predicted to show no regulatory secondary structure (NORS) and harbor the only site (T63) phosphorylated in vivo. The following rubredoxin-like metal-binding motif (RD, ∼74–147) makes tight interactions with catalytic domain (∼148–420), which is C-terminally flanked by a tetratricopeptide repeat domain (TPRD). Initially, we prepared new expression plasmids for the N-terminal regions by a mutagenesis approach [1] and assigned the 1H, 15N and 13C nuclei [2] to characterize the backbone dynamics and structural properties of the NORS and of the RD in the reduced, metal bound and the oxidized, metal free forms by NMR and MD simulations. Complementary we monitored PknG kinase activity. The NORS region is as predicted natively disordered and the target of autophosphorylation, which does not induce folding or interactions with the RD. Our data further suggest that oxidation induced unfolding of the RD regulates substrate access to the catalytic domain and thereby PknG function under different redox conditions, e.g. increased levels of reactive oxidative species (ROS) in host macrophages. The RD can further interact with membrane mimetic micelles and bicelles, which may play a role for the observed membrane localization.

References [1] M. Wittwer, S. A. Dames, J. Expr. Purif. 111, (2015) [2] M. Wittwer, S. A. Dames, Biomol. NMR Assign. in press, (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 3

Structural and Functional Studies of a 1 MDa Chaperonin in Action by NMR

G. Mas, P. Macek, J.-Y. Guan, E. Colas-Debled, E. Crublet, G. Schoehn, P. Gans, P. Schanda, J. Boisbouvier Institute de Biologie Structurale CEA/CNRS/UGA – Grenoble -France E-mail: [email protected]

NMR spectroscopy offers a near unique ability to monitor structural and dynamic changes in real-time and at atomic resolution. Historically, the application of real-time 2D NMR techniques has been limited to the study of small proteins on the second to minute timescale. We have established that, with a combination of specific isotope labeling schemes of methyl groups [1-2] and optimized NMR spectroscopy [3], similar real-time NMR studies can also be applied to study the self-assembly process of large macromolecular assemblies [4]. In this presentation, I will show that a combination of methyl specific labeling approaches and optimized NMR spectroscopy, can be used to probe different functional states and the refolding cycle of a 1 MDa active chaperonin. To decipher this mechanism, we have reconstituted the functional assembly specifically labeled on Methionine and Valine methyl groups [5]. The methyl group frequencies have been assigned using an efficient approach based on mutagenesis [6-7], and used to probe the chaperonin structure allowing the identification of NMR spectra corresponding to the intermediate states and the active species of the chaperonin functional cycle. NMR allowed us to investigate the structural rearrangement corresponding to the different states during the functional cycle of this large biological machinery and to characterize the interaction between the chaperonin and an unfolded substrate protein. Using a clever combination of different NMR approaches, it has been possible to follow the refolding of the unfolded protein by the chaperonin and the effects of the unfolded protein on the functional cycle of the chaperonin in action.

References [1] Gans et al. Angewandte Chemie International Edition 49, 1958-1862 (2010). [2] Kerfah et al. Current Opinion in Structural Biology 32, 113-122 (2015). [3] Amero et al. Journal of the American Chemical Society 131, 3448-9 (2009). [4] Macek et al. (submitted). [5] Mas et al. Journal of Biomolecular NMR 57, 251-262 (2013). [6] Amero et al. Journal of Biomolecular NMR 50, 229-236 (2011). [7] Crublet et al. Methods in Molecular Biology 1091, 229-244 (2014).

Plenary Lectures & Parallel Sessions

Parallel Session 3

Studying intrinsically disordered proteins under true in vivo conditions by combined cross polarization and carbonyl detection NMR Juan Lopez1,2, Robert Schneider1, François-Xavier Cantrelle1, Isabelle Huvent1, and Guy Lippens1,3 1NMR & Molecular Interactions, UMR 8576 UGSF, CNRS / Université Lille, Lille, France 2Departamento de Ciencias-Quimica, Pontificia Universidad Catolica del Peru, Lima, Peru 3Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France E-mail: [email protected]

Under physiological conditions, studies of intrinsically disordered proteins (IDPs) by conventional proton-detected NMR methods are severely limited by fast amide proton exchange with water. 13C detection has been proposed as a solution to the exchange problem, but is hampered by low sensitivity. We propose a new pulse sequence combining proton-nitrogen cross polarization and carbonyl detection to record high-resolution, high- sensitivity NMR spectra of IDPs at physiological conditions. We demonstrate the efficacy of this approach by recording a high-quality N-CO spectrum of alpha-synuclein in bacterial cells at 37°C.

Plenary Lectures & Parallel Sessions

Parallel Session 3

β-Hairpin motifs of disease-related amyloidogenic intrinsically disordered proteins H. Shaykhalishahi1, Ewa A. Mirecka1, Sophie Feuerstein2, Michael M. Wördehoff1, Lothar Gremer1,2, Gunnar F. Schröder1,2, Torleif Härd3, Matthias Stoldt1,2, Dieter Willbold1,2 and Wolfgang Hoyer1,2 1Institute of Physical Biology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany 2Institute of Complex Systems (ICS-6), Research Centre Jülich, 52425 Jülich, Germany 3Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences (SLU), 750 07 Uppsala, Sweden E-mail: [email protected]

The aggregation and toxicity of amyloidogenic IDPs are fundamental to the pathogenesis of several human degenerative diseases. The conformational ensembles of the IDPs contain substantial fractions of conformers that exhibit long-range intramolecular interactions. Applying NMR spectroscopy, we have identified β-hairpin conformations of islet amyloid polypeptide (IAPP), α-synuclein, and amyloid-β peptide (Aβ) in complex with engineered binding proteins [1,2]. Here we present the structure of IAPP bound to the β-wrapin (β-wrap protein) HI18 and investigate the substoichiometric inhibition of IAPP aggregation entailed by HI18. We furthermore highlight the important role of the β-hairpin formed in the sequence region 35-59 of α-synuclein, which contains the β-strand segments β1 and β2 of amyloid fibril models and most disease-related mutations. We show by disulfide engineering, biophysical techniques, and cell viability assays, that intramolecular tertiary interactions between the β1 and β2 segments of α-synuclein interfere with its aggregation, and moreover inhibit aggregation of IAPP and Aβ [3]. Our results reveal a common preference of different amyloidogenic proteins for formation of β-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. References [1] E. Mirecka et al., Angew. Chem. Int. Ed. Journal. 53, (2014) [2] H. Shaykhalishahi et al., ChemBioChem 16, (2015) [3] H. Shaykhalishahi et al., Angew. Chem. Int. Ed. 54, (2015)

Plenary Lectures & Parallel Sessions

7.8 Parallel Session 4

Plenary Lectures & Parallel Sessions

Parallel Session 4

Defects in inorganic solids J. Schmedt auf der Günne 1, Wenyu Li, J. Weber, W. Sauskojus, R. Trettin, 1Department of Chemistry and Biology, Siegen University, Siegen Germany

E-mail: [email protected]

Point defects are responsible for function in many crystalline materials, like light-conversion or charge transport in ionic conductors. We show how defects can be identified and their doping homogeneity can be judged from magnetic resonance experiments. Depending on the nature of dopants being paramagnetic or diamagnetic different strategies are available to study the defect structure by magnetic resonance. On the example of TiO2:F, ZnO:Al,

SnO2:F we demonstrate the potential of defect identification by a combination of quantum chemical calculations and NMR. The importance of using different techniques like XRD, NMR and ESR is shown on TiO2:F which depending on the chemical treatment transforms its defect structure, which explains the differing results which had been found about its photochemical activity. An emphasis of the presentation will be how doping homogeneity [1] of paramagnetically doped host structures can be judged from quantitative NMR experiments.

References [1] W. Li, V. R. Celinski, J. Weber, N. Kunkel, H. Kohlmann, J. Schmedt auf der Günne, Phys. Chem. Chem. Phys. 18 (2016), 9752.

Plenary Lectures & Parallel Sessions

Parallel Session 4

EPR spectroscopy of spin labeled protein single crystals: insights into structure and dynamics T. Risse1 1 Institute of Chemistry and Biochemistry, Physical Chemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany E-mail: [email protected]

The line shape of CW EPR spectra of spin labeled proteins has been extensively used to elucidate structural and dynamic information such local dynamics, backbone fluctuations and structural rearrangements. A detailed analysis of the CW line shape is intricate, because of a lack of sufficient spectral information. In this contribution spin labeled T4 lysozyme single crystals will be used as an exploratory example to show how EPR spectroscopy in combination with high resolution X-ray crystallography and quantum chemical calculations can be used to acquire additional spectral information, which can be used to gain insight not only into the dynamics of the protein, but also into the internal dynamics of the spin label itself. We will highlight some of the possible insights by discussing a reversible conformational exchange process in T4 lysozyme both in terms of the structural properties of the states as well as their thermodynamic properties. To this end it turns out that an understanding of the internal structure and dynamics of the spin label plays an important role for the understanding of the observed phenomena. The latter aspects will be discussed in more detail as well.

Plenary Lectures & Parallel Sessions

Parallel Session 4

Reconstructing the morphology of noncrystalline solids from NMR of noncovalent interactions I.G. Shenderovich1 1Faculty of Chemistry and Pharmacy, University of Regensburg, Regensburg, Germany E-mail: [email protected]

The mechanical and chemical properties of solids depend on their morphology. There are many methods that can provide the composition of very complex materials. There is a lack of methods suitable to study intermolecular interactions even in simple noncrystalline solids. Magnetic resonance spectroscopy (NMR) is the method of choice for such applications. The only critical requirement of NMR is the presence in the system of interest of spin-labels that will be suited to the task. This presentation will cover the following aspects: (i) what nuclei can serve as the suitable spin-labels; (ii) the effect of noncovalent interactions on NMR parameters [1,2]; (iii) what one can learn about the morphology of surfaces using solid-state NMR [3]; (iv) time-efficient DFT calculations as a complement to solid-state NMR [2,4]; (v) reconstruction of the morphology of noncrystalline organic solids from NMR parameters.

References [1] P. Lorente, I.G. Shenderovich, N.S. Golubev, G.S. Denisov, G. Buntkowsky G., H.-H. Limbach. Magn. Reson. Chem. 39, (2001) [2] I.G. Shenderovich, J. Phys. Chem. C. 117, (2013) [3] I.G. Shenderovich, G. Buntkowsky, A. Schreiber, E. Gedat, S. Sharif, J. Albrecht, N.S. Golubev, G.H. Findenegg, H.-H. Limbach. J. Phys. Chem. B. 107, (2003) [4] G.U. Begimova, E.Yu. Tupikina, V.K. Yu, G.S. Denisov, M. Bodensteiner, I.G. Shenderovich, J. Phys. Chem. C. 120, (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 4

Local order in Intermetallics resolved by NMR F. Haarmann1,2 1 Max-Planck-Institut für Chemische Physik fester Stoffe, 01187 Dresden, Germany 2 Institute of Inorganic Chemistry, RWTH Aachen University, 52074 Aachen, Germany E-mail: [email protected]

Intermetallic compounds are a fascinating class of materials with respect to structural chemistry and technological applications. Our interest focuses on chemical bonding and local ordering of the atoms. A strategy to investigate local order in intermetallics combines NMR, quantum mechanical (QM) calculations, and diffraction techniques [1,2]. The quasi-binary section of the intermetallic phases MAl4 and MGa4 with M = Sr, Ba was characterized by means of X-ray diffraction, NMR, and QM calculations. The compounds form a solid solution M(Al1-xGax)4 with M = Sr, Ba in the BaAl4 type structure with two crystallographic positions for the triel elements, X(4b) and X(5b). Two types of local ordering of the Al atoms are evident from 27Al NMR experiments: First a preferred occupation of the 4b sites by Al and second a formation of Al pairs with small interatomic distances. This model is supported by QM efforts. Super lattice structures were derived to model the varying local atomic arrangements within periodic boundary conditions. These were used to probe the electric field gradient (EFG), the formation energies, and lattice parameter being in good agreement with the experiments. References [1] F. Haarmann, In R. K. Harris, R. E. Wasylishen, Editors, Enzyclopedia of Magnetic Resonance, John Wiley & Sons, Ltd, Chichester, (2011). [2] O. Pecher, B. Mausolf, K. Lamberts, D. Oligschläger, C. Niewieszol, U. Englert, F. Haarmann, Chem. Eur. J. 21(40), 13971-13982, (2015).

Plenary Lectures & Parallel Sessions

7.9 Plenary Lectures 3

Plenary Lectures & Parallel Sessions

Plenary Lectures 3

Protein Structure and Dynamics from Single-particle Cryo-EM Data G. Schröder1 1Institut für Physikalische Biologie2ICS-6, Forschungszentrum Jülich, 52425 Jülich, Germany E-mail: [email protected]

One of the biggest challenges in the analysis of cryo-EM images is the heterogeneity and flexibility of the molecules, which on the one hand severely limits the achievable resolution but on the other hand yields valuable information on conformational dynamics. A computational approach will be presented to reduce the conformational variance of a set of single-particle images with the goal of increasing the resolution. We reconstruct conformational variance of a molecule from the variance of the density and use this information for image classification. In a next step, the individual 3D density reconstructions from all classes are recombined into one single (higher resolution) reconstruction by a novel elastic map alignment procedure. The goal is to improve the resolution and at the same time to gain a complete picture of the conformational variance of a macromolecule.

Plenary Lectures & Parallel Sessions

Plenary Lectures 3 hybridFRET - deciphering biomolecular structure and dynamics C. A. M. Seidel1 1 Chair of Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Germany E-mail: [email protected]

The recent advances in fluorescence spectroscopy and microscopy as well as in multi-scale modelling of complex biochemical systems set the stage to apply an integrated approach for achieving ultimate resolution in space (sub-nanometer) and time (picoseconds) for characterizing the structure and dynamics of proteins and their complexes. Considering the huge success of NMR spectroscopy in describing dynamic biomolecules, NMR determines a number of parameters which are also accessible to fluorescence techniques such as rotational diffusion times and local order parameters. Moreover, a number of parameters and processes exist for both spectroscopies, which have a similar information type, if the different distance dependencies around the probe (nucleus or dye label) are taken into account. Illustrative examples for NMR-fluorescence analogies are: (1) dipolar coupling (NOE - FRET), (2) conformational dynamics in the ms time scale (Relaxation dispersion experiments - dynamic Photon Distribution Analysis [1], (3) unique information on the local probe environment (chemical shift - fluorescence quenching). I will present hybrid studies using NMR-, EPR-, SAXS- and high-precision FRET- measurements [2,3] combined with molecular dynamics and coarse grained simulations for two prototypic proteins differing in size and number of domains - the small 14 kD protein GABARAP with one and the large 65kD human Guanylate Binding Proteins with three domains.

[1] S. Kalinin, et al., J. Phys. Chem. B. 114, 7983–7995 (2010) [2] E. Sisamakis, Methods Enzymol. 475, 455 (2010) [3] S. Kalinin, Nat. Methods 9, 1218-1225 (2012)

Plenary Lectures & Parallel Sessions

7.10 Plenary Lectures 4

Plenary Lectures & Parallel Sessions

Plenary Lectures 4

Fully protonated proteins and proton-detected NMR at > 100 kHz magic-angle spinning G. Pintacuda1 1Institute of Analytical Sciences (CNRS / ENS Lyon / Université Claude Bernard Lyon 1), Villeurbanne, France E-mail: [email protected]

Protein structure determination by proton detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side-chains is extremely limited. Here we show in fully protonated proteins that at 100 kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1H-1H proximities that define a protein structure. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes and improved magnetization transfer offset the reduced sample size at 100 kHz spinning and above. Less than two weeks of experiment time and a single 0.5 mg sample is sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and structure determination. These findings increase the impact of solid-state NMR to samples that cannot easily be deuterated, and for samples that can only be produced in sub milligram quantity, and we show examples of a microcrystalline protein, a protein dimer in an intact viral capsid assembly, and a membrane protein in lipid bilayers. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of samples of high biological relevance.

References [1] L. B. Andreas, K. Jaudzems, J. Stanek, D. Lalli, A. Bertarello, T. Le Marchand, D. Cala-De Paepe, S. Kotelovica, I. Akopjana, B. Knott, S. Wegner, F. Engelke, A. Lesage, L. Emsley, K. Tars, T. Herrmann, and G. Pintacuda, Proc. Natl. Acad. Sci. USA, in press (2016)

Plenary Lectures & Parallel Sessions

Plenary Lectures 4

Exploring conformational equilibria of heterodimeric ABC exporters by EPR E. Bordignon Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum, Universitätsstr. 150, 44801 Bochum, Germany E-mail:[email protected]

ATP binding cassette (ABC) exporters are found in all phyla of life and play a key role in the transport of a wide variety of molecules across cell membranes. Around half of the forty human ABC exporters are heterodimers encompassing two functionally non-equivalent ATP binding sites, which includes transporters of major clinical importance such as CFTR, SUR1 and TAP1/2. The field agrees that a typical ABC exporter adopts two principal states, namely an inward-facing (IF) state with NBDs fully or partially separated and an outward-facing (OF) state with nucleotides bound at the closed NBD dimer interface, which is coupled to substrate extrusion at the transmembrane domains (TMDs). Yet, the molecular events leading from an open to a closed NBD dimer having two nucleotides sandwiched at the interface (the so called power stroke event) is still under debate. Here we present a comprehensive Double Electron-Electron Resonance study on two heterodimeric exporters, namely TM287/288 [1,2] and BmrCD as well as on the homodimeric MsbA. The response of the transporters during turnover conditions and upon binding of different nucleotides and nucleotide analogues highlight novel mechanistic features related to the energy landscape of TM387/288. The model presented is based on the catalytic cycle of the wildtype transporters and it is corroborated by an analysis of catalytically inactive mutants. Our results call a recent study on the heterodimeric ABC exporter BmrCD into question [3], which claimed that transition of the TMDs from an IF to an OF conformation is driven exclusively by ATP hydrolysis in heterodimeric ABC exporters. The EPR data obtained allow to build a unified mechanistic model, which reconciles all data available for heterodimeric exporters and highlights differences with respect to the homodimeric counterparts [4].

References [1] M. Hohl et al., Nat Struct Mol Biol. 19(4):395 (2012) [2] M. Hohl et al., Proc Natl Acad Sci. 111(30):11025 (2014) [3] S. Mishra et al., eLife 16;3:e02740 (2014) [3] M.H. Timachi, C.A. Hutter, M. Hohl, T. Assafa, S. Boehm, A. Mittal, M.A. Seeger and E. Bordignon, submitted.

Plenary Lectures & Parallel Sessions

Plenary Lectures 4

From nanodiscs to isotropic bicelles: a procedure for solution NMR studies of detergent sensitive integral membrane proteins

Aisha Laguerre, Frank Löhr, Erik Henrich, Christopher Hein, Beate Hoffman, Frank Bernhard and Volker Dötsch Institute of Biophysical Chemistry, Goethe University, Max-von-Laue Str. 9, 60428 Frankfurt, Germany E-mail: [email protected]

A significant challenge for the use of liquid state NMR spectroscopy for the structure determination of membrane proteins is sample preparation of the membrane proteins in environments that are suitable for investigation by solution NMR and keep the protein in a functional state. Detergents have long been the mainstay of researchers but often to the detriment of protein function. Recent work has seen an expansion in the number of membrane mimetics such as the nanodiscs or bicelles. The large size of nanodiscs, however, hinders their broad application in solution NMR characterization of membrane proteins with integral membrane proteins resulting in broad and overlapping signals. We have developed a detergent titration method that significantly enhances the spectral quality combined with a combinatorial labeling strategy that facilitates the backbone assignment of the integral membrane protein lipoprotein signal peptidase II. Our method thereby facilitates NMR investigations while preserving the central benefit of nanodisc technology i.e. the presence of a lipid core around the MP necessary for function.

Plenary Lectures & Parallel Sessions

7.11 Parallel Session 5

Plenary Lectures & Parallel Sessions

Parallel Session 5

Ultrafast 2D NMR: from reaction monitoring to quantitative metabolomics Patrick GIRAUDEAU1,2 1CEISAM, Université de Nantes, Nantes, France 2Institut Universitaire de France, Paris, France E-mail: [email protected]

Ultrafast (UF) multidimensional NMR spectroscopy –a generic approach that makes it possible to record nD spectra in a single scan[1]– has recently evolved into a powerful analytical tool. [2] UF NMR is particularly useful in situations where the experiment duration is critical such as reaction monitoring or the coupling with other techniques, but it is also recognized for its high precision in quantitative analysis. [3] Hybrid multi-scan techniques based on UF 2D NMR also offer an interesting alternative to conventional acquisitions while being more sensitive than pure single-scan experiments. [4] We will describe some of the most recent applications of UF 2D NMR in various fields of analytical chemistry: the real- time monitoring of electrochemical reactions at high field, [5] as well as the first implementation of UF 2D NMR on a benchtop spectrometer to monitor an organic synthesis in line. [6] Then, we will highlight the potential of UF 2D NMR for high-throughput quantitative metabolomics of biological samples. [7] Finally, we will discuss the metabolomics perspectives arising from the first hyperpolarized UF 2D experiments on biological extracts, relying on dissolution DNP assisted by cross-polarization. [8]

References [1] L. Frydman, T. Scherf, A. Lupulescu, Prod. Natl. Acad. Sci. USA 99, (Year), 15858. [2] P. Giraudeau, L. Frydman, Annu. Rev. Anal. Chem. 7, (2014), 129. [3] A. Le Guennec, I. Tea, I. Antheaume, E. Martineau, B. Charrier, M. Pathan, S. Akoka, P. Giraudeau, Anal. Chem. 84, (2012), 10831. [4] S. Akoka, P. Giraudeau, Magn. Reson. Chem. 53 (2015), 986. [5] R. Boisseau, U. Bussy, P. Giraudeau, M. Boujtita, Anal. Chem. 87, (2015), 372. [6] B. Gouilleux, B. Charrier, S. Akoka, F.-X. Felpin, M. Rodriguez-Zubiri, P. Giraudeau, Trends Anal. Chem. in press, (2016), doi: 10.1016/j.trac.2016.01.014 [7] T. Jézéquel, C. Deborde, M. Maucourt, V. Zhendre, A. Moing, P. Giraudeau, Metabolomics 11, (2015), 1231. [8] J.-N. Dumez, J. Milani, B. Vuichoud, A. Bornet, J. Lalande-Martin, I. Tea, M. Yon, M. Maucourt, C. Deborde, A. Moing, L. Frydman, G. Bodenhausen, S. Jannin, P. Giraudea, Analyst 140, (2015), 5860.

Plenary Lectures & Parallel Sessions

Parallel Session 5

Elucidating the Nanoscale of Ionic Liquids with EPR spectroscopy Ulrike Seifert,1 Andreas Roos,1 Yasar Akdogan,2 Daniel R. Kattnig,3 and Dariush Hinderberger1 1Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany 2Materials Science and Engineering, Izmir Institute of Technology, Izmir, Turkey 3Department of Chemistry, University of Oxford, Oxford, UK email: [email protected]

Magnetic resonance methods such as nuclear magnetic resonance (NMR) or electron paramagnetic resonance (EPR) can give valuable insights into materials that show long range disorder and some short range or intermediate range order, e.g. synthetic and biological soft matter. In particular, EPR spectroscopy with its high sensitivity and selectivity is complementary to the well-established methods of soft matter characterization (e.g. light-, X-ray, neutron scattering) and delivers information that is otherwise not accessible.[1] In particular, multifrequency continuous-wave (CW) and echo-detected EPR allows studying the direct environment surrounding spin probes and rotational dynamics in the microsecond to picosecond regime. Ionic liquids (ILs) are one type of “soft” matter system that is intrinsically difficult to study. ILs are salts that are fluid at ambient temperatures and constitute unique class of “designer solvents” that have great potential for use in many fields of chemistry. Here, it will be shown using examples from that EPR spectroscopy on spin probes of suited chemical nature (polarity, hydrophobicity, size, etc.) can help illuminating – on the molecular scale - the complex, dynamic self-ordered structures formed by (ILs) and aqueous mixtures of ILs.[2-8] References [1] R. Graf, M. R. Hansen, D. Hinderberger, K. Münnemann, H. W. Spiess, Phys. Chem. Chem. Phys. 16, 9700-9712 (2014) [2] V. Strehmel, S. Berdzinski, H. Rexhausen, J. Mol. Liq., 192, 153–170 (2014) [3] Y. Akdogan, J. Heller, H. Zimmermann, D. Hinderberger, Phys. Chem. Chem. Phys. 12, 7874-7882 (2010) [4] Y. Akdogan, M. J. N. Junk, D. Hinderberger, Biomacromolecules 12, 1072-1079 (2011) [5] D. R. Kattnig, D. Hinderberger, Chem. Asian J. 7, 1000-1008 (2012) [6] D. R. Kattnig, Y. Akdogan, C. Bauer, D. Hinderberger, Z. Phys. Chem. 226, 1363-1378 (2012) [7] D. R. Kattnig, Y. Akdogan, I. Lieberwirth, D. Hinderberger, Mol. Phys. 111, 2723-2737 (2013)

Plenary Lectures & Parallel Sessions

Parallel Session 5

New chiral alignment media based on polyglutamates and polyaspartates S. Hansmann, M. Köberle, F. Roth, D. Herold, V. Schmidts and C. M. Thiele Clemens Schöpf Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Germany. E-mail: [email protected]

Homopolypeptides are known to form lyotropic liquid crystalline (LLC) phases due to their α-helical backbone. These homochiral LLC phases can be used as alignment media which allow for enantiodifferentiation.1 Commonly used alignment media are poly-γ-benzyl-L/D- glutamate (PBLG or PBDG) or the corresponding ethyl ester (PELG).1, 2 Comparison of the enantiodiscriminating properties of PELG and PBLG emphasized that the side chain and therefore the spatial closeness of the analyte to the helical backbone has an impact on the effect.3 Thus we were wondering how an additional chiral center in the side- chain of the polyglutamate will effect enantiodiscrimination as the resulting alignment media are diastereomers. Furthermore going from the glutamate backbone to the aspartate backbone also is beneficial for enantiodiscrimination: These are known to allow for helix reversal upon temperature change, giving access to two alignment-media in one. Recent (unpublished) results on our new chiral alignment media poly-γ-2-methylbutyl-L/D- glutamate and poly-β-phenethyl-L-aspartate (PPLA) are presented.

References [1] Reviews: a) M. Sarfati, P. Lesot, D. Merlet, J. Courtieu, Chem. Comm. 2000, 2069-2081; b) B. Böttcher, C. M. Thiele, eMagRes, 2012, 1, 169–180. [2] a) C. Aroulanda, M. Sarfati, J. Courtieu, P. Lesot, Enantiomer 2001, 6, 281-287; b) A. Marx, V. Schmidts, C. M. Thiele, Magn. Res. Chem. 2009, 47, 734-740. [3] S. Hansmann, T. Larem (née Montag), C. M. Thiele, Eur. J. Org. Chem. 2016, 7, 1324– 1329.

Plenary Lectures & Parallel Sessions

Parallel Session 5

CLIP-COSY: A Clean In-Phase Experiment for the Rapid Acquisition of COSY-type Correlations

M. R. M. Koos1, J. D. Haller1 and B. Luy1

1Institute of Organic Chemistry and Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany.

E-mail: [email protected]

The COSY-experiment is one of the oldest 2D NMR experiments; yet it is still invaluable for the assignment of signals in a homonuclear spin system as it only shows coherences between directly coupled spins, peaks of high explanatory power. The signal build-up in the conventional COSY (DQF-COSY), however, is dependent on t1, starting at zero intensity. This leads to the well-known anti-phase line shape and high acquisition time in the indirect dimension. We present a clean in-phase COSY (CLIP-COSY [1]) which reduces the required experiment time and yields simple in-phase cross-peaks. Another essential and widely used pulse sequence for determination of spin systems is the TOCSY-experiment where polarization is transferred to every spin within the system. Different mixing sequences for the transfer are well known (DIPSI, MOCCA, MLEV), however, they are associated with high power consumption. Average Hamiltonian Theory shows that the Perfect Echo sequence provides a method for low power broadband planar mixing which can be used for the TOCSY-experiment.

References [1] M. R. M. Koos, G. Kummerlöwe, L. Kaltschnee, C. M. Thiele and B. Luy, Angew. Chem. Int. Ed. 55, (2016)

Plenary Lectures & Parallel Sessions

7.12. Parallel Session 6

Plenary Lectures & Parallel Sessions

Parallel Session 6

W-band Pulse EPR: A Biophysical Toolbox for the Study of the biological water splitting catalysis Nick Cox,1,2 Yury Kutin,2 Maria Chrysina,2 Dimitrios A. Pantazis,2 Wolfgang Lubitz,2 Anton Savitsky2 1Research School of Chemistry, Australian National University, Acton ACT, Australia 2Max Planck Institute for Chemical Energy Conversion, D-45470 Mülheim (Ruhr) Germany E-mail: [email protected]

EPR spectroscopy and related double resonance techniques represent versatile tools for the study of transition metal catalysis.1 Historically the investigation of such systems has been hampered by the intrinsic low sensitivity of these methods and sample related issues including fast magnetic (T1) relaxation. We have overcome these limitations by performing EPR measurements at higher magnetic fields (3 T, W-band) and through the development of ELDOR-detected NMR (EDNMR), a double resonance technique with intrinsic high sensitivity and large bandwidth.1 This can be demonstrated on nature’s water splitting catalyst, a pentaoxygen tetramaganese calcium (Mn4O5Ca) cofactor that is found in a unique pigment–protein complex known as Photosystem II.2,3 Key to understanding its activity is the identification of the catalyst’s two 17 substrate water binding sites. Using EDNMR coupled with H2 O labelling, we have identified that the cofactor contains a unique exchangeable oxygen bridge (O5).4,5 Freeze-quench measurements indicate that this bridge exchanges with the same rate as observed for one of the substrate waters as seen by mass spectrometry, identifying O5 as a substrate of the water splitting reaction. In addition, EDNMR has allowed us to examine the electronic structure of the four manganese ions of the cofactor in the last metastable intermediate of 6 the reaction cycle (S3). It is observed that the four Mn ions in this state must all be six coordinate requiring the cofactor to bind an additional water molecule. Together these experimental results suggest that the O-O bond forms between two manganese-bound activated oxygens. References [1] N Cox, A Nalepa, ME Pandelia, W Lubitz, A Savitsky Methods in enzymology. 563, (2015) [2] N Cox, DA Pantazis, F Neese, W Lubitz Acc. Chem. Res. 46, (2013) [3] Y Umena, K Kawakami, JR Shen, K Kamiya Nature 473, (2011) [4] L Rapatskiy, N Cox, A Savitsky, WM Ames, J Sander, MM Nowaczyk, M Rögner, A Boussac, F Neese, J Messinger, W Lubitz J. Am. Chem. Soc. 134, (2012) [5] M Pérez Navarro, WM Ames, H Nilsson, T Lohmiller, DA Pantazis, L Rapatskiy, M Nowaczyk, F Neese, A Boussac, J Messinger, W Lubitz, N Cox Proc. Natl. Acad. Sci. U.S.A 110, (2013) [6] N Cox, M Retegan, F Neese, DA Pantazis, A Boussac, W Lubitz Science. 345, (2014)

Plenary Lectures & Parallel Sessions

Parallel Session 6

EPR-Based Metal Ion Trilateration Dinar Abdullin, Andreas, Meyer, Gregor Hagelüken, Olav Schiemann Institute of Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany E-mail: [email protected]

Metal ions are important for the folding, structure and catalysis of biomolecules. We will present an EPR-based metal ion positioning system for paramagnetic ions in such biomolecules1. In this approach site directed spin labeling with nitroxides or trityl radicals is used and the label coordinates are determined by means of in silico methods. Then the label-to-ion distances are measured by Pulsed Electron-Electron Double Resonance (PELDOR or DEER) or Relaxation Induced Dipolar Modulation Enhancement (RIDME)2,3,4. Finally the ion site is localized within the fold of the biomolecule by means of trilateration. This approach was successfully tested on Cu(II) in azurin yielding a deviation of 2 Å with respect to the crystallographic site. The source of this error is mainly related to the error of the label coordinates. The imperfection of the in silico labeling methods will be shown by localizing the label positions by X-ray crystallography and PELDOR measurements5.

References [1] D. Abdullin, N. Florin, G. Hagelueken, O. Schiemann, Angew. Chem. Int. Ed. 2015, 54, 1827. [2] D. Abdullin, F. Duthie, A. Meyer, E.S. Müller, G. Hagelueken, O. Schiemann, J. Phys. Chem. B 2015, 119, 13534. [3] A. Meyer, O. Schiemann, J. Phys. Chem. A 2016, 120, 3463. [4] A. Meyer, D. Abdullin, G. Schnakenburg, O. Schiemann, Phys. Chem. Chem. Phys. 2016, 18, 9262.G. [5] Hagelueken, D. Abdullin, O. Schiemann, Phys. Chem. Chem. Phys. 2016, 18, 10428.

Plenary Lectures & Parallel Sessions

Parallel Session 6

Light-induced conformational changes of the sensory module of phytochrome Cph2 T. Assafa1,2, K. Anders3, L. Essen3, E. Bordignon1,2 1 Berlin Joint EPR Laboratories, Department of Experimental Physics, Free University of Berlin, Germany 2 Department of Chemistry and Biochemistry, Ruhr-University Bochum, Universitätstsr. 150, 44801 Bochum 3Department of Chemistry, Philipps University, Marburg, Germany E-mail: [email protected]

Phytochromes are light sensitive proteins with possible applications in synthetic biology. They exist in one of two photoactive states which absorb either red (Pr state) or far-red light (Pfr state). The crystal structures of the sensory domain dimer of DrBphP in both photoactive states have been published [1]. The structures show a large change in secondary structure of the tongue motif and a kinking of the long helical spine connecting the PAS-GAF and PHY domains. This kink has been further modeled to fit SAXS data suggesting the conformational change of the DrBphP dimer is larger than suggested by the crystal structures. Our aim is to resolve the structural changes of the helical spine and the tongue region of Cph2 in solution with site directed spin labelling and double electron-electron resonance (DEER).

DEER experiments show detectable conformational changes in the helical spine and the tongue domain. Via a multilateration approach we are able to distinguish two coexisting conformations of the helical spine, supported by deuterium exchange data. In addition, we observe a conformational change upon light activation comparable to the homologous crystal structures, strongly ruling out the exaggerated kink in the Pfr state of DrBphB obtained by molecular dynamics. From our data, we have the first hints of an equilibrium of conformations detailing the translocation of the activation signal from the chromophore to the C-terminus of Cph2.

[1] H. Takala, Nature. 509, (2014)

Plenary Lectures & Parallel Sessions

Parallel Session 6

Micro-Resonators for Electron Paramagnetic Resonance Spectroscopy of Size Limited Samples at 9.5 GHz. J.W. Sidabras1, D. Suter2, E.J. Reijerse1, A. Savitsky1 and W. Lubitz1 1Max-Plank-Institute for Chemical Energy Conversion, Mülheim a.d. Ruhr, DE 2Technical University Dortmund, Dept. of Physics, Dortmund, DE E-mail: [email protected]

We present the development of micro-resonators for X-band (9.5 GHz) Electron Paramagnetic Resonance (EPR) for volume-limited samples [1]. Such samples have nanoliter volumes or limited crystal geometries that minimize their effective size. In order to increase signal-to-noise ratio (SNR), one must design new probes that concentrate the magnetic field over a finite sample space. Two challenges exist in micro-resonator design: i) magnetic field inhomogeneity over the sample volume and ii) degraded Q0-value due to large surface currents and dielectric losses.

In order to increase the Q0-value of the micro-resonator, two new geometries have been fabricated on a sapphire substrate. We also present a new broad-side coupled micro- resonator geometry that increases the uniformity over the sample region up to 50%.

Additionally, a micro-helix structure is introduced to provide a SNR benefit due to higher Q0- values and more homogeneous fields. Simulation and experimental data outlining three novel micro-resonator designs is presented: sapphire based 0.5 mm ID omega-loop resonator, sapphire based 0.1 mm ID radial-loop resonator, and a micro-helix design at 0.4 mm ID. Modeling, design, and simulations were performed using Ansys High Frequency Structure Simulator. Experimental concentration sensitivity analysis is presented as a comparison with commercial EPR probes.

References [1] R. Narkowicz, D. Suter, R. Stonies, Planar microresonators for EPR experiments, J. Magn. Reson. 175, 2005

Plenary Lectures & Parallel Sessions

7.13 Parallel Session 7

Plenary Lectures & Parallel Sessions

Parallel Session 7

Solid-state NMR studies of functional dynamics in a half- megadalton enzyme complex

P. Schanda1 1Institut de biologie structurale (IBS)- CEA-CNRS-UGA, Grenoble, France

E-mail: [email protected]

Dynamics are often a key requirement for proteins to fulfill their biological functions. NMR is ideally suited to probe motions at atomic resolution. Here we use various solid-state NMR approaches to address dynamics in a large dodecameric aminopeptidase complex. We obtained full assignment of this 12x39 kDa-large enzyme, and used recently developed proton-detected experiments to probe motions from picoseconds to seconds. Our results provide insight into substrate binding and release; we show how substrate interactions are allosterically modulated by the substrate entry pore region, which is >20 Å apart from the active site. Finally, we demonstrate the role of loop regions in controlling catalytic activity.

Taken together, our data establish that solid-state NMR can resolve functional mechanisms of large biological machines even in cases where solution-NMR is severely challenged by their size.

Plenary Lectures & Parallel Sessions

Parallel Session 7

CHARACTERIZING TRANSIENT STRUCTURES OF RNA USING NMR Katja Petzold1 1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden, Email: [email protected]; http://petzoldlab.com

Many functions of RNA depend on rearrangements in secondary structure that are triggered by external factors, such as protein or small molecule binding. These secondary structural switches require the breaking and reconfiguration of many base-pairs and occur at second or faster timescales in nascent RNA chains. These transitions can feature on one hand localized changes in base-pair alignments in and around non-canonical base-pairs1, such as internal loop and bulges, or can be presented by a complete change in chemical identity of a nucleobase2. 3,4 Here, we describe R1ρ-relaxation-dispersion NMR methods for characterizing transient structures of RNA that exist in low abundance (populations <10%) and that are sampled at 1 three orders of magnitude faster timescales. A new H R1ρ-relaxation-dispersion sequence will be presented that allows for elucidation of dynamics of 1H attached to 13C in RNA and proteins (unpublished). The characterization of two different types of transient structures is presented. 1) The HIV-1 dimerization initiation site (DIS) undergoes large secondary structure rearrangements, that provide the basis for a molecular zipper, which can be crucial for genome packaging.1 2) The GU wobble base-pair undergoes a change from standard wobble geometry to appear like a Watson-Crick base-pair stabilized by Keto-Enol tautomerization.4

References [1] Dethoff, E. A.; Petzold, K.; Chugh, J.; Casiano-Negroni, A.; Al-Hashimi, H. Nature. 2012, pp 724–728. [2] Kimsey, I. J.; Petzold, K.; Sathyamoorthy, B.; Stein, Z. W.; Al-Hashimi, H. Nature. 2015, pp 315–320. [3] Hansen, A. L.; Nikolova, E. N.; Casiano-Negroni, A.; Al-Hashimi, H. JACS. 2009, pp 3818–3819. [4] Nikolova, E. N.; Gottardo, F. L.; Al-Hashimi, H. JACS. 2012, pp 3667–3670.

Plenary Lectures & Parallel Sessions

Parallel Session 7

Proton transverse relaxation as a sensitive reporter on microsecond motions in solid-state P. Rovo1,2 , C. Smith2, K. Giller2, S. Becker2, B. de Groot2 and R. Linser1 1Ludwig-Maximilians-Universität, Munich, Germany 2Max Planck Institute for Biophysical Chemistry , Göttingen, Germany E-mail: [email protected]

SH3 protein domains are small polyproline-recognition modules mediating protein-protein interactions in various signalling pathways which regulate cell growth, apoptosis or transcription, and thus these domains are linked to many pathological diseases [1]. Here we present a novel solid-state NMR method for the reliable determination of stochastic motions occurring on the microsecond timescale. We show that 1H transverse relaxation of perdeuterated and partially reprotonated proteins at fast magic angle spinning allows the investigation of both amplitude and timescale of µs range conformational fluctuations. Our relaxation data of chicken α-spectrin SH3 suggest that the loops around the ligand binding site (RT- and N-src loops) exhibit spontaneous high-amplitude conformational fluctuation on the μs timescale in the absence of bound ligand and the lowly-populated SH3 conformation consists primarily of holo-like structures which exist in equilibrium with the ground state apo conformation. This inherent plasticity between interconverting macrostates is compatible with the conformational-preselection model [2] and provides new insight into the recognition mechanisms of SH3 domains. The presented methods have important implications for dynamic investigations of microcrystalline, membrane embedded or fibrillar solid proteins.

References [1] B.K. Kay FEBS Lett. 586, 2606–2608. (2012) [2] E.Z. Eisenmesser et al. Nature 438, 117–121. (2005)

Plenary Lectures & Parallel Sessions

Parallel Session 7

Microsecond protein dynamics probed by solid-state NMR using 15 N R1ρ relaxation experiments under fast MAS (60-110 kHz)

Nils-Alexander Lakomek1, Susanne Penzel1, Alons Lends1, Riccardo Cadalbert1, Matthias Ernst1, Beat H. Meier1

1 ETH Zurich, Laboratory of Physical Chemistry, Department of Chemistry and Applied Biosciences, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland

E-mail: [email protected]

15 N R1ρ relaxation experiments in solid-state NMR are sensitive to time scales and amplitudes of internal protein motions in the ns-µs time window, difficult to probe with solution-state. 15 Using N R1ρ relaxation experiments with variable MAS frequency under fast MAS (60-110 kHz) and a model-free analysis, we find for the protein ubiquitin uniform small amplitude dynamics on a time scale of about 1µs across the entire protein, and larger amplitude motions on the same 1µs time scale for several sites, including the first loop region connecting β-strand 1 and 2, as well as Glu 24, Asn 25 and Asp 52, which are known to be involved in a hydrogen-bond reordering process, however on a two orders of magnitude slower time scale. According to our analysis, the ns-µs backbone protein backbone dynamics occur in a narrow time window around 1µs, but are of lower amplitude than concluded in previous solid-state NMR studies. Our study adds to the debate about the presence and/or amplitude of protein backbone dynamics in the ns-µs time range, which would directly impact our understanding about the protein energy landscape and molecular recognition processes. Our data experimentally establish, with residue-specific resolution, the 1µs time scale for protein backbone dynamics in microcrystalline ubiquitin. Amplitudes of motion in microcrystalline ubiquitin (for a time window <100 µs) appear however reduced compared to previous RDC-based studies in solution-state NMR (which are sensitive for a time window <10 ms).

Plenary Lectures & Parallel Sessions

7.15 Parallel Session 8

Plenary Lectures & Parallel Sessions

Parallel Session 8

Ultra-Precise NMR-Magnetometers for High Fields

A. Maul, P. Blümler, E. Otten, W. Heil

Institute of Physics, University of Mainz, Mainz, Germany

E-mail: [email protected]

While low magnetic fields (< 10-2 T) can be measured extremely precisely (ca. 10-13) by SQUID or SERF, NMR offers the highest precision at higher fields. Furthermore, it can be shown that the highest metrological accuracy is reached for continuous measurements of frequencies. Therefore one needs a sample with long coherence times, as it is the case for motionally averaged signals of gases. To obtain such extremely long lasting signals the sample has to be kept in suitable containers, low pressure and very homogeneous fields. Another prerequisite is hyperpolarization to overcome the low spin density in gases. We have developed such magnetometers for fields ranging from micro- to several Tesla.

The high field magnetometer uses 3He, which ideally must be hyperpolarized in-situ in high magnetic fields. To achieve this, a special variant of metastability optical pumping had to be developed and special care had to be taken for the choice of materials in its direct vicinity due to magnetic susceptibility effects. Ideally the gas container has to be a perfect sphere 3 containing only 1-3 mbar of He [1]. In spheres with inner diameters of 8 mm we observe T2* times up to 5 min in standard NMR magnets, which already allows for ultra-precise (< 10-12) field measurements at several tesla [2]. We developed this instrument especially for field monitoring during high precision mass-spectroscopy using Penning traps to test for the standard model of physics.

References

[1] A. Maul, P. Blümler, W. Heil, et al., Review Scientific Instruments. 87 (2016) 015103 1-8 [2] A. Nikiel, P. Blümler, W. Heil, et al., The European Physics Journal D 68 (2014) 1-12.

Plenary Lectures & Parallel Sessions

Parallel Session 8

Classical and dynamic phase transitions of supercooled ionic liquids in bulk and in confinement

Amin Ordikhani-Seyedlar, Carlos Mattea, Siegfried Stapf Dept. of Technical Physics II, TU Ilmenau, PO Box 100 565, 98684 Ilmenau, Germany E-mail: [email protected]

Ionic liquids reveal properties different from molecular liquids mainly due to their ionic character, in particular close to the glass transition, and in the presence of surfaces such as in porous materials or organogels. NMR relaxation studies provide an opportunity to observe the dynamics of anions and cations individually [1,2].

In this contribution, the calorimetric and relaxation dispersion (T1(ω)) behaviour of several imidazolium based ionic liquids with different anions, in bulk [3] as well as confined in porous media, is presented with a focus on the supercooling regime. The degree of supercooling varied substantially between the different sidechain lengths, and T1 relaxation times were measured at temperatures that cover the range from ambient conditions to the glass transition of the corresponding IL. The relaxation experiments reveal pronounced changes in the dynamics, relative to the bulk, of both ions when confined in nm-scale pores. Rotational and translational correlation times were obtained reflecting different temperature dependences, i.e. super-Arrhenius and heterogenous dynamics in bulk, while Arrhenius dynamics is found under confinement, as a consequence of the physical restriction to the correlation length. Diffusion coefficients obtained from the relaxation experiments follow the Vogel-Fulcher-Tammann (VFT) law above a dynamic transition temperature Td≈1.28 Tg, below which a further deviation is observed.

References [1] A. Wulf, R. Ludwig, P. Sasisanker, H. Weingärtner, Chem. Phys. Lett. 439, 323 (2007) [2] D. Kruk, R. Meier, A. Rachocki, A. Korpala, R.K. Singh, E.A. Rössler, J. Chem. Phys. 140, 244509 (2014) [3] A. Ordikhani-Seyedlar, S. Stapf, C. Mattea, Phys Chem Chem Phys 17, 1653 (2015)

Plenary Lectures & Parallel Sessions

Parallel Session 8

Intra-aneurysmal flow and thrombosis J. Flohr1, J. Viess1, J. Clauser2, G. Cattaneo3, M. Küppers1, B. Blümich1 1RWTH Aachen University, Institut für Technische und Makromolekulare Chemie, Worringerweg 2, 52074 Aachen, Germany. 2RWTH Aachen University, Helmholtz-Institute for Biomedical Engineering, Pauwelsstraße 20, 52074 Aachen, Germany. 3Acandis GmbH & Co. KG, Theodor-Fahrner-Str. 6, 75177 Pforzheim, Germany. E-mail: [email protected]

An often occurring disease pattern of blood vessels is an aneurysm, a spatially limited enlargement of an artery. In addition to this health threat blood clots can develop in the damaged blood vessel [1]. To prevent the rupture of an aneurysm, a stent can be inserted into the vessel. A stent is usually a metallic lattice, which may trigger the formation of a blood clot in the lumen of the aneurysm [2]. For determining the relaxation times of thrombi with varying amounts of blood cells and plasma a 0.22 T tomograph was used [3]. Furthermore, flow patterns in a silicon phantom of a saccular aneurysm were examined by applying the FLIESSEN [4] pulse sequence in high and low magnetic fields.

References

[1] H. Rieger et al., Klinische Angiologie, Spinger-Verlag, Berlin, Heidelberg, (1998). [2] J. R. Cebral et al., Am. J. Neuroradiol. 32, (2011) 27-33. [3] E. Danieli et al., J. Magn. Reson. 198, (2009) 80-87. [4] A. Amar et al., ChemPhysChem 11, (2010) 2630-2638.

Plenary Lectures & Parallel Sessions

Parallel Session 8

Dynamics in thin polymer films and at interfaces U. Lappan1, U. Scheler1 1Leibniz-Institut für Polymerfoschung Dresden e.V, Dresden, Germany E-mail: [email protected]

Organic-inorganic hybrid materials appear in nature and man-made materials. Functional properties are to a large extend determined by the interfaces. For the investigation of near-interface species systems with a high surface-to-volume ratio like coated nanoparticles from silica or hydroxyapatite or thin fluorpolymer films have been used. If the carrier does not contain protons (silica or fluoropolymer) direct measurements are feasible, otherwise selective excitation by chemical shift or in combination with relaxation filters is applied. For the polymer dynamics T1rho or T2 have been measured with chemical shift resolution. Based on the chemical shift polymer and solvent signals are separated in swelling experiments exhibiting significantly different effects for polymer brushes and bulk polymers of the same molecular weight. In polyelectrolyte multilayers of 13 more than 50 layers C detection of T1rho becomes feasible, which enables to determine differences of the dynamics in the polycation and the polyanion. In-situ low-field NMR under uniaxial load complements these studies, in which the strength of the interaction with the inorganic filler as the basis of the reinforcement is demonstrated [1]. Spin-label EPR enables selective experiments with the inherent higher sensitivity of EPR. Continuous-wave EPR and line shape fitting in EasySpin [1, 2]. Thus the influence of the external medium (pH and ionic strength) on polyelectrolyte multilayers congaing weak polyanions has been investigated. Increasing the pH more acid groups dissociate leading to stringer interaction with the water and thus higher mobility in all layers.

References [1] U. Böhme, U. Scheler, AIP Conference Proceedings 1713 (2016) ArticleID: 090009 [2] U. Lappan, B. Wiesner, U. Scheler Macromolecular Chemistry and Physics 215 (2014) 1030-1035 [3] U. Lappan, B. Wiesner, U. Scheler, Macromolecules 48 (2015) 3577-3581

Plenary Lectures & Parallel Sessions

7.16 Plenary Lectures 5

Plenary Lectures & Parallel Sessions

Plenary Lectures 5

Conformational dynamics of the phototaxis membrane protein complex revealed by EPR spectroscopy H.-J. Steinhoff Department of Physics, University Osnabrück, 49069 Osnabrück, Germany E-mail: [email protected]

Recent developments including pulse techniques and spin labeling methods make electron paramagnetic resonance (EPR) spectroscopy an attractive approach for studying the structure and conformational dynamics of large biomolecular complexes, e.g., of membrane proteins in their native environment [1]. We will outline new strategies for bio-orthogonal site directed spin labeling which open the path for in vivo EPR and DEER spectroscopy. We will review our time resolved studies on the light induced signal transfer mechanism in the halobacterial phototaxis sensory rhodopsin-transducer complex, SRII-HtrII [2, 3]. Inter-spin distance changes in the light-gated ion channel channelrhodopsin-2, which is related to SRII, reveal a light induced movement of the transmembrane helix B [4], which opens the ion pathway, and provides a new mode of rhodopsin conformational change, which adds to the known displacement of helix F observed in bacteriorhodopsin and SRII.

References [1] I. Hänelt, D. Wunnicke, E. Bordignon, H.J. Steinhoff, and D.J. Slotboom. Nature Structural & Molecular Biology 20, (2013) [2] D. Klose, N. Voskoboynikova, I. Orban-Glass, C. Rickert, M. Engelhard, J.P. Klare, H.J. Steinhoff. FEBS Letters 588 (2014) [3] P.S. Orekhov, D. Klose, A.Y. Mulkidjanian, K.V. Shaitan, M. Engelhard, J.P. Klare, H.J. Steinhoff. PLoS Comput Biol. 11:e1004561. (2015) [4] T. Sattig, C. Rickert, E. Bamberg, H.J. Steinhoff, C. Bamann. Angewandte Chemie Int. Edition 52 (2013)

Plenary Lectures & Parallel Sessions

Plenary Lectures 5

Dual DNP and EPR capabilities at 7 Tesla enable mechanistic studies to new applications Songi Han1,2, Ilia Kaminker1, Alisa Leavesley1, Alicia Lund1 and Ting Ann Siaw1 1Department of Chemistry and Biochemistry University of California, Santa Barbara, CA 93106 2Department of Chemical Engineering, University of California, Santa Barbara, CA 93106 E-mail: [email protected]

We present a versatile homebuilt DNP-EPR instrument operating at a magnetic field of 7T designed for investigating DNP mechanisms at cryogenic temperatures.1 Homebuilt modules were installed as add-ons to a commercial 7 T Bruker NMR system to enable dual DNP-EPR operation in frequency, time-domain, CW and pulsed mode. The broad band (10 GHz) quasioptical bridge operating at 200 GHz enables acquisition of EPR spectra in frequency-stepped mode in a constant-field NMR magnet. We showcase electron spin relaxation (T1e, T2e) and electron spectral diffusion measurements enabled by dual-frequency pump-probe Electron- Electron Double Resonance (ELDOR) capabilities, precisely under DNP conditions. The ELDOR data reveals an extreme extent of EPR line saturation, offering direct evidence for “oversaturation” seen at 4K, >50mW MW and high radical concentration. Recent advances in microwave pulse shaping (amplitude and phase) at 200 GHz will also be presented, bringing us a step closer towards pulsed, time-domain, DNP capabilities. These new technical capabilities present new powerful opportunities for basic studies of DNP mechanisms towards a rational design of DNP experiments, as well as new materials science opportunities from the study of catalyst surfaces, Li-ion battery electrolyte systems to semiconductors, enabled by DNP at a broad temperature range new to NMR, ranging from room temperature down to < 4 Kelvin.

References [1] Siaw, T. A. et al. Effect of electron spin dynamics on solid-state dynamic nuclear polarizationperformance. Phys. Chem. Chem. Phys. 16, 18694 (2014).

Plenary Lectures & Parallel Sessions

7.17 Parallel Session 9

Plenary Lectures & Parallel Sessions

Parallel Session 9

Protein intrinsic disorder in the MAPK cell signalling pathways J. Kragelj1, E. Delaforge1, A. Palencia2, M. Nanao2, M. Blackledge1 and M.R. Jensen1 1Institut de Biologie Structurale, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, France 2EMBL Grenoble Outstation, Grenoble, France E-mail: [email protected]

Intrinsically disordered proteins (IDPs) are highly abundant in the human proteome and play key regulatory roles in biology. NMR spectroscopy is uniquely suited to characterize IDPs at atomic resolution, and ensemble descriptions have emerged as the preferred tool for capturing both the structure and dynamics of IDPs [1, 2]. Characterization of IDP complexes is an entirely different challenge as exchange between free and bound conformation(s) often occurs on the micro- to millisecond time scale leading to extensive line broadening of the NMR signals. Here, methods will be presented for characterizing the structure, dynamics and kinetics of IDP complexes using a combination of nuclear relaxation rates, relaxation dispersion and chemical exchange saturation transfer experiments. The approaches are applied to IDPs within the mitogen-activated protein kinase (MAPK) cell signalling pathways that assemble specific kinases into highly dynamic signalling complexes. Our results reveal how IDPs in the MAPK pathways rely on linear motifs for governing signalling specificity, and how different IDP binding modes may induce different functional states of the folded, binding partners [3].

References [1] M.R. Jensen, M. Zweckstetter, J.-R. Huang, M. Blackledge, Chem. Rev. 114, (2014). [2] J. Kragelj, V. Ozenne, M. Blackledge, M.R. Jensen, Chemphyschem. 14, (2013). [3] J. Kragelj, A. Palencia, M. Nanao, D. Maurin, G. Bouvignies, M. Blackledge, M.R. Jensen, Proc. Natl. Acad. Sci. 112, (2015).

Plenary Lectures & Parallel Sessions

Parallel Session 9

Exploring RNA Polymerase Regulation by NMR Spectroscopy Stefan H. Knauer, Johanna Drögemüller, Martin Strauß, Birgitta M. Wöhrl, Kristian Schweimer, and Paul Rösch Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, Bayreuth, Germany E-mail: [email protected]

Multisubunit RNA polymerases (RNAPs) transcribe all cellular genomes and are highly regulated by numerous transcription factors. Only little information about dynamics, conformational rearrangements, and transient interactions of RNAPs is available, which, however, is crucial for a complete understanding of transcription regulation in atomic detail. Here we present approaches to study E. coli RNAP (400 kDa, five subunits) by NMR spectroscopy in solution to gain insights into the protein's dynamics and interaction with transcription factors. First, we developed an efficient protocol to assemble active RNAP from separately produced subunits, allowing specific labeling of individual constituents, and we established experiments to identify the RNAP subunit to which a transcription factor binds [1]. We recorded [C,H] correlation spectra of [1H,13C]-labeled Ile, Leu, and Val methyl groups in deuterated RNAP and of methyl group labeled β’ subunit within reconstituted, deuterated RNAP [1]. Further, we developed approaches to determine the RNAP binding surfaces of transcription factors and to follow conformational changes within those factors upon RNAP binding using [1H,13C]-labeled methyl groups of Val, Leu, and Ile in deuterated transcription factors as probes [2].

References

[1] J. Drögemüller, M. Strauß, K. Schweimer, B.M. Wöhrl, S.H. Knauer, and P. Rösch, Sci Rep. 5, 10825 (2015) [2] J. Drögemüller, M. Strauß, K. Schweimer, M. Jurk, P. Rösch, and S.H. Knauer, Sci Rep. 5, 16428 (2015)

Plenary Lectures & Parallel Sessions

Parallel Session 9 The Nedd4-1 WW domain recognizes the PY motif peptide through coupled folding and binding equilibria

Vineet Panwalkar1, Philipp Neudecker1,2, Michael Schmitz3, Justin Lecher1,2, Marianne Schulte1,2, Dieter Willbold1,2 and Andrew Dingley1

1IC6-6 (Strukturbiochemie), Forschungszentrum Jülich, Jülich, Germany 2Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany 3School of Chemical Sciences, The University of Auckland, Auckland, New Zealand E-mail: [email protected]

The four WW domains of human Nedd4-1 interact with poly-proline (PY) motifs of the epithelial Na+ channel (ENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We investigated the structure and dynamics of hNedd4-1 WW3* to understand the high affinity. The solution structure of the apo-WW3* resembles the domain in complex with the α-ENaC peptide; although, 3J couplings analysis revealed side-chain χ1 rotameric averaging for residues in the apo-WW3* domain, which was further investigated by molecular dynamics simulations. Modelfree analysis of 15N spin relaxation data revealed that the apo- and peptide-bound states of WW3* domain have similar backbone ps-ns times-scale dynamics. However, the apo-WW3* domain exhibits pronounced chemical exchange on the millisecond time-scale which gets quenched upon peptide binding. 1HN and 15N CPMG relaxation dispersion experiments at five different temperatures reveal that apo- WW3* domain exists in equilibrium between the natively folded peptide-binding competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium, determined by fitting a thermal denaturation profile monitored by CD spectroscopy in combination with the CPMG data, reveals that the unfolded state is populated to ~20% at 37°C. The results show that the binding of hNedd4-1 WW3* domain to the α-ENaC peptide follows a coupled folding-binding equilibrium [1].

References [1] Panwalkar V et al. Biochemistry. 55, (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 9

EPR Spectroscopy on Membrane Proteins in Native Environments: New Developments B. Joseph and T.F. Prisner Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Frankfurt/M, Germany E-mail: [email protected]

An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron−electron double resonance (PELDOR, also known as DEER) is a well-established technique to follow conformational changes in purified membrane protein complexes. We demonstrate the application of PELDOR to obtain structural information [1] and to observe conformational changes [2] in an outer membrane transporter in intact E. coli and native membranes. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. Thus structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment. To further extent these studies to hetero-oligomeric complexes in whole cells or native membranes multiple spin labels are required. In this direction we also demonstrate the application of an orthogonal labeling strategy employing a triarylmethyl (TAM) radical with a nitroxide spin label to observe a membrane protein-ligand complex in native membranes. This approach provides higher sensitivity and total selectivity in the complex membrane environments having huge background signals [3].

References [1] B. Joseph, Angew. Chem. Int. Ed. 54, (2015) [2] B. Joseph, J. Am. Chem. Soc. 138, (2016) [3] B. Joseph, Submitted, (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 9

NMR Fragment Screening for Challenging Targets Markus Schade1 1Grünenthal GmbH, Drug Discovery Technologies, 52099 Aachen, Germany E-mail: [email protected]

Protein-protein-interactions, several proteases, peptide-binding GPCRs and protein phosphatases are often categorized as challenging drug targets, because borderline drugability complicates their modulation by small molecule drugs. Covalent small molecule inhibitors can boost the drugability for such targets, but frequently bring along the risks of immunogenicity and off-target adverse effects. Here we utilized NMR spectroscopy for the identification of novel, low Mw (fragment) modulators of a challenging class of protease targets. We demonstrate that the NMR assay was more robust in detecting such fragment modulators than state-of-the-art biochemical screening. Compared with the biophysical techniques surface plasmon resonance and microscale thermophoresis, the NMR assay produced reliable binding curves for fragments that interfered with the detection of those former two techniques. The crystal structures of three fragment modulators complexed with protein show that these fragments bind to a new hot-spot sub-pocket of the protease which was not yet targeted by any crystallography-defined inhibitor in the literature. We show how we use these fragment-protein starting points for the chemical synthesis of more potent, noncovalent, drug-like modulators.

References [1] Vargas C. et al., Chem. Med. Chem. (2014) 9, 1458. [2] Scheich C. et al., J. Med. Chem. (2010) 53, 8362. [3] Phillips C. et al., J. Am. Chem. Soc. (2011) 133, 9696.

Plenary Lectures & Parallel Sessions

Parallel Session 9

Chaperonin stabilizes aggregation-prone proteins and interferes with fibrillation of amyloids E. Colas-Debled1 and JY. Guan1, G. Mas1, E. Crublet1, P. Macek1, C. Moriscot1, G. Schoehn1, P. Gans1, L. Gremer2, W. Hoyer3, D. Willbold2,3, P. Schanda1, J. Boisbouvier1 1Institut de Biologie Structurale, Grenoble, France 2Institute of Complex Systems, Jülich, Germany 3Institute for Physical Biology, Dusseldorf, Germany E-mail: [email protected]

Chaperonins are huge ATP-dependent molecular chaperones that are able to refold misfolded proteins in the cells. Many studies show that the malfunction of chaperonins and accumulation of misfolded proteins play an important role in aging-related diseases and therefore have raised great pharmaceutical interest. However, the mechanism of chaperonin function and the process of protein misfolding at an atomic level remain unclear. We studied the interaction between a 1 MDa chaperonin, the Thermosome, and client proteins of various sizes and functions, including aggregation-prone proteins and amyloidogenic proteins (such as amyloid-beta and alpha-synuclein). To overcome the size limitation in NMR spectroscopy, we used advanced isotope labeling of methyl groups[1-3] coupled with liquid- and solid-state NMR to probe the actions of the chaperonin. We also applied complementary biophysical techniques to monitor the changes in the aggregation and fibrillation processes of the client proteins. We observed that the chaperonin prevents aggregation of different substrates and also interferes in the fibrillation mechanisms of amylogenic proteins by slowing down or preventing the fibrils formation process.

References [1] G. Mas et al., J. Biomol. NMR. 57, 251–262 (2013). [2] E. Crublet et al., Methods Mol. Biol. 1091, 229–244 (2014). [3] R. Kerfah et al., Curr. Opin. Struct. Biol. 32, 113–122 (2015).

Plenary Lectures & Parallel Sessions

7.18 Parallel Session 10

Plenary Lectures & Parallel Sessions

Parallel Session 10

High Field DNP enhanced NMR Spectroscopy: Higher efficiency, limiting factors and applications to nanomaterials Ümit Akbey 1,2 1 Interdisciplinary Nanoscience Centre (iNANO), Aarhus University, Aarhus, Denmark 2 Aarhus Institute for Advanced Studies (AIAS), Aarhus University, Aarhus, Denmark Email: [email protected]

Dynamic Nuclear Polarization (DNP) increases sensitivity of NMR by several orders of magnitude. The method exploits the transfer of large initial Boltzmann polarization of electron spins to those of neighboring nuclei. Theoretical nuclear signal enhancements of 1 5-6 ( γe/ γI) ~660 can be obtained for H nuclei, which could result up to a remarkable ~10 reduction in NMR experimental time. In practice, at 9.4 T and 100 K, DNP enhancements are obtained lower much than the theoretical maximum, between 30-200.

New approaches to increase the efficiency of DNP hyperpolarization will be explained by utilizing deuteration and other. The factors affecting the efficiency of the method will be discussed, including MAS effects. Moreover, applications of DNP enhanced NMR on materials will be summarized on several interesting systems.

Plenary Lectures & Parallel Sessions

Parallel Session 10

Cryptochrome model compound F10T: photo-CIDNP NMR studies on low and Earth’s magnetic field S. Paul1, L. Meng2, A. S. Kiryutin3,4, K. L. Ivanov3,4, A. V. Yurkovskaya3,4, X. Wang2 and J. Matysik1 1Institut für Analytische Chemie, Universität Leipzig, Leipzig, Germany 2National University of Defense Technology, Changsha, China 3International Tomography Center, Novosibirsk, Russia 4Novosibirsk State University, Novosibirsk, Russia E-mail: [email protected]

Migratory birds have the remarkable ability to sense the Earth’s magnetic field for navigation. A light-dependent quantum effect in the photoreceptor protein cryptochrome allows for such an iron-free spin-chemical compass. However, the low magnetic field effect in cryptochrome is poorly understood.

Inspired by the structure of cryptochrome, we have synthesized a flavin-linker-tryptophan dyad called F10T.1 DFT calculations show different conformations of the compound which are verified with 2D NMR correlation studies.1 Here we present the first 1H photo-CIDNP (photo-chemically induced dynamic nuclear polarization) studies2 on the artificial cryptochrome F10T at low as well as at Earth’s magnetic field. Magnetic field-dependence of photo-CIDNP has been obtained2 by changing the polarizing magnetic field using a home- build NMR shuttle-system3. This study explored the mechanism for S-T interconversion in low magnetic field. Theoretical modeling of the magnetic field-dependence of photo-CIDNP provided the relevant parameter as exchange interaction, spin diffusion, recombination rates etc. Furthermore, the spin-dynamics at low magnetic fields has been revealed. Hence, the understanding of spin-dynamics in F10T might provide a key to the functional mechanism of cryptochrome at low magnetic field.

References [1] S. Paul et al., manuscript submitted [2] S. Paul et al., manuscript in preparation [3] A. S. Kiryutin et al., JMR, 263, (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 10

Dynamic nuclear polarization enhanced solid state NMR using 3+ bis-Gd polarizing agents Monu Kaushik, 1 Mian Qi, 2 Adelheid Godt, 2 Björn Corzilius, 1

1 Institute of Physical and Theoretical Chemistry, Institute of Biophysical Chemistry, and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von- Laue- Str. 7-9, 60438 Frankfurt am Main, Germany. 2 Faculty of Chemistry and Center for Molecular Materials, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany. Email: [email protected]

Dynamic nuclear polarization (DNP) drastically improves sensitivity of MAS NMR thereby furthering its applicability to new areas of research. A variety of polarizing agents (PAs) have been developed for efficient DNP for high sensitivity gains. This work is based on a relatively new class of PAs: high-spin transition metal ions such as Gd3+, Mn2+. These PAs can substitute intrinsically bound diamagnetic ions in biomolecules with no (or insignificant) effect on their structure, enabling the possibility to obtain site-specific information. However, the polarization transfer mechanisms for these PAs are yet to be understood. Gd3+ is a high-spin (S=7/2) system featuring a strong zero-field (electron quadrupolar) interaction which leads to non-trivial consequences. Gd-DOTA invokes solid effect (SE) at low concentration owing to its narrow EPR linewidth. Deviation from pure SE and contribution of cross effect (CE) is observed for high PA concentration (shorter inter-molecular distance). In our attempts to shed light on underlying polarization transfer mechanisms, bis-Gd rigid model complexes are studied. We are able to probe inter-spin distance dependence of DNP field profiles and enhancements by varying molecular tether length between the chelator moieties. Eventually, a theoretical model for CE DNP via high- spin PAs is developed. This research equips us to comment on designing complexes for efficient CE DNP.

Plenary Lectures & Parallel Sessions

Parallel Session 10

EPR study of assembly mechanisms of the Mn/Fe cofactors in R2lox and R2c proteins Yu. Kutin1, V. Srinivas2, M. Fritz3, R. Kositzki4, H.S. Shafaat5, J. Birrell1, E. Bill1, M. Haumann4, W. Lubitz1, M. Högbom2,6, J.J. Griese2, and N. Cox1,7 1Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany 2Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden 3Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France 4Institut für Experimentalphysik, Freie Universität Berlin, Berlin, Germany 5Department of Chemistry and Biochemistry, The Ohio State University, Columbus, USA 6Department of Chemistry, Stanford University, Stanford, United States 7Research School of Chemistry, Australian National University, Canberra ACT 2601, Australia E-mail: [email protected]

A manganese/iron cofactor that performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins: the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of MnII and FeII. Using EPR spectroscopy, complemented by X-ray anomalous dispersion and Mössbauer spectroscopy, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems [1]. In both cases, excess MnII promotes heterobimetallic cofactor assembly. In the absence of FeII, R2c cooperatively binds MnII at both metal sites, whereas R2lox does not readily bind MnII at either site. Heterometallic cofactor assembly is favored at substoichiometric FeII concentrations in R2lox. FeII and MnII likely bind to the protein in a stepwise fashion, with FeII binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of MnII by FeII at site 2.

References [1] Yu. Kutin et al., J. Inorg. Biochem. http://dx.doi.org/10.1016/j.jinorgbio.2016.04.019 (2016)

Plenary Lectures & Parallel Sessions

Parallel Session 10

NMR spectroscopy as a novel tool in hydrogenase research S. Rumpel1, C. Farès2, E. J. Reijerse1 and W. Lubitz1 1 Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany 2Max Planck Institute for Coal Research, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany E-mail: [email protected]

[FeFe] hydrogenases catalyze the reversible reaction of electrons and protons to molecular hydrogen. They are complex metalloenzymes whose active site consists of a [4Fe-4S]-cluster connected via the thiolate of a cysteine to a unique [2Fe] site. The smallest identified and spectroscopically intensely studied [FeFe]-hydrogenase is the 48 kDa HydA1. Apo-HydA1, containing only the [4Fe-4S]-cluster [1], was shown to be activated by addition of a [2Fe] site mimic [2]. We show here the first NMR study of a hydrogenase. The contact shifts of the protons proximate to the metal center contain intimate details about the metal center’s magnetic state at room temperature which are not available by any other technique. Consistent with presence of a reduced [4Fe-4S]-cluster about eight hyperfine shifted resonances were found for apo-HydA1 between 12-60 ppm. After activation and formation of the CO-inhibited state of HydA1 four new contact shifted signals appear between -2 to -35 ppm. These new signals are assigned to the four methylene protons of the azadithiolate bridge as the [2Fe] site contains two low spin irons and no other non-exchangeable protons. Currently, we use selective amino acid labelling and 13C-direct detection to assign the hyperfine shifted resonances. Thereby important new information about the catalytic mechanism of hydrogenases under native-like conditions may become available.

References [1] D. W. Mulder, Nature. 465, (2010) [2] J. Esselborn, Nat Chem Biol. 9, (2013)

Plenary Lectures & Parallel Sessions

Parallel Session 10

Advanced EPR spectroscopy at high fields/frequencies sheds light on the radical transfer in E. coli ribonucleotide reductase Müge Kasanmascheff,1 Wankyu Lee,2 Thomas U. Nick,1 JoAnne Stubbe,2 Marina Bennati1 1Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. 2Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Email: [email protected]

Ribonucleotide reductases (RNRs) are essential in life because they catalyze the synthesis of DNA building blocks in all living organisms. The E. coli RNR uses a catalytic cycle that involves proton-coupled electron transfer (PCET) over 35 Å and a specific pathway of redox active amino acids present both in α and β subunits.1 The nature of PCET steps at the α2β2 subunit interface is elusive. Therefore, multi-frequency (9, 34, 94 and 263 GHz) EPR, ENDOR, DEER and rapid-freeze-quench (RFQ)-EPR spectroscopies in combination with biochemical methods are used to investigate the subunit interface. Multi-frequency EPR suggested reorientation of the essential pathway tyrosine 731 that is trapped during catalysis.2 Furthermore, ENDOR revealed distortion of the stacked conformation of the two pathway tyrosines 731 and 730. Ultimately, DEER demonstrated a large conformational change of the trapped residue 731 towards subunit interface. This might be the key to drive the radical transfer chemistry at the subunit interface.2 Thus, we further investigated the interaction of

Y731 with the next redox active interface residue 356 via RFQ- and 263 GHz EPR techniques. Due to utility of these state-of-the art techniques, we, for the first time, could demonstrate the interaction between the two essential pathway residues over the subunit interface that involves a conformational change. References [1] U. Uhlin et. al., Nature, 370, 1994. [2] M. Kasanmascheff et. al., Chemical Science, 7, 2016.

Plenary Lectures & Parallel Sessions

7.19 Plenary Lectures 6

Plenary Lectures & Parallel Sessions

Plenary Lectures 6

Structure and dynamics of protein complexes by solid-state NMR J. R. Lewandowski University of Warwick, Coventry, UK E-mail: [email protected]

Solid-state NMR is quickly becoming a powerful approach, complementary to solution NMR, for studying structures and dynamics of protein complexes [1-5]. The absence of isotropic tumbling in the solid state removes the intrinsic size limitation, enabling experiments on even very large biomolecular complexes. However, to fully realize this potential further methodological developments are necessary to maximize the sensitivity and spectral resolution and enable site-specific quantitative applications. In this presentation we discuss a few recent developments in methodology and applications to study structures and dynamics of large protein complexes. We focus on applications involving fast magic angle spinning in the range from 60 to 100 kHz that enable quantitative studies on >300 kDa protein complex involving a few nanomole quantities of monomer domains.

References [1] J. M. Lamley, C. Öster, H. J. Sass, M. Rogowski, A. Oss, J. Past, A. Reinhold, S. Grzesiek, A. Samoson, J. R. Lewandowski, J. Am. Chem. Soc. 136, 16800 (2014) [2] J. M. Lamley, C. Öster, R. A. Stevens, J. R. Lewandowski, Angew. Chem. 54, 15374 (2015). [3] E. Barbet-Massin, C. T. Huang, V. Daebel, S. T. Hsu, B. Reif, Angew. Chem. 54, 4376 (2015). [4] P. Schanda, S. Triboulet, C. Laguri, C. M. Bougault, I. Ayala, M. Callon, M. Arthur, J. P. Simorre, J. Am. Chem. Soc. 136, 17852 (2014). [5] H. R. W. Dannatt, M. Felletti, S. Jehle, Y. Wang, L. Emsley, N. E. Dixon, A. Lesage, G. Pintacuda, Angew. Chem. 55, 6638 (2016).

Plenary Lectures & Parallel Sessions

Plenary Lectures 6

Intermediates and Interactions in Photo- and Organocatalysis Ruth M. Gschwind Universität Regensburg, Fakultät für Chemie und Pharmazie, Institut für Organische Chemie, Universitätsstr. 31, 93040 Regensburg E-mail: [email protected]

The detection and characterization of intermediates in catalytic reactions is crucial for the understanding of mechanisms and the rational optimization of reaction conditions. However, in many rapidly expanding fields of asymmetric catalysis, mechanistic studies as well as structural investigations on intermediates or intermolecular interactions are scarce compared to new synthetic applications. Therefore, in this talk mechanistic and structural studies about topics in photo- and organocatalysis will be discussed. The talk will start with our recent results in the field of photocatalysis. First our LED based NMR illumination device for mechanistic studies on photocatalytic reactions will be introduced, which turned out to be versatile and simple, yet surprisingly powerful.1 Next as applications of this LED device mechanistic studies about photocatalytic reactions with flavin2 and N-aryltetrahydroisoquinolines3 will be discussed. Both NMR reaction profiles and CIDNP studies allow for new insights, which are not only complementary to UV/Vis studies but crucial for the understanding of the mechanism. Both examples show that downstream intermediates and processes accessible on the NMR time scale can be decisive for the mechanistic pathway of photoreactions. In addition, these studies are the first examples revealing one- versus two-electron processes (flavin) or proton versus H-radical abstraction mechanisms in chemical photocatalysis. The second topic is ion pairing catalysis. There is a general lack of experimental structural data about small ion pairs in solution hampering the further development of this booming field in catalysis. On the example of Brønsted acid catalysis not only our recent results about the activation mode in achiral phosphoric acid/imine complexes4 will be presented but also new insights into the structures determining techniques, as well as the structures and hydrogen bond properties of famous chiral phosphoric acid/imine complexes.5

References [1] C. Feldmeier, H. Bartling, E. Riedle, R. M. Gschwind, J. Magn. Res., 232, (2013), 39-44. [2] C. Feldmeier, H. Bartling, K. Magerl, R. M. Gschwind, Angew. Chem. Int. Ed., 54, (2015), 1347-1351. [3] H. Bartling, A. Eisenhofer, B. König, R.M. Gschwind, J. Am. Soc. Chem. under revision. [4] Fleischmann, M.; Drettwan, D.; Sugiono, E.; Rueping, M.; Gschwind, R.M. Angew. Chem. Int. Ed. 2011, 50, 6364-6369. [5] Greindl, J.; Sorgenfrei, N.; Morana, F.; Hioe, J.; Gschwind, R.M. in preparation.

Plenary Lectures & Parallel Sessions

8. Poster Presentations

Biomolecules in liquid state P1 - P29

Relaxation and Dynamics P30 - P33

Biosolids P34 – P45

Hyperpolarization P46 – P50

Materials and Polymers P51 – P58

Small Molecules and Catalysis P59 – P75

Methods and Theory P76 – P83

Hardware, Instrumentation, Methodology & Imaging P84 – P87

Poster presentations

Biomolecules in liquid state Poster Presentation P1

NMR study of full-length HIV-1 Nef interacting with autophagy related proteins Ş. Akgül1,2, A. Boeske1,2, S. Hoffmann1, M. Stoldt1,2, P. Ma1,2 M. Schwarten1, D. Willbold1,2 1 Forschungszentrum Jülich ICS-6: Strukturbiochemie, Jülich Germany 2 Heinrich-Heine-Universität Düsseldorf Institut für Physikalische Biologie, Düsseldorf, Germany E-mail: [email protected]

Human immunodeficiency virus type 1 (HIV-1) protein Nef is a crucial accessory protein during disease progression and responsible for many pathogenic effects in acquired immunodeficiency syndrome (AIDS). It is able to interact with a number of host proteins, including such serving important roles in vesicular trafficking. We observed that Nef is able to directly interact with the GABA receptor associated protein family (GABARAPs), which are members of the of the human autophagy related protein 8 (ATG8) family. Interestingly, Nef is not able to interact with proteins of the LC3 family, another subfamily of the ATG8 protein family. Further we have data showing that Nef secretion requires the expression of GABA receptor associated proteins (GABARAPs). Our data suggests that tryptophan 13 in the N- terminal domain of Nef is crucial for the interaction with GABARAPs, pointing towards a sequence motif including tryptophan 13. Our aim is to reveal the binding site for the interaction within Nef and determine other important residues for this interaction on the molecular level by using liquid-state NMR spectroscopy. We started to further optimize purification and NMR-buffer conditions of the Nef-GABARAP complex. These studies will allow us to understand from the structural sight of view how Nef distinguishes the different ATG8 proteins, if a sequence motif is mediating the interaction and how to specifically interfere the interaction.

Poster Presentations

Biomolecules in liquid state Poster Presentation P2

Conformational changes in the ABC exporter TmrAB during nucleotide cycle probed with PELDOR

K. Barth1, R. Tampé2, T. F. Prisner1 and B. Joseph1 1Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue- Straße 7, 60438 Frankfurt am Main, Germany. 2Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany. E-mail: [email protected]

ATP-binding cassette (ABC) transport complexes use ATP binding and subsequent hydrolysis to translocate substrates across membranes.[1,2] The energy of ATP hydrolysis in the nucleotide binding domains (NBDs) gets converted into conformational changes in the transmembrane domains (TMDs) to make substrate uptake and release possible. ABC proteins are important targets in clinical research as their dysfunction causes various disease in humans. Despite a conserved architecture consisting of two nucleotide binding domains (NBDs) linked to two transmembrane domains (TMDs) in both ABC importers and exporters, recent studies suggest that there could be considerable mechanistic differences. Here we investigated the heterodimeric ABC exporter TmrAB[1,2] from Thermus thermophilus by electron paramagnetic resonance (EPR) spectroscopy. Nitroxide spin labels were attached at key positions in the transporter and allosteric coupling between NBDs and the TMDs as modulated by the nucleotides was monitored using PELDOR (DEER). Our results reveal that ATP hydrolysis, not just ATP binding is necessary to flip TmrAB from an inward-facing to outward-facing conformation. The extent of opening of the periplasmic gate in the outward- facing conformation is influenced by the surrounding environment, showing a larger opening in proteoliposomes.

References [1] A. Zutz, et al., J. Biol. Chem. 286, (2011) [2] J. ter Beek, et al., J. Gen. Physiol. 143, (2014)

Poster Presentations

Biomolecules in liquid state Poster Presentation P3

Photoresponsive formation of an intermolecular minimal G- Quadruplex motif J. Thevarpadam1, I. Bessi2, O. Binas2, D.P.N. Goncalves1, C. Slavov3, H. Jonker2, C. Richter2, J. Wachtveitl3, H. Schwalbe2, A. Heckel1

1Heckel group, Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt am Main 2Schwalbe group, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt am Main 3Wachtveitl group, Institute for Physical and Theoretical Chemistry, Frankfurt, Germany

E-mail: [email protected]

Intermolecular G-Quadruplex structures can act as building blocks for nanotechnological applications, ligating guanine-rich DNA strands in hoogsteen-bond G-tetrads.[1] The incorporation of an Azobenzene moiety into a DNA strand enables light control of the system, through isomerization of the Azo-group.[2] Screening different Azobenzene-DNA constructs, GG-Az1-GG could be identified as promising system, forming one specific G-Quadruplex structure, that unfolds under UV- irradiation. The construct was therefore structurally investigated, utilizing NMR-methods involving NOE- and through-bond correlation, showing a highly symmetric two tetrad Quadruplex structure with two lateral loops. An NMR-structure of GG-Az1-GG could be derived from NOE data and angular restraints, obtained from E.COSY and HSQC experiments. To show the capability of the system, to couple larger DNA, a model system with a 20 nt Duplex attached to GG-Az1-GG was analyzed by 1D-NMR screening.

References [1] G. Mayer, L. Kröck, V. Mikat, M. Engeser, and A. Heckel, ChemBioChem, 6, (2005) [2] Junji Zhang, Jiaxing Wang and He Tian, Mater. Horiz., 1, (2014)

Poster Presentations

Biomolecules in liquid state Poster Presentation P4

Characterization of Inverse Agonist Structure and Function in MC4R Signalling J. Borggräfe1,2, S. Arens1, A. Viegas1, M. Falke1, and M. Etzkorn1,2 1Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Jülich, Germany

E-mail: [email protected]

The melanocortin receceptor 4 (MC4R) is expressed in the hypothalamus and plays a keyrole in the regulation of food intake and energy homeostasis. Proopiomelanocortin (POMC)- derived agonists of MC4R trigger anorexigenic signals whereas its inverse agonist Agouti- related protein (AgRP) and the homolog Agouti signalling protein (ASIP) trigger orexigenic signals. Down-stream signal transduction of MC4R normally involves coupling with different G-proteins, but recent studies also suggest a G-protein independent pathway [1]. AgRP is a 132 aa protein with an unstructured N-terminus and a cysteine-rich C-terminal domain [2]. Deletion or substitution of single loops between AgRP and ASIP alters the function of the inverse agonists and their selectivity to different melanocortin receptors [3]. We are developing recombinant large scale production of (isotope labelled) agouti proteins and their variants using expression into inclusion bodies with subsequent refolding. Several biophysical methods are applied to test protein folding and function, including HPLC, CD, Raman and NMR spectroscopy, as well as fluorescence polarisation and downstream signalling assays. We anticipate that further structural investigations of the ligands and their receptor binding using solution NMR spectroscopy can provide new insights in the complex molecular processes of MC4R signalling.

References [1] Ghamari-Langroudi et al., Nature Chemical Biology, 520, (2015) [2] Rosenfeld et al., Biochemistry, 37, (1998) [3] Patel et al., Journal of Molecular Biology, 404, (2010)

Poster Presentations

Biomolecules in liquid state Poster Presentation P5

NMR Studies into Binding of N-Acetylneuraminic Acid to Norovirus Coat Protein R. Creutznacher1, A. Mallagaray1, R. J. Woods2, A. Sood2 and T. Peters1 1Institute of Chemistry, University of Lübeck, Germany 2Complex Carbohydrate Research Center, University of Georgia, USA E-mail: [email protected]

Human Noroviruses (NV) are the main cause of acute gastroenteritis worldwide, being directly responsible for more than 750.000 deaths a year [1]. NV recognize histo blood group antigens (HBGAs) as cellular attachment factors. The host-cell entry mechanism is not yet known. We have recently demonstrated that the binding of NV to HBGAs is a complex multi- step process allowing fine-tuning of NV – host-cell interaction. L-fucose has been identified as minimal binding motif [2,3]. There is evidence that N-acetylneuraminic acid (Neu5Ac) as part of e.g. gangliosides could also serve as recognition element for NV [4]. Here, we show that Neu5Ac indeed binds to NV coat protein. Modeling predicts Neu5Ac binding to the L- fucose site. We have used the protruding domain (P-dimers, 70 kDa) of GII.4 NV as target protein. Using a combination of STD NMR, 1H-15N TROSY NMR chemical shift perturbation experiments, and computational docking models we show that Neu5Ac and L-fucose bind to a shared binding site of P-dimers. Competition experiments support overlap of binding pockets for L-fucose and Neu5Ac. These findings will have an impact on understanding the infection process at molecular level.

References [1] S. Ahmed et al., Lancet Infect. Dis. 14: 725-730 (2014) [2] A. Mallagaray et al., Angew. Chem. Int. Ed. 54: 12014-12019 (2015) [3] A. Mallagaray et al., Glycobiology (2016) accepted [4] L. Han et al., JACS 136: 12631-12637 (2014)

Poster Presentations

Biomolecules in liquid state Poster Presentation P6

1H NMR-based Metabonomics of CSF in Post-Operative Cognitive Dysfunction (POCD) M. D. Díaz1, M. K. Parr2, O. Peters3, C. Schipke4, B. Albrecht1 and N. E. Schlörer1 1 NMR facility, Department of Chemistry, University of Cologne, Germany 2Institute of Pharmacy,Free University of Berlin, Germany 3Department of Psychiatry, Charité – Campus Benjamin Franklin, Berlin, Germany 4Institute of Neuropathology, Charité, Berlin, Germany E-mail: [email protected]

The use of NMR spectroscopy as a chemometric tool has opened a wide window of new applications.[1] As an especially attractive field appears its implementation in clinical screening, which is a potential aim of the study presented. A certain group of patients which underwent surgery or electroconvulsive therapy later-on suffers from cognitive deficits known as post-operative cognitive dysfunction (POCD). These consequences can outbalance positive effects of the treatment and assessment of the individual risk for POCD would be therefore an important advantage to plan a treatment. As starting point for a non-targeted analysis of patient and control samples, cerebrospinal fluid (CSF) was selected,[2] which also allows for targeted analysis of biomarkers. Initial results and a new approach for the sample preparation protocol[3] will be presented.

References [1] J. K. Nicholson, J. C. Lindon, Nature 2008, 455, 1054 [2] D. S. Wishart et al., J. Chromatogr. B 2008 871(2), 164 [3] M. Malet-Martino et al., Metabolites 2014, 4, 115

Poster Presentations

Biomolecules in liquid state Poster Presentation P7

The use of amphipols for refolding and NMR structural characterization of 7-TM proteins S. Elter1, S. Arens1, A. Viegas1, V. Gelev4, G. Wagner3 and M. Etzkorn1,2 1Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Jülich, Germany 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA 4Faculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria

E-mail: [email protected]

Amphipols (amphipathic polymers) have demonstrated great potential as suitable membrane substitutes. Among the reasons that favor amphipols over conventional detergent based surfactants, are their ability to increase stability of the embedded membrane protein as well as their good refolding properties, in particular for seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) [1]. In this study we characterize the effects of amphipol assisted refolding of different 7-TM proteins using size-exclusion chromatography, time-resolved absorption spectroscopy and solution-state NMR. The investigated proteins include Bacteriorhodopsin (BR) as well as two class-A GPCRs, i.e. the melanocortin-2 and melanocortin-4 receptors (MC2R and MC4R)[2,3]. A home build cell-free expression system was used to produce mg amounts of the different 7-TM proteins in an unfolded state and the effects of amphipol as well as cofactor concentrations on BR refolding rates were characterized. Additionally, NMR data of unfolded BO and fully folded BR were recorded and allow initial insights into the molecular arrangements of both states. These results will guide our efforts towards a better understanding of membrane protein folding.

References [1] Zoonens and J.L. Popot, J Membr Biol, 247, (2014) [2] Elter et al., J Membr Biol, 247, (2014) [3] Etzkorn et al., Structure, 21, (2013)

Poster Presentations

Biomolecules in liquid state Poster Presentation P8

Expression studies of a GPCR in different insect cells for NMR studies Marcel Falke1, Sabine Arens1, Reza Ahmadian2 and Manuel Etzkorn1 1Institut für Physikalische Biologie, Düsseldorf, Germany 2Institut für Biochemie und Molekularbiologie II, Düsseldorf, Germany E-mail: [email protected]

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signalling proteins, and they are considered major targets of approximately half of all therapeutic agents [1]. Despite their pharmacological importance, detailed information about the structure and function of GPCRs remains insufficient. NMR structural studies on GPCRs are challenging, this is related to low expression yields, difficulties in their refolding and/or lack of stability when incorporated into a non-native membrane mimetic. Melanocortin-4 receptor (MC4R), a prototypical GPCR, plays an important role in the control of energy homeostasis and is directly linked to obesity [2]. So far, no high resolution structural information is available for MC4R. The MC4R is an interesting target for NMR not only because of its important biological function but also because it is one of the smallest GPCRs known. Here, we report on expression results for the high-yield production of MC4R in the two insect cell lines Sf9 and Tnao38, as well as, first purification studies in detergent environment. In addition, first medium tests have been done for establishment of isotope labelling in insect cells.

References [1] A. L. Hopkins, Nat Rev Drug Discov. 1, (2002) [2] D. Huszar, Cell. 88, (1997)

Poster Presentations

Biomolecules in liquid state Poster Presentation P9

NMR Studies of large proteins using specific isotopic labeling of methyl group. P. Gans1 , G. Mas1, R. Kerfah2, I. Ayala1 , R. Sounier1, E. Crublet2, O. Hamelin3 and J. Boisbouvier1 1IBS, Grenoble, France. 2NMR-Bio, Grenoble, France. 3iRTSV/LCBM, Grenoble, France.

E-mail:[email protected]

Large protein complexes are involved in key cellular processes but their study by NMR has been a real challenge for a long time. The strategy of specific isotope labeling of methyl groups in a perdeuterated protein has however significantly extended the frontier of liquid state NMR. Perdeuterated protein with selective protonation of the methyl groups can be produced by adding the specifically labeled metabolic precursors in the fully deuterated culture medium before protein expression. We have exploited metabolic pathways in E. coli and synthesized new isotope-labeled precursors to extend the library of methyl labeling methods. This allows the stereospecific labeling of the prochiral methyl groups of Leucine and Valine as well as the regioselective labeling of Isoleucine methyl probes. New protocols have been developed to extend and optimize specific labeling approaches to methyl groups of Methionine and Threonine residues, as well as Alanine residues. Using these new tools, we have setup robust and scrambling-free protocols to label in targeted proteins any combination of methyl groups. These specific labeling strategies are particularly adapted to extract precise long-range NOE restraints between remote probes separated by up to 12 Å. We will show here uses of these labeling strategies in order to obtain structurally meaningful long-range NOEs in large proteins.

Poster Presentations

Biomolecules in liquid state Poster Presentation P10

Recognition of dimethylarginine by the SMN Tudor domain involves conformational selection Gerd Gemmecker1,2, Shreyas Supekar3, Kostas Tripsianes4, Ville Kaila3, Michael Sattler1,2 1Baverisches NMR-Zentrum, Dept. Chemie, TU München, Garching, Germany 2 Institute for Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany 3Computational Biocatalysis, Chem. Dept., TU München, Garching Germany 4CEITEC, Masaryk University, Brno, Czech Republic

E-mail: [email protected]

Tudor-domains are the only known proteins that bind symmetrically N-dimethylated arginine (sDMA), a post-translationally modified amino acid central for regulating RNA processing in eukaryotic cells. The ligand-binding sites of these proteins are highly specific, comprising a characteristic aromatic cage containing several aromatic side chains.1 Here we discuss the energetics and dynamics of the conformational selection mechanism of sDMA in solution and in the basic Tudor domain of human survival motor neurons (SMN) protein. Using combined dynamic NMR spectroscopy and quantum chemical calculations, we can show that sDMA isomerizes rapidly in solution between the anti-syn and anti-anti configurations of its N-methyl groups. Although the anti-anti conformation is present in <1% in solution, the SMN Tudor domain has the remarkable ability to conformationally select this minor conformation in the recognition process. We propose that the aromatic cage in the SMN leads to a stronger cation–π interaction within the active site, and can result in significant selectivity for anti-anti sDMA vs. the anti- syn conformation. We are now studying mutations in the aromatic cage that could tune the energetics between the two conformations. Our findings reconcile structural differences observed between the anti-anti sDMA-binding basic SMN Tudor domain and the anti-syn sDMA-binding extended Tudor domains.1

References [1] Tripsianes et al., M., Nature Struct. Mol. Biol. 18, 1414–1420 (2011)

Poster Presentations

Biomolecules in liquid state Poster Presentation P11

Analysis of conformational changes in the substrate binding protein of a TRAP transporter from V. cholerae by PELDOR spectroscopy and X-ray crystallography J. Glaenzer1 , M. Peter1, G.H. Thomas2, G. Hagelueken1 1Structure, Function Dynamics of Macromolecules, Institute for Physical and Theoretical Chemistry, University of Bonn 3Department of Biology, University of York E-mail: [email protected]

Tripartite ATP-independent periplasmic (TRAP) transporters are an important group of membrane transporters in bacteria. They are composed of two transmembrane domains and a periplasmic substrate binding protein (SBP). The SBP scavenges the substrate (e.g. sialic acids), undergoes conformational change and delivers it to the transporter. Release of the substrate reverses the SBP into its unbound, open conformation. The molecular details of the interaction between the SBP and the domains are still part of current studies. However, for an efficient transport process, only the substrate loaded (closed) SBP should bind to the transporter. It is thus important to investigate whether the conformational changes of the SBP are strictly substrate induced or if the molecule constantly samples the open-, closed- or possibly intermediate conformations. We aim to answer this question using EPR distance measurements on the SBP SiaP from V. cholerae. We used the “difference distance matrix” feature of mtsslWizard to find optimal spin labeling positions on the molecular surface of SiaP. The amino acid residues of SiaP were then mutated in pairs to cysteines at the optimal positions and labeled with MTSSL. High quality PELDOR data of the spin-labeled SBP were recorded in the presence and absence of its substrate (Neu5Ac). Our results reveal new insights into the conformational changes of the SBP in frozen solution and the interactions which are critical for substrate binding.

Poster Presentations

Biomolecules in liquid state Poster Presentation P12

An artificial amyloid-β dimer A tool to resolve the structures of the earliest assemblies and aggregation processes F. Hasecke1, D. Schölzel2, E. Reinatz1, P. Neudecker2, H. Heise1,2, W. Hoyer1 ¹ Institute of Physical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany ² Institute of Complex Systems, ICS-6: Structural Biochemistry, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany E-mail: [email protected]

The accumulation, aggregation, and patho-mechanisms of the amyloid-β (Aβ) peptide as the most widely accepted causative molecular agent of Alzheimer's disease are subject to intense studies. From a biophysical perspective, besides the exact structure of the large, fibrillar aggregates formed by Aβ, the aggregation-initiating process of dimerization of single Aβ peptides is still not resolved. Here we present an artificial Aβ dimer (diMAβ) as a tool to study the dimerization process and aggregation behavior of Aβ. Two Aβ1-40 subunits were connected head-to-tail via a [G₄S]₄ linker. Analysis by heteronuclear NMR spectroscopy revealed that the subunits of diMAβ act as independent monomers with the characteristics of intrinsically disordered peptides. Furthermore, diMAβ exhibits a dramatically increased aggregation propensity compared to monomeric Aβ40 and forms fibrillar aggregates as seen in AFM. Consequently, dimerization appears to be a critical step in amyloid aggregation.

Poster Presentations

Biomolecules in liquid state Poster Presentation P13

Characterizing and functionalizing the nanodiscs system L. Heid1, T. Viennet1,2, S. Elter1, A. Viegas1 and M. Etzkorn1,2 1Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Jülich, Germany

E-mail: [email protected]

Lipid bilayer nanodiscs can provide a close-to-native environment for the investigation of membrane proteins and offer promising potential for NMR structural studies [e.g. 1,2,3]. Nanodiscs are formed by two copies of a membrane scaffold protein (MSP) and suitable lipids. Several MSP variants are available, including NMR-optimized constructs [4]. Albeit its great potential and increased usage, the structural details of the molecular assembly of nanodiscs is not fully understood. We establish and test a series of selected scaffold proteins with cysteine point mutations aiming to (i) allow a better structural characterization of the system, (ii) identify possible interaction of embedded proteins and the scaffold proteins (e.g. using FRET and paramagnetic relaxation NMR experiments), and (iii) functionalize the nanodiscs (e.g. using fluorescence and targeted-DNP [5] probes).

References [1] Raschle et al., Curr. Opin. Struc. Biol., 20, (2010) [2] Etzkorn et al., Structure, 21, (2013) [3] Kofuku et al., Angewandte Chemie, 53, (2014) [4] Hagn et al., JACS, 135, (2013) [5] Viennet et al. Angewandte Chemie, in press (2016)

Poster Presentations

Biomolecules in liquid state Poster Presentation P14

The Proton Fumarate Symporter SLC26Dg Investigated by EPR Spectroscopy E. A. Jaumann1, Y.-N. Chang2, T. F. Prisner1, E. R. Geertsma2 and B. Joseph1 1Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Frankfurt/M, Germany 2Institute of Biochemistry, Goethe University Frankfurt, Frankfurt/M, Germany E-mail: [email protected]

The solute carrier family 26 (SLC26) comprises secondary anion transporters whose dysfunction in humans leads to a plethora of diseases. Recently, the first crystal structure of a prokaryotic homolog SLC26Dg from D. geothermalis has been determined [1]. SLC26Dg is a H+-fumarate symporter. The membrane domain of SLC26Dg is monomeric and contains 14 transmembrane helices constituting a core and a gate domain. In the crystal structure, the cytoplasmic STAS domain is located at an unnatural position that would place it in the lipid bilayer. We use site-directed spin labeling combined with pulsed EPR spectroscopy (PELDOR, also known as DEER) [2,3] to investigate the mechanism of substrate translocation in SLC26Dg. In this respect, spin labeling of single and double cysteine mutants was optimized and initial PELDOR experiments were performed in detergent micelles and proteoliposomes. Our results suggest the formation of dimers in the lipid bilayer with the gate domains acting as the interface. Interestingly, RT CW-EPR spectroscopy of spin labels on the STAS domain revealed significant changes in mobility between detergent and liposome environments. We are performing further PELDOR measurements to define the dimer interface and the STAS domain orientation in the membrane environment.

References [1] E.R. Geertsma et al., Nat. Struct. Mol. Biol. 22, (2015) [2] A.D. Milov et al., Chem. Phys. Lett. 110, (1984) [3] M. Pannier et al., J. Magn. Reson. 142, (2000)

Poster Presentations

Biomolecules in liquid state Poster Presentation P15

Molecular Basis of Microtubule Regulation by Microtubule- Associated Protein Tau

Harindranath Kadavath1,2, Mariusz Jaremko1, Łukasz Jaremko1,2, Romina Vanesa Hofele1, Jacek Biernat3, Satish Kumar3, Katharina Tepper3, Henning Urlaub1, , Eckhard Mandelkow3, & Markus Zweckstetter1,2,4

1 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 2 German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany 3 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 4 Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center, Göttingen, Germany

Microtubules regulate cell division, cell morphology, intracellular transport and axonal stability and therefore play crucial roles in cell function. The structure, dynamic behavior and spatial organization of microtubules in neurons are regulated by microtubule-associated proteins. The microtubule-associated protein Tau promotes formation and stabilization of axonal microtubules and thus influences intracellular transport, axonal stability and cell morphology. In Alzheimer’s disease the interaction of Tau with microtubules is impaired.

Despite the importance of the regulation of microtubule structure and dynamics by Tau, very little is known about the interaction of Tau and other microtubule-associated proteins with microtubules. To fill this gap, we studied the interaction of Tau with microtubules using a combination of NMR spectroscopy and mass spectrometry. We show that Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules (1). We further show that Tau promotes microtubule assembly by binding to protofilaments at the interface between α-β-tubulin heterodimers using small groups of evolutionary conserved residues (2). The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Collectively, our study establishes a conserved mechanism of microtubule polymerization and thus regulation of axonal stability and cell morphology by microtubule-associated protein Tau.

References 1. Kadavath H, et al. (2015) Folding of the Tau Protein on Microtubules. Angew. Chem. Int. Ed. Engl. 54(35):10347-10351. 2. Kadavath H, et al. (2015) Tau stabilizes microtubules by binding at the interface between tubulin heterodimers. Proc. Natl. Acad. Sci. USA 112(24):7501-7506.

Poster Presentations

Biomolecules in liquid state Poster Presentation P16

Self-diffusion and thermally induced aggregation of eye-lens 1 crystallins studied by H-PFG-NMR L. Löser, S. Link, J. Balbach, K. Saalwächter, A. Krushelnitsky1 1Martin-Luther-Universität Halle-Wittenberg, Faculty of Natural Science II, Betty-Heimann- Str. 7, 06120 Halle (Saale) E-mail: [email protected] a-crystallin is one of the main constituents of mammalian eye lenses. This protein possesses the so-called chaperon-like activity, which means preventing forming large aggregates of other eye lens crystallins (β and g) upon affecting different adverse factors. In this way, a- crystallin keeps lens transparent. Malfunction of a-crystallin leads to aggregation and precipitation of lens proteins known as cataract. The conventional experimental technique for studying chaperon-like activity of a-crystallin is optical spectroscopy that allows detecting aggregation kinetics in real time. In this contribution we present 1H pulsed field gradient (PFG) NMR technique as an alternative approach for studying the aggregation kinetics. PFG NMR provides the self-diffusion coefficients that depend on the size of Brownian particles. Although PFG NMR is a less sensitive experiment, it enables observing the particles of different size separately and to see whether different proteins form stable aggregates or not. This method was applied to studying the thermal denaturation and aggregation of gβ- crystallin in the absence and presence of bovine α-crystallin in solution. These two proteins have very different sizes, they produce different components in the diffusion decay and thus can be observed separately. Our data demonstrate that the mechanism of the α-crystallin chaperon-like activity may depend on the protein concentration.

Poster Presentations

Biomolecules in liquid state Poster Presentation P17

Attachment of Norovirus to Histo Blood Group Antigens: A cooperative multi-step process A. Mallagaray1, J. Lockhauserbäumer2, C. Rademacher3, F. Parra4, G. S. Hansman5, C. Uetrecht2 and T. Peters1 1Center of Structural and Cell Biology in Medicine, Institute of Chemistry, University of Lübeck, Lübeck, Germany. 2Dynamics of Viral Structures, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany. 3Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Postdam, Germany. 4Universidad de Oviedo, Instituto Universitario de Biotecnología de Asturias, Oviedo, Spain. 5Schaller Rearch Group at the University of Heidelberg and the DKFZ & Department of Infectious Diseases and Virology, University of Heidelberg, Heidelberg, Germany. E-mail: [email protected]

Human noroviruses recognize histo blood group antigens (HBGAs) as cellular attachment factors. So far, independent and equivalent multiple binding sites were held responsible for viral attachment. Here we present studies into the binding of GII.4 Saga, GII.4 Ast6139, and GII.10 Vietnam026 norovirus protruding domains (P-dimers) and virus-like particles (VLPs) to HBGAs at atomic resolution. Using NMR experiments and native mass spectrometry we have shown that binding of HBGAs to norovirus P-dimers is a cooperative multi-step process revealing four instead of two HBGA binding sites per P-dimer[1]. A crystallographic study has then localized these extra binding pockets in the case of P-dimers of the GII.10 Vietnam026 strain.[2] It is an obvious question whether similar results will be obtained for virus particles, too. For VLPs, the binding of HBGAs involves more than four cooperatively coupled binding steps, suggesting the presence of additional hitherto unidentified L-fucose binding sites on capside surface.[3] We propose that at least six distinct and cooperatively coupled HBGA binding sites are present on the surface of GII.4 or GII.10 VLPs. These findings should have implications in understanding the first steps of the infection process and in the design of novel entry inhibitors.

References [1] A. Mallagaray et al. Angew. Chem. Int. Ed. 54, 12014-12019 (2015). [2] A. D. Koromyslova et al. Virology 483, 203-208 (2015). [3] A. Mallagaray et al. Glycobiology (2016), accepted.

Poster Presentations

Biomolecules in liquid state Poster Presentation P18

Molecular recognition of cellulose and cellulose fragments carbohydrate-binding modules Marcus Michaelis1, Ruslan Nedielkov1, Robert Creutznacher2, Amir Goldbourt3, and Heiko M. Möller1 1University of Potsdam, Institute of Chemistry, Karl-Liebknecht-Straße 24-25, D-14476 2University of Lübeck, Institute of Chemistry, Ratzeburger Allee 160, D-23562 3Tel Aviv University, School of Chemistry, Ramat Aviv, IL-69978

E-mail: [email protected]

For the degradation of the plant cell wall various enzymes are working synergistically in a multi-enzyme complex known as the cellulosome. Major components of the cellulosome are the carbohydrate-binding modules (CBMs) which have a characteristic fold and possess binding activity towards various carbohydrate structures. The carbohydrate binding property makes CBMs accessible to a broad spectrum of applications like biofuel production, targeting of functional molecules to materials containing cellulose, cell immobilization or characterization of fibers [2]. An attractive group of CBMs is the CBM family 3 that binds crystalline cellulose. Until today no 3D structure of a cellulose-bound carbohydrate-binding module has been solved and the current binding mode is based on the structures of unbound CBMs and molecular dynamics simulations. To better understand the binding mode we investigate the CBM3b domain of cellobiohydrolase 9A from Clostridium thermocellum (CBM3b-Cbh9A) and its carbohydrate binding mutant N126W by a combination of solid state and solution NMR spectroscopy [3]. The resonance assignments of CBM3b-Cbh9A and its mutant as well as initial binding studies will be presented.

References [1] S. Jindou et al., Acta Cryst. F63, 1044-1047 (2007) [2] O. Shoseyov et al., Microbiol Mol Biol Rev. 70(2), 283-295 (2006) [3] H. Ivanir and A. Goldbourt, J Biomol NMR. 59, 185-197 (2014)

Poster Presentations

Biomolecules in liquid state Poster Presentation P19

Focal adhesion targeting mediated by the protein Paxillin A. Prestel1, C. Paone2, C. Hauck2, and H. M. Möller1, 1Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany 2Department of Biology, University of Konstanz, 78457 Konstanz, Germany E-mail: [email protected]

Integrin-mediated cell migration is of key importance for fundamental physiological processes like embryonic development, tissue homeostasis or leucocyte trafficking [1]. Integrin clustering mediated by interactions with the extracellular matrix induces the formation of focal adhesions (FA) that physically link integrins to the actin cytoskeleton [2]. Paxillin is a major component present in early focal adhesions [3] and, as a transducer of integrin signalling, it is important for embryonic development and linked to cancer progression [4]. We combined NMR spectroscopic, microscopic and biochemical approaches in order to dissect the molecular mechanism of how the LIM domains mediate the recruitment of paxillin to FAs. First, we solved the solution structure of a tandem construct comprising of paxillin’s LIM2 and LIM3 domains. We then identified the cytoplasmic domain of β-integrins to interact with paxillin’s LIM domains. These findings are corroborated by cell adhesion assays and fluorescence microscopy studies. Taken together our results provide a structural basis for focal adhesion targeting of paxillin.

References [1] Winograd-Katz SE, Fässler R, Geiger B, Legate KR, Nat Rev Mol Cell Biol 15:273-88 (2014). [2] Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM, Nature 468:580-4 (2010). [3] Scales TM, Parsons M, Curr Opin Cell Biol 23:562-8 (2011). [4] Zhao CJ, Du SK, Dang XB, Gong M, Med Sci Monit 21:1989-95 (2015).

Poster Presentations

100

Biomolecules in liquid state Poster Presentation P20

Temperature dependency of 19F chemical shifts in aqueous solution M. Plaumann1 , R. Ringleb1, F. Euchner1, C. Bruns1, M. Munt1, T. Herrmann1, U. Bommerich1 and J. Bernarding1 1Otto-von-Guericke University Magdeburg, Department for Biometrics and Medical Informatics, Magdeburg, Germany, Country E-mail: [email protected]

The number of fluorinated substrates with pharmaceutical applications strongly increased in the last years.[1] Important topics are the development of new MR contrast agents and the detection of fluorine nuclei in living organisms, e.g. for metabolisms studies of drugs by using MR techniques.[2,3] 19F MR spectroscopy allows the determination of changes in temperature or in pH values because of a wide chemical shift range. These are important sizes in the detection of tumour cells and in cancer therapy. Here, we present novel MR spectroscopic data, which describe the temperate dependency of the 19F chemical shift signals of several fluorinated organic substrates in aqueous solution - D2O as well as isotonic saline solution. As model compounds substrate like trifluoroethanol, 3-fluoropyridinine, 3-fluoro-D,L- tyrosine, 2,6-difluoronicotinic acid and 2-fluoro-4-(trifluoromethyl)-pyridine-3-carboxylic acid were chosen. The temperature was varied in the range of 300 K to 330 K. The results of these measurements show the influence of the presence of ions in aqueous solution. Furthermore, information concerning the relation of molecular structure and 19F chemical shifts were achieved. This will be important i.e. for future hyperthermia studies.

References [1] J. Wang et al. Chem. Rev. 114, 2432−2506 (2014) [2] J. Ruiz-Cabello et al., NMR Biomed. 24(2), 114-129 (2011) [3] Z.-X. Jiang et al., Angew. Chem. Int. Ed. 48, 4755–4758 (2009)

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101

Biomolecules in liquid state Poster Presentation P21

High-pressure NMR and stability of protein deposits in neurodegenerative diseases N. Rezaei-Ghaleh1, M. Amininasab2, S. Kumar3, J. Walter3, and M. Zweckstetter1,4,5 1German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany 2Department of Cell and Molecular Biology, University of Tehran, Tehran, Iran 3Department of Neurology, University of Bonn, Bonn, Germany 4Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 3Department of Neurology, University Medical Center, University of Göttingen, Göttingen, Germany E-mail: [email protected]

Protein aggregation is a pathological hallmark of neurodegenerative diseases. A remarkable feature of neurodegenerative pathology is its rapid spreading in human brains. Recently, we have used high pressure NMR to investigate the stability of amyloid-β (Aβ) aggregates against pressure-induced monomer dissociation [1]. Using Ser-8 phosphorylation as an example of a posttranslational modification, it was shown that the Aβ modification could modulate the stability of Aβ deposits, even when the site of modification was outside the structured core of Aβ fibrils. Quantitative analysis of Aβ monomer release data showed that the Ser-8 phosphorylation increased both the kinetic and thermodynamic stability of Aβ aggregates against pressure-induced monomer dissociation. Molecular dynamics simulation data showed that, in the absence of Aβ monomer release, the phosphorylated Aβ fibrils had higher compressibility than the non-phosphorylated fibrils, suggesting a potential mechanism for their lower susceptibility against pressure-induced monomer dissociation. Our data demonstrate the power of high-pressure NMR for quantitative determination of the stability of neurodegeneration-related protein deposits. In addition, they support an important role of posttranslational modifications in modulating the stability of protein deposits and thereby influencing the initiation and spreading of neurodegenerative pathology.

References [1] N. Rezaei-Ghaleh et al., Nat Commun. 7, (2016)

Poster Presentations

102

Biomolecules in liquid state Poster Presentation P22

Metabolomics in neutrophil activation B. Richer1, N. Salei2, T. Laskay2 and K. Seeger1 1Institute of Chemistry, University of Lübeck, Germany 2Department of Infectious Diseases and Microbiology, University of Lübeck, Germany E-mail: [email protected]

Neutrophil activation is an important part of host defence against pathogens. Chronic inflammation and autoimmunity are often associated with neutrophil abnormalities in phenotype and functions. Since effector functions of immune cells during inflammation are tightly linked to their metabolic state [1], we aim to analyze neutrophil metabolome upon activation. For this purpose, human neutrophils from healthy blood donors (n=6) were treated either with TNF-alpha or with LPS, whereas untreated neutrophils were used as a control. Since apoptotic cells are abundant at the site of inflammation, we additionally aimed to analyze the metabolome of apoptotic neutrophils. The water-soluble metabolites from 20 million neutrophils were extracted and lyophilised.

Noesypr1d-experiments were performed in D2O. The resulting spectra were processed and statistically analysed. Discrimination of the four groups was achieved by PCA in principal components 1 and 2. Preliminary data revealed differences in lactate amount between control and pro- inflammatory-treated neutrophils. In addition, NAD, ATP and lactate contributed to discrimination between apoptotic and non-apoptotic neutrophils. For identification of batch effects and to clarify the differences the study is currently repeated.

Based on our results we would like to further investigate the metabolic pathways behind neutrophil activation.

References [1] Pearce EL, Immunity 38, (2013)

Poster Presentations

103

Biomolecules in liquid state Poster Presentation P23

Structural Insights into Dynamic Processes of Hormone- Melanocortin-4 Receptor Interactions S. Schriek1 , M. Falke1, J. J. Yu2, R. Ahmadian2, G. Wagner3, M. Etzkorn1 1Institute of Physical Biology, Heinrich-Heine-University, Düsseldorf, Germany 2 Institute of Biochemistry and Molecular Biology II, Heinrich-Heine-University, Düsseldorf, Germany 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA E-mail: [email protected]

Melanocortin-4 Receptor (MC4R), a prototypical G protein-coupled receptor, is an important player in regulation of energy homeostasis and directly linked to obesity [1]. High-resolution structural insights into the pharmacological highly relevant ligand binding events between melanocortins and MC4R are, however, still limited. Here we report on progress to understand structural and dynamical details underlying the hormone-MC4R interactions using NMR spectroscopy and biochemical assays. To characterize signaling properties of the receptor expressed in mammalian and insect cells as well as hormones expressed in E.coli, we design and apply ligand binding and downstream activation assays. In addition, NMR results could be obtained on (isotope labeled) hormones and their interaction with membrane mimetics such as conventionally used detergents [2] and different nanodiscs.

References [1] I. S. Farooqi, N Engl J Med. 348, (2003) [2] W. Lee, BBRC. 456 (2015)

Poster Presentations

104

Biomolecules in liquid state Poster Presentation P24

Anti-aggregation Approach to Biotech Proteins M. Schulte1,2, P. Neudecker1,2, P. Skoczinski3, C. Haist3, A. Knapp3, M. Dick4, T. Classen4, K.- E. Jaeger4, J. Pietruszka4 and D. Willbold1,2 1Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf,40225 Düsseldorf, Germany 2Institute of Complex Systems, ICS-6 (Structural Biochemistry), Forschungszentrum Jülich, 52425 Jülich, Germany 3Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52425 Jülich, Germany 4 Institute of Bioorganic Chemistry, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52425 Jülich, Germany

E-mail: [email protected]

Undesired protein aggregation is a major problem hampering production, storage and application of biotech-relevant enzymes. Using various methods, mostly based on NMR spectroscopy, we aim to identify residues that are involved in the self-aggregation “hotspots”. Identification of these residues allowed us to rationally design enzyme variants with reduced or to eliminated aggregation without prior knowledge of the 3D structure. We are currently applying this approach to Lipase A from B. subtilis, a monomeric enzyme that is aggregation prone, and the K58E mutant of deoxyribose-5-phosphate aldolase (DERA) from E. coli as a model system, which exists in a monomer-dimer equilibrium. For the B. subtilis Lipase A we observed that the majority of the aggregation hotspots are hydrophobic amino acids, which suggests that aggregation may be triggered by hydrophobic interactions. Therefore, our mutation strategy aimed to reduce hydrophobicity by mutating large hydrophobic side-chains into small hydrophobic or hydrophilic side-chains. Seventeen point mutations are currently being produced and screened for their aggregation propensity. Mutations that reduce protein aggregation while maintaining enzyme activity will be combined in further rounds of mutation to obtain a stable and active B. subtilis Lipase A variant for aggregation-free biotechnological application.

Poster Presentations

105

Biomolecules in liquid state Poster Presentation P25

Characterization of 2-deoxyribose-5-phosphate aldolase enzyme catalysis using solution-state NMR

M. Schulte1,2, V. Panwalkar2 and D. Willbold1,2

1Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf,40225 Düsseldorf, Germany 2Institute of Complex Systems, ICS-6 (Structural Biochemistry), Forschungszentrum Jülich, 52425 Jülich, Germany

E-mail: [email protected]

The 259 amino-acid homodimer E. coli 2-deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible aldol reaction between acetaldehyde and glyceraldehyde-3-phosphate to generate 2-deoxyribose-5-phosphate. High expression yields and stability make DERA a perfect candidate as industrial organic catalyst. However, a strict dependency on phosphorylated substrates poses a major limitation. Combination of mutagenesis and simulation studies has suggested that interplay between coupled motions, entropic changes and flexibility drives the DERA catalytic activity [1]. Conformational dynamics have been shown to play key roles in enzyme catalysis. To obtain a complete understanding of an enzyme-catalyzed reaction, it is therefore necessary to characterize multi-amplitude motions of the participating protein. We are investigating the role of motion in DERA enzyme catalysis using solution-state NMR spectroscopy by analyzing fast (ps-ns) and slow (µs-ms) timescale motions of a DERA monomer mutant [2] at multiple fields and temperatures; in its free and substrate analog bound states. We have completed a full chemical shift resonance assignment of the monomer DERA mutant using standard triple resonance experiments and non-uniform sampling of data. A thorough understanding of motions and its role in enzyme catalysis could be used to engineer novel DERA mutants, catalyzing new substrates.

References [1] Ma et al. Chemical Science. 7, (2016) [2] Dick et al. Chemical Science. 7, (2016)

Poster Presentations

106

Biomolecules in liquid state Poster Presentation P26

Following Selenium Replacement in [FeFe]-Hydrogenase with EPR and NMR C. Sommer1, S. Rumpel1, S. Roy2, E. Reijerse1, V. Artero2, W. Lubitz1 1MPI Chemical Energy Conversion, Stiftstr. 34-36, Mülheim an der Ruhr, Germany 2 Laboratoire de Chemie et Biologie des Métaux, rue des Martyrs, Bâtiment K', Grenoble, France E-mail: [email protected]

Hydrogenases are metallo enzymes that catalyze the conversion of molecular hydrogen. [FeFe]-hydrogenases contain the so called H-cluster which is built up from a [4Fe4S] cluster 1 and a [2Fe]H cluster containing -CO and -CN ligands as well as a bridging dithiolate ligand.

Within this cluster all sulfur atoms can be replaced with selenium by different techniques. For the cubane cluster a biochemical route including complete unfolding of the protein backbone and addition of iron and selenium ions is used. To replace the bridging dithiolates the complex is made synthetically and inserted2 into in E.coli overexpressed apo protein.3 In this work HydA1 from Chlamydomonas reinhardtii was investigated. In 1D 1H NMR spectroscopy the selenium substituted hydrogenase shows slightly increased downshift compared to wild-type protein due to stronger magnetic interaction. In cw-EPR the [4Fe4Se] apo protein shows a high-spin system with S=7/2. The maturated protein reveals a rhombic spectrum similar to the wild-type.

References [1] W. Lubitz, Chem.Rev. 114, (2014) [2] J. Esselborn, Nat Chem Biol. 9 ,(2013) [3] J. Kuchenreuther, PLoS ONE 5, (2010)

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107

Biomolecules in liquid state Poster Presentation P27

NMR resonance assignment of YuaI and a first look into its 3D structure St. Thomas1 and Heiko M. Möller1 1 University Potsdam, Department of Chemistry, Potsdam, Germany E-mail: [email protected]

YuaI a soluble protein and belongs to the family of GCN5 related N-acetyl transferases. Interestingly, it is encoded in B. subtilis with the gene for an SPFH- protein (yuaG) and a reggie-like NfeD- protein (yuaF) in the operon yuaFGI [1]. Because genes that are combined in one operon are also co-regulated and co-expressed, this indicates a functional relationship between the three components. The yuaFGI operon is under control of the factor σW that is known to be important for the resistance of B. subtilis against certain antimicrobiotica [2] and other stress factors. We present here the current state of the 1H-,13C- and 15N- NMR resonance assignment of the acetyltransferase YuaI and show the results of various homology models (SWISS-Model [3-4], Phyre2 [5]) and their evaluation on the basis of our NMR data including 15N- and 13C- dispersed 3D NOESY-HSQC experiments. It turns out that unusual chemical shift values caused by the magnetic anisotropies of aromatic side chains play a significant role in evaluating homology models. Further refinment of our structural models by using NMRe [6] will be also presented and discussed.

References [1] A. F. Neuwald and D. Landsman. Trends Biochem Sci, 22, 154-155 (1997) [2] J. D. Helmann. Sci Prog, 89, 243-266 (2006). [3] Marco Biasini, Stefan Bienert, Andrew Waterhouse, Konstantin Arnold, Gabriel Studer, Tobias Schmidt, Florian Kiefer, Tiziano Gallo Cassarino, Martino Bertoni, Lorenza Bordoli, Torsten Schwede. Nucleic Acids Research; (1 July 2014) 42 (W1): W252-W258; doi: 10.1093/nar/gku340 (2014) [4] Arnold K., Bordoli L., Kopp J., and Schwede T. Bioinformatics, 22,195-201 (2006) [5] Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T Nucleic Acids Research. 37, D387-D392 (2009) [6] Guex, N., Peitsch, M.C., Schwede, T. Electrophoresis, 30(S1), S162-S173 (2009) [7] Kelley LA et al. Nature Protocols 10, 845-858 (2015) [8] Ryu H, Lim G, Sung BH, Lee J. Bioinformatics 15;32(4):611-3. (2016)

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108

Biomolecules in liquid state Poster Presentation P28

Structural Dynamics of a Heterodimeric ABC Exporter from a Hyperthermophilic Organism Studied by EPR M. Hadi Timachi1, Cedric A. J. Hutter2, Markus A. Seeger2, Enrica Bordignon1

1Department of Chemistry and Biochemistry, Ruhr University Bochum, Germany 2Institute of Medical Microbiology, University of Zurich, 8006 Zürich, Switzerland E-mail: [email protected]

The question concerning the origin of the power stroke for transport is a recurrent matter of debate in the ABC transporter field. In the prevalent ATP-switch model postulated by Higgins and co-workers[1], ATP binding leads to NBD closure and a concomitant switch from the inward- to the outward-facing state. ATP hydrolysis then destabilizes the NBD nucleotide sandwich dimer and the transporter reverts to its inward-facing state with disengaged NBDs. In the ATP-switch model, ATP hydrolysis is not required for substrate release; the role of ATP hydrolysis is to recharge the machinery for the next ATP-binding and substrate transport reaction. In combination with other biophysical techniques, site-directed spin labeling EPR provides a very good insight into protein structure and dynamics under conditions relevant for function. Based on SDSL-EPR studies on BmrCD, a diverging mechanism for heterodimeric ABC exporters was postulated, in which the transition to the outward-facing state strictly requires ATP hydrolysis and not only ATP binding[2], which challenged the ATP-switch model. As a conclusion, ABC exporters were divided in homodimers requiring ATP binding only for the transition to the outward-facing state and heterodimers requiring ATP hydrolysis instead. In the current work, SDSL EPR is applied to the structural-functional study of the complete conformational cycle of the heterodimeric ABC exporter TM287/288 studying the dynamic consequences of a broader set nucleotides and nucleotide analogs than the three conditions (apo, AMP-PNP and ATP-vanadate) used to study BmrCD. References [1] Higgins, C.F., and Linton, K.J. (2004). The ATP switch model for ABC transporters. Nat Struct Mol Biol 11, 918-926. [2] Mishra, S., Verhalen, B., Stein, R.A., Wen, P.C., Tajkhorshid, E., and Mchaourab, H.S. (2014). Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter. Elife 3.

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109

Biomolecules in liquid state Poster Presentation P29

The role of lipid composition and post-translational modification for membrane association and aggregation behaviour of α-synuclein T. Viennet1,2, M. M. W. Wördehoff1, H. Shaykhalishahi1, A. K. Büll1, W. Hoyer1 and M. Etzkorn1,2 1Institute of Physical Biology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Jülich, Germany E-mail: [email protected]

The protein α-Synuclein (αS) is naturally found in presynaptic neurons and involved in Parkinson’s disease through its intracellular abnormal aggregation. This mechanism is thought to consist of a complex interplay between free cytosolic and membrane-bound forms of αS [1]. It is thus of particular importance to understand its interactions with membranes. Following previous studies using micelles and vesicles [2-4], here we report on the comprehensive in vitro study of the interaction of αS with phospholipid bilayer nanodiscs. Usage of this well-defined membrane mimetic allowed us to characterize the influence of the lipid charge density and the membrane fluidity in respect to αS interaction at a residue specific level by solution NMR. In addition the effect on αS aggregation are reported. The N- terminal acetylated form of αS, i.e. its natural state [5], is used and compared with non- acetylated form, revealing specific differences both in terms of membrane affinity and aggregation behaviour.

References [1] S. M. Butterfield and H. A. Lashuel. Angew Chem Int Ed Engl. 49, (2010) [2] T. S. Ulmer et al. J Biol Chem. 280, (2005) [3] G. Fusco et al. Nat Commun. 5, (2014) [4] C. Galvagnion et al. Nat Chem Biol. 11, (2015) [5] I. Dikiy and D. Eliezer. J Biol Chem. 289, (2014)

Poster Presentations

110

Relaxation and Dynamics Poster Presentation P30

Water dynamics on THF clathrate hydrate studied by 2nd-order central-line stimulated-echo 17O NMR M. Adjei-Acheamfour1 , M. Storek, J. Tilly, J. Beerwerth, and R. Böhmer 1Fakultät Physik, Technische Universität Dortmund, 44221 Dortmund, Germany E-mail: [email protected]

Oxygen-17 stimulated-echo spectroscopy is a novel nuclear magnetic resonance (NMR) technique that allows one to investigate the time scale and geometry of ultraslow molecular motions in oxygen containing materials [1]. The method is based on detecting orientationally encoded frequency changes within oxygen's central-transition NMR lines that are predominantly broadened by second-order quadrupolar interactions. As an experimental example, the ultraslow water motion in polycrystals of tetrahydrofuran clathrate hydrate is studied via two-time oxygen-17 stimulated-echo correlation functions. The resulting correlation times and those of hexagonal ice [2] are similar to those from previous deuteron NMR measurements. In addition, the results of two additional experiments are presented which yield information concerning the geometry of reorientation. In the first, a static two- dimensional chemical exchange central-line powder spectrum governed by second-order quadrupolar interactions was obtained [3]. In the second, we determined final-state correlations as a function of the evolution time. Both experiments are compared with calculations based on a six-site jump model for the local process of a water molecule in a tetrahedral environment and it is found that this model describes the present results well if oxygen-proton dipolar interactions are accounted for. We thank Prof. Gerd Buntkowsky and his team (TU Darmstadt) for their hospitality when performing additional experiments in their laboratory. Financial support by the Deutsche Forschungsgemeinschaft under Grant No. BO1301/10-1 is gratefully acknowledged.

References [1] M. Adjei-Acheamfour, and R. Böhmer, Second-order quadrupole interaction based detection of ultra-slow motions: Tensor operator framework for central-transition spectroscopy and the dynamics in hexagonal ice as an experimental example. J. Magn. Reson. 249, 141 (2014) [2] M. Adjei-Acheamfour, J. F. Tilly, J. Beerwerth, and R. Böhmer, Water dynamics on ice and hydrate lattices studied by second-order central-line stimulated-echo oxygen-17 nuclear magnetic resonance.rimental example. J. Chem. Phys. 143, 214201 (2015) [3] M. Adjei-Acheamfour, M. Storek, J. Beerwerth, and R. Böhmer, Two-dimensional second- order quadrupolar exchange powder spectra for nuclei with half-integer spins. Calculations and an experimental example using oxygen NMR. Solid State Nucl. Magn. Reson. 71, 96 (2015)

Poster Presentations

111

Relaxation and Dynamics Poster Presentation P31

Spin diffusion in rotating frame is enhanced by moderate MAS G. Hempel Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Germany E-mail: [email protected]

As shown for spin diffusion in laboratory frame [1], also the rotating-frame spin diffusion might get more intensive if the MAS rate increases. This is demonstrated by means of 1H-13C cross-polarization dynamics. After decay of initial dipolar oscillations and before longitudinal relaxation in the rotating frame will destroy magnetization, exponential-like growth of the 13C magnetization is observed. This can be considered as consequence of polarization transport from remote protons to those which are instantaneously at least partially depolarized because of the magnetization oscillations. The faster this kind of spin diffusion is the smaller is the time constant TIS of magnetization growth. Quantitative analysis requires a model function because at intermediate times, oscillations and growth overlap. An appropriate function is derived similar to the considerations in [2]. Consequent use of Liouville space simplifies the calculations in case of more than two spins. Experiments were performed on alanin, polyethylene and polystyrene. Data analysis reveals acceleration of the exponential growth if the spinning rate is increased up to 3 kHz. This is interpreted as enhancement of spin diffusion in the rotating frame during sample spinning.

References [1] G. Hempel, submitted to J. Magn. Res. [2] J. Hirschinger, J. Raya, Molecular Physics. 113 (2015) 3161

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112

Relaxation and Dynamics Poster Presentation P32

Experimental Detection of Spin-Diffusion Anisotropy A. Wurl, M. Roos, K. Saalwächter, and G. Hempel Martin-Luther-Universität, Institut für Physik, Halle, Germany E-mail: [email protected]

Spin diffusion is based on dipolar interaction within a spin ensemble. The fact that this interaction is anisotropic leads to the question if spin diffusion is anisotropic, too. Analytical calculations by modelling the diffusion process as a result of a multitude of spin flips show that such anisotropy may indeed be present. One experimental approach concerns proton spin diffusion in the rotating frame detected during 13C-1H cross polarization (CP). We focus on the exponential-like part of the CP build- up curve after the decay of initial dipolar oscillations. This part of the CP build-up curve is ascribed to polarization transport from remote protons to protons close to 13C positions, in other words, to spin diffusion which re-equilibrates the proton magnetization profile locally disturbed by CP. Anisotropic rotating-frame spin diffusion could be detected in polyethylene, polystyrene and alanine. Another experimental scheme is based on lab-frame proton spin diffusion in samples consisting of a stack of macroscopically oriented, alternating layers of polystyrene and polybutadiene. After creating magnetization differences between neighboured layers (for instance, by use of a double-quantum filter), and after a variable waiting time, the relative amount of the magnetization in both phases was estimated. Quantifying spin diffusion rates for different orientations of the sample relative to the magnetic field, a weak anisotropy of spin diffusion has been revealed.

Poster Presentations

113

Relaxation and Dynamics Poster Presentation P33

Fingerprinting systems to identify fake paintings and assign wall paintings for cultural heritage C. Rehorn1, W. Zia1, T. Meldrum2, C. Kehlet3, E. Del Federico3, Giuseppe Zolfo4, Jane Thompson5, B. Blümich1 1RWTH-Aachen University, Aachen, Germany 2The College of William and Mary, Williamsburg, Virginia 3Pratt Institute, New York, USA 4Restauration and Conservation, Ercolano, Italy 5The Herculaneum Conservation Project, Ercolano, Italy E-mail: [email protected]

Non-destructive analysis of arbitrarily sized samples with the NMR-MOUSE [1, 2], a unilateral stray-field NMR sensor, has proven to be a valuable procedure in many branches of cultural heritage as insights into the stratigraphy of intact objects can be gained while information about material properties such as molecular mobility is provided. As the MOUSE selectively detects signal only from a slice a few millimeters away from the surface, depth profiles can be acquired by mounting the device on a stepper-motor controlled sled. Surface and sub-surface layers of mock-ups and forged paintings were analyzed in situ with unilateral NMR relaxometry. We investigate structural changes within the paint layers which are invoked by different methods of cleaning and restoration. Furthermore, a correlation map of T1 and T2 relaxation times similar to [3] is presented that can serve as a fingerprinting chart for paints employed by a painter or art forger. Wall paintings from the remains of ancient villas in Herculaneum are known to exhibit layered structures of mortar underneath the painted surface. [4] Ongoing work concerns the stratigraphy of such wall paintings and the possibilities in identifying the time and area of origin from destruction-free NMR analysis.

References [1] G. Eidmann, R. Savelsberg, P. Blümler, B. Blümich, J. Magn. Reson. A 122 (1996) 104–109. [2] B. Blümich, J. Perlo, F. Casanova, Prog. Nucl. Magn. Reson. Spectrosc. 52 (2008) 197–169. [3] F. Presciutti et al., Appl. Phys. Lett. 93 (2008) 033505. [4] A. Haber, B. Blümich, D. Souvorova, E. Del Federico, Anal Bioanal Chem 401 (2011) 1441– 1452.

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114

Biosolids Poster Presentation P34

Colour tuning of cyanobacteriochromes S. Altmayer1, L. Scarbath-Evers2, S. Jähnigen2, C. Song1, W. Gärtner1, D. Sebastiani2, J. Matysik1

1 Institut für Analytische Chemie, Universität Leipzig, Linnéstr. 3, Leipzig 2 Institut für Chemie, MLU Halle, von-Danckelmann-Platz 4, Halle (Saale) E-Mail: [email protected]

In 2001 Ohmori et al.1 discovered a new class of tetrapyrrole-binding photoreceptor proteins called cyanobacteriochromes (CBCRs). They form a subgroup of the well-studied phytochromes which are red/far-red-sensing photoreceptors2. Phytochromes operate in narrow spectral windows around 660 nm and 730 nm, whereas CBCRs access the entire spectral range from UV to near infrared3. Here we aim for understanding the factors controlling the colour transition, i.e., the HOMO-LUMO gap, in phytochromes and CBCRs. To this end, we apply solid-state MAS NMR on Cph1 (classical phytochrome) and the GAF2 domain of AnPixJ (CBCR) to study the modification of double bond conjugation and the dynamics of charge changes in the pocket as an effect of photoisomerisation . Molecular dynamics simulations and quantum chemical calculations will elucidate the influence of steric and electrostatic protein-chromophore-interactions on NMR and UV/vis spectra. In particular, we will address the dynamics of water around and inside the binding pocket on the picoseconds’ time range which has a notable effect on the hydrogen bond network of the chromophore and its spectroscopic response. The results should allow to draw conclusions on the colour-tuning mechanism as an important parameter for optogenetically applications.

References [1] M. Ohmori, DNA res. 8, (2001) [2] N.C. Rockwell, Chemphyschem. 11(6), (2010) [3] M. Ikeuchi, Photochem. Photobiol. Sci. 7, (2008)

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115

Biosolids Poster Presentation P35

Trityl Spin Labels for EPR Distance Measurements A. Berndhäuser1, J. J. Jassoy1, and O. Schiemann1 1Institue for Theoretical and Physical Chemistry, 53115 Bonn, Germany

E-mail: [email protected]

EPR based distance measurements have become an effective tool in for the analysis of the structure of large biomolecules, e.g. proteins and DNA strands. As most of the biomolecules do not have native spin centers, and those that do rarely more than one, the distance measurement require the introduction of spin labels into the biomolecule. The most common type of spin labels are nitroxide radicals such as the commercially available MTSSL label. However, these nitroxides suffer from a number of disadvantages, e.g. their short lifespan under in-cell conditions and a short relaxation time at biological relevant temperatures. As a result, new types of spin-labels have been developed in the past years, e.g. triarylmethyl radicals. The latter received some attention in the last two years due to the first reported distance measurements at biologically relevant temperatures, but suffered from their rather demanding synthesis and resulting low availability. Here, we present the synthesis of new triarylmethyl labels for the labelling of proteins and their application in distance measurements.

Poster Presentations

116

Biosolids Poster Presentation P36

Solid-State NMR of membrane-bound and free Nitrophorin 7 C. Beumer1,2, S. Varghese1,2,3, E. Decaneto4,5, M. Knipp4,5 and H. Heise1,2 1Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany 2Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf Germany 3current address: Department of Chemistry, Lancaster University, Lancaster, United Kingdom 4Biophysical Chemistry, Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany 5Department of Physical Chemistry II, Ruhr University Bochum, Bochum, Germany E-mail: [email protected]

The 21 kDa protein Nitrophorin 7 (NP7) belongs to a class of heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus [1]. It reversibly binds nitric oxide (NO), coordinated to an iron atom of the heme b cofactor. It releases it during blood feeding following a change in pH from the insect saliva (pH 5-6) to the host blood (pH 7.4). Here, NO acts as a vasodilator and blood-coagulation inhibitor [2]. The importance of the signaling molecule NO in the cardiovascular regulation led to the aim to understand the molecular mechanisms of the storage and delivery of NO in naturally occurring NO-delivery systems, like nitrophorins [3]. NP7, in contrast to the other isoforms (NP1-4), can bind to negatively charged cell surfaces [4]. It tends to oligomerize and precipitate at higher concentrations in solution [5]. Here, we report on solid-state NMR investigations on NO-bound NP7 in its diamagnetic state. We compare spectra of V,L,F,Y-reverse-labeled NP7 bound to DMPC/DMPS liposomes with free, precipitated NP7. The spectra exhibit a high degree of similarity, suggesting that membrane binding does not affect the overall fold of the protein. Partial sequential assignments cold be obtained from CC and NCOCX/NCACX correlation spectra.

References [1] M. Knipp, Protein Express Purif. 54 (2007) [2] M. Knipp, Biochemistry. 46 (2007) [3] S. Abbruzzetti, J Am Chem Soc. 134 (2012) [4] J. F. Andersen, Biochemistry. 43 (2004) [5] S. Varghese, Biochemistry. 52 (2013)

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117

Biosolids Poster Presentation P37

13C, 2H NMR Studies of Structural and Dynamical Modifications of Glucose-Exposed Porcine Aortic Elastin

1M.C. Silverstein, 1K. Bilici, 1S.W. Morgan, 2Y. Wang, 2,3Y. Zhang, and 1G.S. Boutis

1Department of Physics, Brooklyn College, The City University of New York, 2900 Bedford Avenue, 11210, Brooklyn NY, USA 2Department of Mechanical Engineering and 3Department of Biomedical Engineering, Boston University, 110 Cummington Mall, 02215, Boston MA, USA. Email:[email protected], [email protected] Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid- state NMR spectroscopic and relaxation methodologies. Results from macroscopic stress- strain tests are also presented and indicate that glucose-treated elastin is mechanically stiffer than the same tissue without glucose treatment. These measurements show a large hysteresis in the stress-strain behavior of glucose-treated elastin—a well-known signature of viscoelasticity. Two-dimensional relaxation NMR methods were used to investigate the correlation time, distribution, and population of water in these samples. Differences are observed between the relative populations of water, whereas the measured correlation times of tumbling motion of water across the samples were similar. 13C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after glucose treatment. Although some overall structure is preserved, the process of glucose exposure results in more heterogeneous structures and slower mobility. The correlation times of tumbling motion of the 13C-1H internuclear vectors in the glucose-treated sample are larger than in untreated samples, pointing to their more rigid structure. The 13C cross- polarization spectra reveal a notably increased α-helical character in the alanine motifs after glucose exposure. Results from molecular dynamics simulations are provided that add further insight into dynamical and structural changes of a short repeat, [VPGVG]5, an alanine pentamer, desmosine, and isodesmosine sites with and without glucose. The simulations point to changes in the entropic and energetic contributions in the retractive forces of VPGVG and AAAAA motifs. The most notable change is the increase of the energetic contribution in the retractive force due to peptide-glucose interactions of the VPGVG motif, which may play an important role in the observed stiffening in glucose-treated elastin. References [1] C.M. Kielty, M.J. Sherratt, and C.A.Shuttleworth, Journal of cell science, 115-14 (2002) 2817–2828.

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118

Biosolids Poster Presentation P38 Mechanical, structural, and dynamical modifications of cholesterol exposed porcine aortic elastin

1K. Bilici, 1S.W. Morgan, 1C.M. Silverstein, 2Y. Wang, 2H. J. Sun, 2,3Y. Zhang, and 1G.S. Boutis

T1-T2 NMR techniques, we measured the correlation time, distribution, and population of water in cholesterol treated samples. The measured correlation times of tumbling motion of water were similar; however, there were differences between the relative populations of water which correlate with macroscopic changes in the swelling of the tissue following cholesterol exposure. 13C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after cholesterol treatment. These measurements indicate that cholesterol treated aortic elastin cross polarizes less than the control samples. The measured correlation times of the tumbling motion of the 13C-1H internuclear vectors in the cholesterol treated sample are smaller than in untreated samples pointing to increased mobility. Simulations on a short elastin repeat VPGVG in the presence of cholesterol are used to investigate the energetic (dU/dr) and entropic (-TdS/dr) contributions to the retractive force, in comparison to the same peptide in water. Peptide stiffness reduces for the peptide in cholesterol in comparison to the peptide in water due to a decrease in the entropic force, in qualitative agreement with macroscopic stress-strain measurements.

Reference [1] C.M. Kielty, M.J. Sherratt, and C.A.Shuttleworth, Journal of cell science, 115-14 (2002) 2817–2828.

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119

Biosolids Poster Presentation P39

Solid-state Photo-CIDNP Effect Observed in Aureochrome LOV- C287S by 13C MAS NMR Yonghong Ding,1 Saskia Bannister,2 Tilman Kottke,2 and Jörg Matysik1 1Institute of Analytical Chemistry, University of Leipzig, Linnéstr. 3, 04103 Leipzig, Germany; 2Physical and Biophysical Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany. E-mail: [email protected]

Aureochromes constitute a class of biological blue-light receptors that contain flavin mononucleotide (FMN) as chromophore in its light-oxygen-voltage (LOV) domain. FMN absorbs UV-A/blue light and undergoes a cyclic photoreaction which involves an intermediate flavin-cysteinyl adduct formation. The mutation in aureochrome-LOV (C287S) abolishes the adduct formation and shows a photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect in solid-state MAS NMR. Previously this effect was confined to photosynthetic systems and the flavoprotein phototropin LOV1-C57S [1]. The mutation in the LOV domain leads to an increased lifetime of 3FMN and further induces a less competitive electron transfer from a distant tryptophan (Trp) to FMN. A tentative photocycle involving the generation of a spin-correlated radical pair [FMN•-–Trp•+] will be proposed. Possible mechanisms that cause the solid-state photo-CIDNP effect are presently investigated by employing aureochrome LOV-C287S as a model system. References [1] Thamarath, S. S., et al, J. Am. Chem. Soc., 132 (2010) 15542.

Poster Presentations

120

Biosolids Poster Presentation P40

Interaction between N-terminal human Prion protein and Amyloid beta oligomers studied by solid-state MAS-NMR A.König1,2, N.Rösener1,2, L.Gremer1,2, D.Schölzel1,2, D. Willbold1,2 and H.Heise1,2 1Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Jülich, Germany 2Institut für physikalische Biologie der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany E-mail: [email protected]

Both prion protein (PrP) and Amyloid beta (Aβ) play an important role in the pathogenesis of neurodegenerative disorders as they misfold or form amyloid fibrils. Misfolded PrP is responsible for a number of diseases, e. g. bovine spongiform encephalopathy and Creutzfeldt-Jakob disease [1]. Aβ oligomers have been identified as neurotoxic factor of the pathogenesis of Alzheimer's disease (AD), whereas fibrils are known to be the main component of insoluble plaques [2]. In contrast to the secondary structure of fibrils, secondary structure elements of the oligomers are relatively unknown, due to their transient nature [3], [4]. Recently, it has been shown that oligomeric Aβ binds human PrP (hPrP) with high affinity. First models use ionic interactions between both species including two Lys-rich parts (aa 23 to 27 and 95 to 110) of the hPrP. As a result the C-terminus can be deleted without preventing the binding between hPrP and Aβ [5], [6]. We show first MAS NMR experiments on species-specifically uniformly labeled samples, with N-terminal hPrP (aa 23 to 144).

References [1] J. J. Helmus et al., Journal of the American Chemical Society. 132, (2010) [2] D. J. Selkoe, Behavioural Brain Research. 192, (2008) [3] M. T. Colvin et al., Journal of the American Chemical Society. 137, (2015) [4] S. Parthasarathy et al., Journal of the American Chemical Society. 137, (2015) [5] S. Chen et al., Journal of Biological Chemistry. 285, (2010) [6] B. R. Fluharty et al., Journal of Biological Chemistry. 288, (2013)

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121

Biosolids Poster Presentation P41

Structural studies on scrapie seeded ovine PrP amyloids T. Piechatzek1,2, C. Pohl2, B. Esters2, H. Heise1,2 1ICS-6 FZ-Jülich, Jülich, Germany 2IPB HHU Düsseldorf, Düsseldorf, Germany E-mail: [email protected]

Neurodegenerative diseases such as transmissible spongiform encephalopathies are not always due to inheritance, but can also be caused by proteinaceous infectious particles (prions), consisting of misfolded prion protein (PrP). A detailed structure of fibrillary PrPSc is yet unknown, however, different structural models have been proposed1,2. In our contribution, we will report on our experimental progress in characterization of seeded amyloids of full-length ovine recombinant PrP by biochemical methods and high resolution solid-state NMR3. We will show our results investigating the structure of seeded amyloid PrP, which will include the identification of secondary structure elements in the amyloid core region based on partial preliminary sequential assignments. Further, we will present our results on a following generation derived from a former parental generation, which also includes the identification of secondary structure elements by partial preliminary sequential assignments. These findings are supported by results obtained for amyloids prepared with different isotopic labeling schemes. Subsequently, we will state on the comparison between these amyloid generations based on the results of the secondary structure elements.

References [1] Cobb, et al. JBC 283(50), (2008) [2] Govaerts, et al. PNAS 101(22), (2004) [3] Müller et al. Prion 8(5), (2014)

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122

Biosolids Poster Presentation P42

Solid-state NMR investigations of lipid membranes interacting with amphiphilic triblock copolymers Ruth Bärenwald1, Anja Achilles1, Mark Jbeily2, Jörg Kressler2, Kay Saalwächter1 1Institut f. Physik, Martin-Luther-Univ. Halle-Wittenberg, 06099 Halle (Saale), Germany 2Institut f. Chemie, Martin-Luther-Univ. Halle-Wittenberg, 06099 Halle (Saale), Germany E-mail: [email protected]

Pluronics are triblock copolymers consisting of hydrophilic end blocks and a hydrophobic block, and are widely used in the pharmacological industry. Depending on the lengths of the different blocks, Pluronics can influence membrane properties in a variety of ways, e.g., increase or decrease membrane permeability. This can be explained by different ways of incorporation [1], i.e., spanning the membrane or just penetrating it as hairpin.

We focus on Pluronics and chemically related variants with higher propensity to act as hydrogen bond donor and acceptor, potentially increasing the interaction with the lipid head group [2]. Using solid-state MAS NMR, we investigate structure and dynamics of phosphatil- dylcholine membranes with variable bilayer thickness and saturation, mixed with copolymers of variable block length. 1H-1H 2D-NOESY experiments were applied to confirm the insertion of the copolymer into the bilayer, and motionally averaged residual dipolar couplings (RDCs) were measured to obtain information on molecular dynamics. For the lipids, 13C-1H RCDs were determined with the R-PDLF experiment, obtaining similar results for all samples and the pure lipids, confirming a weak influence of the polymers on lipid dynamics. 1H-1H RDCs were measured with the BaBa-xy16 DQ recoupling pulse sequence [3], which is suitable for measuring rather small RDCs related to the guest molecules. In this way, we were for the first time able to quantify the relative amount of inserted vs. non-inserted polymer and conclude on its conformation.

References [1] H. Rabbel et al., Macromolecules 48, 4724 (2015) [2] C. Schwieger et al., Soft Matter 10, 6147 (2014) [3] K. Saalwächter et al., J. Magn. Reson. 212, 204 (2011)

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123

Biosolids Poster Presentation P43

Conformational dynamics in the beta-barrel transporter FhaC investigated by solid-state NMR Robert Schneider1, Xavier Hanoulle1, Jérémy Guérin2, Maëlenn Païline Delcourt2, and Françoise Jacob-Dubuisson2 1NMR & Molecular Interactions, UMR 8576 UGSF, CNRS / Université Lille, Lille, France 2Institut Pasteur de Lille, CIIL, Lille, France E-mail: [email protected]

The Two-Partner Secretion (TPS) pathway in Gram-negative bacteria is dedicated to the export of proteins serving notably as virulence factors. TpsB transporters are transmembrane β-barrel proteins located in the outer membrane. In the whooping cough agent Bordetella pertussis, the TpsB transporter FhaC mediates secretion of the adhesin FHA. Its resting-state crystal structure, a 16-stranded beta-barrel preceded by two periplasmic POTRA domains, is known, but its mechanism of protein transport has so far remained elusive [1]. FhaC is characterized by considerable dynamics. It has been shown that crucial elements of its structure populate a dynamic equilibrium already in the absence of FHA. Notably, helix H1 and loop L6, which block the translocation pore in the resting state, may be displaced from the pore [2,3]. Such movements are likely implicated in the transport mechanism. Our aim is to characterize the conformational dynamics of FhaC in molecular detail and to elucidate its role in transport. We have performed 1H-detected solid-state NMR experiments on perdeuterated, Ile-δ1 methyl-labeled FhaC in lipid bilayers. We report on progress in resonance assignment of methyl signals in this 61.5 kDa protein, as well as spectral signatures of mutants known to affect the open-close equilibrium of the protein.

References [1] Clantin, B. et al. Science 317, 957–961 (2007). [2] Guérin, J. et al. Mol. Microbiol. 92, 1164–1176 (2014). [3] Guérin, J. et al. Mol. Microbiol. 98, 490–501 (2015).

Poster Presentations

124

Biosolids Poster Presentation P44

Aβ(1-42) fibrillar structure studied by high resolution solid-state MAS NMR D. Schölzel1,2 , F. Weirich1,2, J. Schönborn1,2, A. König1,2, L. Gremer1,2 and Henrike Heise1,2 1 Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Jülich, Germany 2Institut für Physikalische Biologie der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany E-mail: [email protected]

The progress of Alzheimer’s disease is believed to be related to the formation of amyloid beta (Aβ) aggregates in the human brain. Although differing only slightly in the amino acid sequence, compared to Aβ 1-40, Aβ 1-42 is more prone to aggregation, causing a higher toxicity [1]. Here we present the preliminary site specific resonance assignment of one conformation of Aβ 1-42 fibrils, obtained by solid state magic angle spinning (MAS) NMR [2][3]. Interestingly, specific differences to previously described fibrillar Aβ 1-42 secondary structure can be found.

References [1] D.J. Selkoe et al., Physiol. Rev. 81 (2001) [2] M.T. Colvin et al., J. Am. Chem. Soc. 137 (2015) [3] Y. Xiao et al., Nat. Struct. Mol. Biol. 22 (2015)

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125

Biosolids Poster Presentation P45

Abstract Dr. C. Song1, M. Sc. C. Lang2, Prof. Dr. M. A. Mroginski3, Prof. Dr. L.-O. Essen4, Prof. Dr. W. Gärtner5, Prof. Dr. J. Hughes2 and Prof. Dr. J. Matysik1 1Institut für Analytische Chemie, Universität Leipzig, Leipzig, Germany 2Pflanzenphysiologie, Justus-Liebig-Universität, Gießen, Germany 3Institut für Chemie, Technische Universität Berlin, Berlin, Germany 4Fachbereich Chemie, Philipps-Universität, Marburg, Germany 5Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, Germany E-mail: [email protected]

Recently, solid-state NMR enabled us to resolve the molecular structure of the chromophore-binding pocket of phytochrome A3 (phyA3) from oat in the red-light-absorbing Pr state [1]. Extending this work, we here present the application of several 2D solid-state NMR methods to the corresponding far-red-light-absorbing Pfr state of the complete sensory module of phyA3 (PAS–GAF–PHY), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pfr state of this protein was studied aiming to understand the electronic structure of the chromophore and its interactions with the proximal amino acids. A complete set of 13C and 15N assignments for the chromophore were obtained. Additionally, a large number of 1H–13C distance restraints between the C chromophore and its binding pocket were revealed. The general Pr → Pfr δ changing pattern of oat phyA3 is found to be similar to that of Cph1Δ2, particularly for those carbons associated with the A–B and B–C methine bridges as well as C17 of the ring D. The overall similarity shows that the Pr → Pfr electronic differences of the π-conjugations between oat phyA3 and Cph1Δ2 are small. Our data support the 15Ea configuration adopted by the Pfr chromophore. Also we provide unequivocal evidence for the presence of a helical tongue in the phyA3 Pfr photoproduct whereas, by contrast, this protein region is extended in the Pr state, closing the chromophore-binding pocket.

References [1] C. Song, L.-O. Essen, W. Gärtner, J. Hughes and J. Matysik, Mol. Plant. 5, (2012).

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126

Hyperpolarization Poster Presentation P46

Dynamic Nuclear Polarization enhanced solid state NMR studies of surfactant systems as novel green solvents S. Bothe1 , S. Hadjiali1, M. M. Hoffmann2, T. Gutmann1 and G. Buntkowsky1 1TU Darmstadt, D-64287 Darmstadt, Germany 2Department of Chemistry and Biochemistry, The College at Brockport, State University of New York, Brockport, NY, 14420, USA E-mail: [email protected] ; [email protected]

Polyethylene glycol (PEG) and related surfactants have been employed as efficient “green solvents” for synthetic chemistry [1, 2]. However, there is a lack of physicochemical studies aimed at gaining an understanding of their role as solvents. In the present studies we propose to employ high field Dynamic Nuclear Polarization spectroscopy (DNP) [3] as a tool to study solute solvent interactions. As model systems we used three surfactants (C10E6, C10E6P1 and Triton X-100) and PEG with three different radicals as solutes. Enhancement by solid state DNP was obtained and interestingly the experiments turned out to show a superposition of positive and negative signals, which have not been reported before. Inspection of the magnetic field dependence (DNP enhancement profile) indicates that two different hyperfine coupling constants are present, each representing a distinct spatial orientation of radical to surfactant. Additional investigations by NMR, electron paramagnetic resonance spectroscopy and differential scanning calorimetry were employed in order to interpret these findings.

References [1] J. Chen et al., Green Chemistry 7, (2005) [2] M. M. Hoffmann, PCT Int. Appl., (The Research Foundation of the State University of New York, USA). Wo, (2006), p 13 pp. [3] T. Maly et al., J. Chem. Phys. 128, (2008)

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127

Hyperpolarization Poster Presentation P47

Quantum Rotor Induced Polarization in Crystals C. Dietrich1, S. Berger1, S. Khazaei2, D. Sebastiani2 and J. Matysik1 1Universität Leipzig, Institut für Analytische Chemie, Linnéstr. 3, Leipzig, Germany 2MLU Halle, Institut für Chemie, Von-Danckelmann-Platz 4, Halle (Saale), Germany E-mail: [email protected]

Named after his discoverer the Haupt-effect or quantum rotor induced polarization (QRIP) is a relatively unexplored hyperpolarization mechanism, since it was found only in a few substances so far. It is most prominent in γ-picoline, where the methyl group has a particularly small hindering barrier for rotation.[1] When cooled down to liquid helium temperature (4.2 K) and then suddenly heated up by adding solvent at room temperature an increase of the methyl carbon signal with an unusual signal pattern can be observed.[2] The phenomenon is explained by a non-Boltzmann distribution of rotational and spin quantum states at low temperatures and transfer by a cross relaxation to observable magnetization. To this end the tunnel splitting of the methyl group appears to be crucial.[3] Presently, it is investigated whether the effect in fact correlates to tunnel energies or to geometrical constraints in the crystal. For this matter further substances, similar to γ-picoline are synthesized, measured in the same way and the crystal structures are compared. Especially interesting are the intermolecular distances and angles between methyl groups.

References [1] J. Haupt, Phys. Lett. A 38, (1972) [2] M. Icker, J. Magn. Reson. 219 (2012) [3] B. Meier, J. Am. Chem. Soc. 135 (2013)

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128

Hyperpolarization Poster Presentation P48

Selective protein hyperpolarization in cell lysates using targeted DNP T. Viennet1,2, A. Viegas1, A. Küpper3, S. Arens1, V. Gelev4, O. Petrov4, T. N. Grossmann3, H. Heise1,2 and M. Etzkorn1,2 1Institute of Physical Biology, Heinrich Heine University, Düsseldorf, Germany 2Institute of Complex Systems, Forschungzentrum Jülich, Germany 3Chemical Genomics Center of the Max Planck Society, Dortmund, Germany 4Faculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria E-mail: [email protected]

NMR spectroscopy has the intrinsic capabilities to investigate proteins in native environments. Though such studies remain challenging, mainly due to low sensitivity and large background signals. While Dynamic Nuclear Polarization (DNP) can overcome sensitivity issues, it conventionally enhances background and target the same way. In line with previous localized DNP approaches [1-7], we here report on the usage of a covalently biradical-labeled ligand that, after binding to the targeted protein, can selectively direct hyperpolarization to the desired protein instead of the whole sample. Using this targeted-DNP approach we could selectively filter out our target protein directly from crude cell lysate obtained from only 8 ml of fully isotope enriched cell culture. Our approach is simple and applicable to the study of proteins with atomic resolution in increasingly native concentrations and environments.

References [1] V. Vitzthum et al. Chemphyschem. 12, (2011) [2] T. Maly et al. J Phys Chem B. 116, (2012) [3] C. Fernandez-de-Alba et al. Chemistry. 21, (2015) [4] A. N. Smith et al. Angew Chem Int Ed Engl. 54, (2015) [5] E. A. van der Cruijsen et al. Chemistry. 21, (2015) [6] M. A. Voinov et al. J Phys Chem B. 119, (2015) [7] B. J. Wylie et al. J Biomol NMR. 61, (2015)

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129

Hyperpolarization Poster Presentation P49

Conformational ensembles of (disordered) proteins studied by DNP- NMR B.Uluca1,2, H.Shaykhalishahi1,2, T. Viennet 1,2, M. Etzkorn1,2, W.Hoyer1,2 and H.Heise,1,2 1Research Centre Jülich, Institute of Complex Systems 6, Wilhelm-Johnen-Straße, 52425 Jülich, Germany 2Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany [email protected]

DNP overcomes the inherently low sensitivity of magnetic resonance methods by transferring high polarization of unpaired electrons to nuclei. Low temperature NMR spectra usually suffer from severe line broadenings due to freezing out different conformations [1]. While this is usually accounted for as an unwanted side-effect of DNP-NMR, these inhomogeneously broadened lines also contain valuable information about conformational ensembles of (disordered) proteins.

We have studied in different conformational ensembles of the intrinsically disordered protein α-syn which is known to bind lipid membranes. Sparse labeling [2] allowed us to quantify the conformational distribution of monomeric α-syn, fibrillar α-syn and monomeric α-syn in complex with nanodiscs in the frozen state. Additionally, we have investigated the amyloid binding protein “β-wrapin AS69” [3] which is designed to specifically bind α-syn in the free form as well as in fibrillar form by DNP-NMR. Low temperature studies allowed us to probe the effect of binding on the conformational ensembles of β-wrapin AS69. Different NMR line widths and enhancement factors are observed for free and bound state.

References [1] Tycko R. NMR at Low and Ultralow Temperatures. Accounts of chemical research 2013;46:1923-32. [2] Hong M, Jakes K. Selective and extensive 13C labeling of a membrane protein for solid- state NMR investigations. J Biomol NMR 1999;14:71-4. [3] Mirecka EA, Shaykhalishahi H, Gauhar A, Akgül S, Lecher J, Willbold D, et al. Sequestration of a beta-hairpin for control of alpha-synuclein aggregation. Angewandte Chemie 2014;53:4227-30.

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130

Hyperpolarization Poster Presentation P50

13C Photo-CIDNP MAS NMR observed in the reaction center of the diatom Cyclotella meneghiniana J. Zill1, M. Kansy2, R. Goss2, L. Köhler1, A. Alia3, C. Wilhelm2, J. Matysik1 1Institute of Analytical Chemistry, Leipzig, Germany 2Institute of Biology, Leipzig, Germany 3Leiden Institute of Chemistry, Leiden, Netherlands, Institute of Medical Physics and Biophysics, Leipzig, Germany E-mail: [email protected]

Photo-CIDNP MAS NMR presents a unique tool to obtain insight into the photosynthetic reaction centers of bacteria and plants. Using the dramatic enhancement of sensitivity and selectivity of the solid-state photo-CIDNP effect, structural as well as functional information can be obtained from the cofactor molecules forming a light-induced spin-correlated radical pair (SCRP) in a given reaction center.[1] Here we demonstrate that the effect can be observed in a further species, which belongs neither to the plant nor the bacteria kingdom. Cyclotella meneghiniana is a member of the diatom phylum and, therefore, belongs to the kingdom of chromista. Chromista are some of the most productive organisms in nature, even in comparison to trees and terrestrial grasses.[2] The observation of the effect in chromista indicates that the effect occurs in all photosynthetic organisms and completes the list with the last phototrophic kingdoms. Our data also demonstrate that the photo- and spin- chemical machineries of photosystem I of plants and chromista are very similar with respect to structure as well as function.

References [1] M. Najdanova et.al., J. Photochem. Photobiol. B: Biology, 152, (2015) [2] D. Werner (Ed.), The Biology of the diatoms, Blackwell Scientific Publications, (1977)

Poster Presentations

131

Materials and Polymers Poster Presentation P51

Analysis of technical polymers by high- and low-field NMR A. Adams, Y. Teymouri, J. Zhang, M. Wagemann, A. Görges and B. Blümich Institut für Technische und Makromolekulare Chemie, RWTH Aachen University, Worringerweg 2, D-52074 Aachen, Germany E-mail: [email protected]

Solid-state NMR is today one of the most versatile and powerful analytical tools for the detailed characterization of the morphology of solid polymers. The information gained is of great importance for establishing proper structure-property relationships required for the rational design of polymers for particular applications and for an improved understanding of the molecular changes leading to the deterioration of the macroscopic properties. With this understanding in mind a survey of recent work on technical polymers from our laboratory will be presented. In particular, it will be shown that NMR methods working at high and low magnetic field, alone or in combination with other analytical methods, can help to gain novel insights into changes of the molecular network of various semi-crystalline polymers under the impact of different loads [1-6]. Moreover, a new strategy for non-destructive quantification of the mechanical status of crosslinked polyethylene exposed to chemical aging will be presented [5]. In addition, it will be shown that the single-sided NMR is the key non-destructive analytical tool for acquiring detailed information about the kinetics of the temporal and spatial molecular changes in polymer products with the loading time [2]. Furthermore, a novel methodology for quantifying the local plasticizer concentration in polyvinyl chloride products with the possibility to identify the type of plasticizer without the need of spectroscopic techniques will be introduced [4]. The presented results are of fundamental importance due to the current lack of reliable non-destructive analytical tools and are seen as a significant step forward towards improving existing lifetime-prediction models.

References [1] A. Adams, TrAc xxx (2016) xxx. [2] J. Zhang and A. Adams, Polym. Degrad. Stab. (under revision). [3] Y. Teymouri, A. Adams, and B. Blümich, Eur. Polym. J. 80 (2016) 48. [4] A. Adams, R. Kwamen, B. Woldt, and M. Graß, Macromol. Rapid Comm. 36 (2015) 2171. [5] A. Adams, A. Piezeck, G. Schmitt, and G Siegmund, Anal. Chim. Acta, 887 (2015) 163. [6] N. Sun, M. Wenzel, and A. Adams, Polymer, 55 (2014) 3792.

Poster Presentations

132

Materials and Polymers Poster Presentation P52

Time-resolved EPR and theoretical investigations of metal-free dual singlet-triplet OLED emitters H Matsuoka1, L. Schmitt2, M. Retegan3, F. Neese3, S. Höger2, and O. Schiemann1 1 Institute für Physikalische und Theoretische Chemie, University of Bonn, Bonn, Germany 2 Kekulé-Institut für Organische Chemie und Biochemie, University of Bonn, Bonn, Germany 3 Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, Germany E-mail: [email protected]

In organic light-emitting diodes (OLEDs) using fluorescent emitters, internal quantum efficiency is limited to about 25%. Introduction of phosphorescent emitters allows to take advantage of spin statistics and to raise this limit. There are two principal routes to controlling the yield of phosphorescence in molecular emitters: by raising radiative rates or by suppressing non-radiative decay. To enhance radiative decay from triplet states via phosphorescence, normally metal–organic emitters are used in OLEDs. Recently, we have explored triplet harvesting without heavy atoms, in which thiophene-decorated phenazines are used as the basic building block of dual emitters. They showed both room-temperature electrofluorescence and electrophosphorescence. We have investigated the excited triplet state of a series of such phenazines by time-resolved EPR. Molecular properties such as zero- field splittings were characterized in frozen solutions. The zero-field splittings, which depend on substituents and sulfur positions in the molecules, were also investigated by quantum chemical calculations in order to elucidate the influence of pi-conjugation pathway within the emitter structure on the non-radiative triplet decay channel. The lifetime of the triplet state determined by time-resolved EPR demonstrated that the pi-conjugation pathway controls the non-radiative triplet decay channel.

Poster Presentations

133

Materials and Polymers Poster Presentation P53

Adjustable Dynamic Hydrophobic Attachment of Fatty Acids in Amphiphilic Core-shell Polymers J. Reichenwallner1, A. Thomas2, L. Nuhn2,4, T. Johann2, A. Meister1,3, H. Frey2 and D. Hinderberger1 1Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany 2Institute of Organic Chemistry, Johannes Gutenberg-University, 55128 Mainz, Germany 3Institute Institute of Biochemistry and Biotechnology, Martin-Luther-Universität Halle- Wittenberg, 06120 Halle, Germany 4Department of Pharmaceutics, Ghent University, 9000 Ghent, Belgium

E-mail: [email protected]

We report the dynamic self-assembly properties of synthetic amphiphilic core-shell polymers[1] and small amphiphilic ligand molecules. The individual brush-like graft copolymer structures (k) are obtained by controlled radical addition-fragmentation chain transfer [2] (RAFT) polymerization of click-coupled linear polyglycerol (linPPGm) chains linked to alkylene methacrylate forming a hydrophobic core surrounded by a hydrophilic shell. We employ continuous wave electron paramagnetic resonance (CW EPR) spectroscopy targeting the amphiphilic fatty-acid spin probe 16-DSA (16-doxyl stearic acid) to polymers of different alkylene and polyglycerol chain lengths. Upon binding, 16-DSA reveals polymer-based dynamic effects such as temperature response, phase transitions and ligand binding properties. In this regard, the increase of the hydrophobic alkylene chain length of the polymers alters the physical properties of the core region significantly, so that the number

NL,k of binding partners and dissociation constants KD,k can be adjusted. We furthermore suggest an intrinsic dynamic hydrophobic attachment mechanism of those potential drug carrier systems towards fatty acids that is furthermore characterized by highly nonlinear Van´t Hoff plots[3].

References [1] A. Thomas, K. Niederer, F. Wurm, H. Frey, Polym. Chem., 5, (2014). [2] J. Chiefari, Y. K. Chong, F. Ercole, et al., Macromolecules. 31, (1998). [3] R. I. Boysen, A. J. O. Jong, J. A. Wilce, et al., J. Biol. Chem. 277, (2002).

Poster Presentations

134

Materials and Polymers Poster Presentation P54

Identification and Characterization of Side Products in the Gilch Synthesis of Poly(ortho-Phenylene Vinylenes) V. Schmidts,1 V. Rittscher,2 S. Immel,2 M. Rehahn,2 and C. M. Thiele1 1Clemens-Schöpf-Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, D-64287 Darmstadt, Germany. 2Ernst-Berl-Institut für Technische und Makromolekulare Chemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, D-64287 Darmstadt, Germany. E-mail: [email protected]

The class of poly(phenylene vinylene) based polymers (short: PPVs) are of particular interest as materials for organic light-emitting diodes (OLEDs). The Gilch synthesis of para- substituted PPVs has been studied extensively over the last decade.[1-3] Recently, a new class of PPVs based on poly(ortho-phenylene vinylene)s (ortho-PPVs) were introduced.[4] The new polymer showed the absorption maximum at λ = 394 nm, while the maximum fluorescence intensity was found at λ = 498 nm. These properties hint at a possible application as blue-emitting polymeric materials — whereas para-PPVs emit from deep red to yellowish-green.[5] We herein present a deeper look into the polymerization mechanism and explore possible reaction pathways leading to unwanted side products. By cooling the NMR tube to ‒80°C and slowly heating up, we were able to follow the Gilch synthesis as the reaction progressed. We were able to identify and characterize intermediate species, which indicate reaction pathways different from those observed for para-PPVs. The observations are in line with competing Diels-Alder-type reactions of ortho-quinodimethane species as proposed by theoretical modelling.[6]

References [1] T. Schwalm, Macromol. Rapid Commun. 30, 1295 (2009). [2] T. Schwalm, Macromolecules 40, 8842 (2007). [3] A. Fleissner, Chem. Mater. 21, 4288 (2009). [4] V. Rittscher, Macromol. Rapid Commun. 37, 814 (2016). [5] A. C. Grimsdale, Chem. Rev. 109, 897 (2009). [6] S. Immel, unpublished results.

Poster Presentations

135

Materials and Polymers Poster Presentation P55

Thermal Evolution of 4- and 5-fold coordinated Al-sites in Aluminum Hydroxide Fluorides with low Fluorination Degree L. Ahrem1, G. Scholz1, R. Bertram2, E. Kemnitz 1 1 Humboldt-Universität zu Berlin, Department of Chemistry, Berlin, Germany 2 Institut für Kristallzüchtung, Berlin, Germany

A modified approach of the fluorolytic sol-gel synthesis was used to synthesize pure, amorphous aluminum hydroxide fluorides with very low and easily adjustable fluorination degrees, as well as controllable surface areas. The careful investigation of their thermal behavior revealed that these aluminum hydroxide fluorides undergo dehydroxylation reactions already at very low temperatures (i.e. below 80 °C), which is in contrast to all crystalline aluminum hydroxide (fluoride) phases. 27Al and 19F MAS NMR experiments unambiguously demonstrated that this dehydroxylation is clearly linked to a drastic reorganization of local structures and leads to a remarkable evolution of sub-coordinated Al- species. A maximum intensity of about 45 % for 5-fold (AlV) and 54 % for 4-fold (AlIV) coordinated Al-sites was found for sample AlF0.25(OH)2.75, calcined at elevated temperatures. The investigations also revealed that the relative intensity of AlIV - and AlV – species essentially depends on the fluorination degree [1].

References [1] L. Ahrem, G. Scholz, R. Bertram, E. Kemnitz, J. Phys. Chem. C 120 (2016) 9236-9244.

Poster Presentations

136

Materials and Polymers Poster Presentation P56

Morphology of polyethylene exposed to solvents and elevated temperatures by compact NMR: A comparative study Y. Teymouri, A. Adams, and B. Blümich Institut für Technische und Makromolekulare Chemie, RWTH Aachen University, Worringerweg 2, D-52074, Aachen, Germany E-mail: [email protected]

Many polyethylene (PE) products are used today in long-term applications, which require the use of elevated temperatures and/or the contact with different solvents. Both, exposure to heat and solvents can result in morphological changes and impact the mechanical stability of the material. Consequently, it is important to investigate the changes in phase composition and chain mobility of the different domains in the semi-crystalline material due to temperature [1] and solvent-induced crystallization (SINC) [2] in order to understand how the properties of semi-crystalline polymer change over time. While DSC and XRD provide valuable information about crystalline domains, the primary changes upon swelling and thermal aging arise in the amorphous domains, which can be probed only indirectly with these methods [2, 3]. The aim of this current study is to compare the temperature- and solvent-induced morphological changes in polyethylene by compact low-field NMR coupled with other analytical methods. It could be shown that elevated temperatures and the presence of a solvent at lower temperatures kinetically favor morphological rearrangements, which increase the crystallinity. The changes of polymer morphology observed at elevated temperature in air result from both physical and chemical aging, while chemical changes are absent in solvent- induced crystallization (SINC) but may be impacted by wax leaching from the polymer matrix into the solvent. The relaxation data measured with compact NMR at low-field are supported by data from proton wide-line NMR spectroscopy at high-field as well as FTIR, SEC, and DSC data. In particular, the increase in the fraction of the rigid domains with the swelling and aging time is confirmed by wide-line NMR spectroscopy measurements, and DSC measurements revealed a subtle difference between temperature and solvent-induced crystallization. In addition, the FTIR and SEC measurements proved the onset of thermal degradation by oxidation and chain scissions at short times for thermal aging. References [1] Y. Teymouri, R. Kwamen, B. Blümich, Macromol. Mater. Eng. 300 (2015) 1063. [2] Y. Teymouri, A. Adams, and B. Blümich, Eur. Polym. J. 80 (2016) 48. [3] C. Hedesiu, D. E. Demco, R. Kleppinger, G. V. Poel, W. Gijsbers, B. Blümich, K. Remerie, V. M. Litvinov, Macromolecules 40 (2007) 3977.

Poster Presentations

137

Materials and Polymers Poster Presentation P57

High Field 51V MAS Dynamic Nuclear Polarization NMR on V-Mo-W Mixed Oxide Catalysts

A. S. Lilly Thankamony1,3, S. Knoche2, A. Drochner2, H. Vogel2, T. Gutmann1, G. Buntkowsky1

1Institute of Physical Chemistry, Technical University Darmstadt, Germany 2 Technical Chemistry, Technical University Darmstadt, Germany 3 Present Address : Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt am Main, Germany Email: [email protected]

Heterogeneous V-Mo-W mixed metal oxide catalysts are important technical catalysts used for the partial oxidation of acrolein to acrylic acid. Because of their high catalytic activity and selectivity, these have been used in industry for several years.[1-2] Solid state NMR (ssNMR) provides structural information on the local environments of NMR active nuclei for characterizing complex or disordered systems. However it suffers from its low sensitivity especially when characterizing systems with low surface areas or small number of spins as it is in the case of V-Mo-W mixed oxide catalysts (surface areas in the range of 1- 40 m2/g). Currently, the most efficient method to increase the sensitivity of ssNMR experiments is high field Dynamic Nuclear Polarization (DNP) combined with Magic Angle Spinning (MAS).[3] Here we apply high field DNP-enhanced 51V MAS ssNMR spectroscopy to V-Mo-W mixed oxide technical catalysts. DNP experiments are supported by EPR studies of V-Mo-W mixed oxide impregnated with different polarizing agents. The structure-reactivity relationship of the catalysts are also explored using 51V MAS DNP.

References [1] S. Endres, Applied Catalysis A: General, 325, 2007 [2] P. Kampe, Phys. Chem. Chem. Phys, 9, 2007 [3] T. Maly, J. Chem. Phy. 128, 2008

Poster Presentations

138

Materials and Polymers Poster Presentation P58

Influence of DNP spin probes on the NMR relaxation and diffusion properties of water molecules in Nafion 1 2 1,3 4 2 1 T. Überrück , O. Neudert , J. Granwehr , S. Han , S. Stapf , B. Blümich 1RWTH Aachen University, Institut für Technische und Makromolekulare Chemie, Worringerweg 1, 52074 Aachen, Germany 2Technical University Ilmenau, Institut für Physik, Unterpörlitzer Straße 38, 98693 Ilmenau, Germany 3Forschungszentrum Jülich, Institut für Energie- und Klimaforschung, Ostring O10, 52425 Jülich, Germany 4 University of California Santa Barbara, Department of Chemistry and Biochemistry, Santa Barbara, CA 93106, USA E-mail: [email protected]

Recently Han et al. showed that Overhauser dynamic nuclear polarizitation (ODNP) is able to analyse proton conduction pathways in Nafion membranes by using different TEMPO derivates as paramagnetic spin-probes [1]. Although their results are promising, the effect of the spin-probes on the general properties of the membrane is not known in detail. In this work, fully hydrated Nafion membranes were studied, containing the paramagnetic nitroxide molecules 4-Hydroxy-TEMPO or 4-Amino-TEMPO, which have previously been used as spin-probes in the ODNP measurements by Han et al. Due to their different functional groups they reside in different molecular environments within the membranes. Both thermal and DNP polarized NMR measurements of the nuclear spin relaxation and diffusion were performed at various magnetic field strengths and different relative radical concentrations. Furthermore, temporal aspects were studied, regarding the equilibration time of the membrane/spin-probe system and the activity of the spin-probes over time. In order to differentiate between paramagnetic and diamagnetic relaxation effects caused by the spin- probes, they were compared to their diamagnetic equivalents 4-Amino-Tetramethyl- piperidine and 4-Hydroxy-Tetramethyl-piperidine. With the in depth investigation of the para- and diamagnetic effects of DNP spin probes on Nafion this work helps to understand the complex interactions in such systems and will be the basis for future DNP enhanced experiments on PEMs or even fuel cells.

References [1] J. Song, O.H. Han, S. Han, Angew. Chem. Int. Ed., 54, 3615-3620, (2015).

Poster Presentations

139

Small Molecules and Catalysis Poster Presentation P59

Band-Selective 2D NMR combined with CASE for Solving the Structure of Symmetrical Helical Molecules John P. Lowe1, David R. Carbery1, Arvin Moser2, Dimitris Argyropoulos3

1Department of Chemistry, University of Bath, Bath, U.K. 2Advanced Chemistry Development, Inc., Toronto, ON, Canada 3Advanced Chemistry Development UK Ltd., Bracknell, U.K. E-mail: [email protected]

High molecular symmetry creates substantial challenges in structure elucidation by NMR. The combination of modern, efficient NMR experiments together with Computer Assisted Structure Elucidation (CASE) could give an answer to such problems. In this case two isomeric, highly-symmetrical helical molecules were synthesized by Rh(I)- catalysed cycloisomerisation of a suitable triyne substrate. The isomers were separated by preparative scale liquid chromatography before 10 mg of each isomer was dissolved with deuterated chloroform and placed in an NMR tube in order to confirm their structures. Here we describe the advanced computation method used for confirming the structures by NMR experiments only. Due to the presence of molecular symmetry the NMR spectra of the resulting compounds exhibited very few peaks relative to the size of the molecule. That is, half the number of expected signals are observed in the 13C NMR spectrum and, in addition, several of the signals are extremely closely spaced. Moreover the deficient hydrogen content of the compounds resulted in a low number of correlations in the 1H- 13C HMBC spectra. CASE together with band-selective versions of the HSQC and HMBC experiments were employed to determine the structures and assign unequivocally the NMR data and thus confirm the new molecules. The final assignment of the two isomeric structures was accomplished by using 1H- 1H 2D NOESY experiments.

Poster Presentations

140

Small Molecules and Catalysis Poster Presentation P60

Application of 1H-NMR-Spektroscopy for analysis of the geographical origin of Hazelnuts R. Bachmann1,2, S. Klockmann2, M. Fischer2, T. Hackl1,2 1 University of Hamburg, Institute of Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg 2 University of Hamburg, Hamburg School of Food Science, Grindelallee 117, 20146 Hamburg

Considering the increasing globalization and the growing interest in local produced products, the determination of the origin of food becomes more and more important. Today Turkey is the largest hazelnut producer with about 75% of the world production. The second largest hazelnut producer is Italy with significant differences in production conditions and qualitative marketing. Therefore a unique method of determination of the products origin would be desirable. In this study different hazelnut samples from Turkey, Italy, Georgia and Germany were analyzed by 1H-NMR-spectroscopy and the multivariate data analysis methods such as PCA and PLS-DA. The PCA showed a good clustering of samples according to their geographic origin. Especially Georgian samples differed significantly from Italian and German samples. The corresponding Loadings-Plots show the important buckets which are responsible for separation of the sample groups. In the next step a discriminant analysis was performed, which showed the total separation of the samples by their origin. It appears that the approach of using 1H-NMR-Spectroscopy together with PCA and PLS-DA delivers a fast and accurate determination of the geographical origin of hazelnuts.

Poster Presentations

141

Small Molecules and Catalysis Poster Presentation P61

Competitive study of the basic suitability of 13C projections from HMBC type experiments to replace 1D 13C spectra Johanna Becker1,2, Martin R.M. Koos1,2, David Schulze Sünninghausen2 and Burkhard Luy1,2 1Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces 4 – Magnetic Resonance, 76344-Eggenstein-Leopoldshafen, Germany 2Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, 76131-Karlsruhe, Germany E-mail: [email protected]

In this work we consider a combination of the two fast scanning approaches ASAP (Acceleration by Sharing Adjacent Polarization) [1] and ALSOFAST (ALternate SOFAST) [2] with HMBC type experiments. A comparative analysis of the different species of HMBC sequences is presented with special attention on minimizing artefacts of different origin. Be it t1-Noise or the distortion of signals by undesired coupling evolution. In a first attempt the applicability of these experiments to compete with the 13C-1D experiment is tested by which the 13C projection should serve as an analogon to the one-dimensional 13C spectrum. In this context speeding up the acquisition is an indispensable need. Facing ultimate resolution, the synergy of the acquisition with NUS (Non Uniform Sampling) is used. In contrast to the direct detection of 13C resonances the two-dimensional proton detected experiments have a much higher intrinsic sensitivity. Furthermore, relaxation times which are crucial in the fast pulsing regime are considerably shorter for proton detected experiments compared to carbon detected ones. So far the acquisition of high resolved two-dimensional spectra is very time consuming. In this work we want to make an attempt to explore these limits.

References [1] (a) Ē. Kupče, R. Freeman. Magn. Reson. Chem. 2007, 45, 2-4; (b) D. Schulze- Sünninghausen, J. Becker, B. Luy. J. Am. Chem. Soc. 2014, 136, 1242-1245. [2] L. Mueller. J. Biomol. NMR 2008, 42, 129-137.

Poster Presentations

142

Small Molecules and Catalysis Poster Presentation P62

NMR Investigations of Photocatalytic Olefin Hydroamidation by Proton-Coupled Electron Transfer Nele Berg, Andreas Seegerer, Ruth Gschwind University of Regensburg, Universitätsstraße 31, 93053 Regensburg E-mail: [email protected]

The catalytic olefin hydroamidation is an efficient process to generate biologically active nitrogen-containing compounds without prior prefunctionalization of the amide substrate.[1] Previously, Knowles et al. published an optimized catalyst system for the photocatalytic intramolecular hydroamidation of unactivated aryl amide derivatives. Therefore, the amide was transferred into the amidyl radical via proton-coupled electron transfer (PCET) by using an excited iridium photocatalyst and a Brønsted base. Subsequent olefin addition using thiophenol as most effective hydrogen-atom donor generates the cyclic product. Surprisingly, the amide substrate can be selectively transferred into the radical in presence of thiophenol although there is a large difference in bond dissociation energies between these functional groups. Although the N-H-bond (99 kcal/mol) is much stronger than the S-H- bond (79 kcal/mol) a thiophenol radical is not formed. The postulated explanation is a difference in hydrogen bond donor abilities of the amide and thiol. Hence, a stronger hydrogen bond between amide and Brønsted base is formed.[2][3] In order to investigate the hydrogen bond donor abilities, low temperature NMR spectroscopic studies of simple 15N-labeled amides, thiophenol and phosphate base were conducted. Thus, 1H, 31P and 15N chemical shifts and 2D experiments of several combinations were investigated to identify different hydrogen bond strengths.

References [1] D. Banerjee, K. Junge, M. Beller, Angew. Chem. Int. Ed. Engl. 53, (2014) [2] D. C. Miller, G. J. Choi, H. S. Orbe, R. R. Knowles, J. Am. Chem. Soc. 137, (2015) [3] G. J. Choi, R. R. Knowles, J. Am. Chem. Soc. 137, (2015)

Poster Presentations

143

Small Molecules and Catalysis Poster Presentation P63

Multiple conformations of histamine in solution: An NMR study in organic- and water-based ordering media M. E. Di Pietro1,2, G. De Luca2, G. Celebre2 and B. Luy1 1 Institute for Biological Interfaces 4 - Magnetic Resonance, Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany 2 Lab. LXNMR_S.C.An., Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, via P. Bucci, 87036 Rende (CS), Italy E-mail: [email protected]

The 2-aminoethylimidazole, known as histamine, is a small flexible biogenic amine neurotransmitter with a key biological role. To rationalize its mechanism(s) of action and design suitable analogues with enhanced activity, a thorough understanding of the molecular spatial arrangement in solution is crucial.[1] To this end, NMR spectroscopy combined to the use of weakly ordering media may represent a valuable strategy, since the anisotropic dipolar interactions (RDCs) that influence the experimental spectra give access, via a proper theoretical treatment, to unique structural and conformational details.[2] In this contribution, we report and compare the conformational features obtained for histamine in three different orienting solvents: (a) a chiral nematic lyotropic liquid crystalline [3] phase composed of PBLG and the organic co-solvent CDCl3, (b) a stretched gel system [4] obtained from gelatin swollen in D2O, and (c) a novel alignment medium composed of the [5] ionic liquid crystal C12MImBF4 slightly doped with water.

Poster Presentations

144

Small Molecules and Catalysis Poster Presentation P64

NMR-Based Metabolomics of Urine and Comparison of Body Fluids in Peritoneal Dialysis

P. Eisenmann1, M. Himmelsbach1, B. Luy1,3, J. Westhoff2, C. Muhle-Goll1,3 1Karlsruhe Institute of Technology, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany 2Heidelberg University Hospital, Centre of Paediatrics and Youth Medicine, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany 3Karlsruhe Institute of Technology, Institute for Biological Interfaces 4, P.O. Box 3640, 76021 Karlsruhe, Germany E-mail: [email protected]

NMR metabolomics has a high potential to detect pathological profiles and to identify biomarkers, as it is highly automatized with simple sample preparation and it is furthermore highly reproducible and quantitative over a broad range. The diagnosis of acute kidney injury (AKI) depends on serum creatinine (SCr), which is a delayed and indirect indicator of AKI only. In addition, SCr cannot differentiate between prerenal and intrinsic AKI and does not allow for the prediction of renal recovery or adverse outcome including the need for renal replacement therapy and mortality. Since significant morbidity and mortality is associated with AKI, there is a great need for novel biomarkers in early diagnosis, differentiation and prognostic outcome for pediatric and adult patients with AKI. Hence, identification of urinary markers and disease-specific metabolic fingerprints by NMR spectroscopy can influence clinical decision-making and presumably improve patient health. After the kidney had recovered, the first urine of young people with AKI was examined. The study investigated indicators of AKI in the foreground and the metabolome in different forms of AKI. In another study, urine, serum and dialysate from patients were studied, which require a peritoneal dialysis.

References [1] Kromke, M.; Palomino-Schätzlein, M.; Mayer, H.; Pfeffer, S.; Pineda-Lucena, A.; Luy, B.; Hausberg, M.; Muhle-Goll, C., Transl Res., 171, 2106, 71-82

Poster Presentations

145

Small Molecules and Catalysis Poster Presentation P65

Alignment of Benzene Derivatives in Stretched Polystyrene and Polybutylacrylate Gels Michael John, Thomas Niklas and Dietmar Stalke Institut für Anorganische Chemie, Georg-August-Universität, Göttingen, Germany E-mail: [email protected]

Since the first observation of alignment of solutes in anisotropic environment,[1] anisotropic NMR parameters have become an important tool in the structure determination of small molecules.[2] Meanwhile, a wide range of media are available for this purpose, the most common ones being nematic liquid crystalline phases[3,4] and cross-linked polymer gels that are either stretched[5] or compressed[6] along the magnetic field axis. While the interaction of solutes with liquid crystalline phases has been studied for a long time,[7] little is known about the mechanism of solute alignment in gels. Simple steric[8] or inertia[9] models for the aligment of biomacromolecules fail for small and often flexible solutes. Only very recently, Luy et al. could reproduce the alignment of strychnine in a polystyrene matrix using MD simulations.[10] 1 By measuring DCH residual dipolar couplings (RDCs), we systematically determined the alignment of a range of symmetric benzene derivatives and polycyclic homologues in stretched polystyrene and polybutylacrylate. Several compounds show drastically different orientations in the two media, which can be explained by a counterbalance of dispersive hydrophobic and polar/H-bond interactions. The results are supported by MD simulations.

References [1] A. Saupe, G. Englert, Phys. Rev. Lett. 11, 462 (1963) [2] e.g. T. Niklas, C. Steinmetzger, W. Liu, D. Zell, D. Stalke, L. Ackermann, M. John, Eur. J. Org. Chem. 6801 (2015) [3] C. M. Thiele, S. Berger, Org. Lett. 5, 705 (2003) [4] L. Verdier, P. Sakhaii, M. Zweckstetter, C. Griesinger, J. Magn. Reson. 163, 353 (2003) [5] B. Luy, K. Kobzar, H. Kessler, Angew. Chem. Int. Ed. 43, 1092 (2004) [6] C. Gayathri, N. V. Tsarevski, R.R. Gil, Chem. Eur. J. 16, 3622 (2010) [7] E.E. Burnell, C.A. de Lange, Chem. Rev. 98, 2359 (1998) [8] M. Zweckstetter, J. Biomol. NMR 23, 127 (2002) [9] H.F. Azurmendi, C.A. Bush, J. Am. Chem. Soc. 124, 2426 (2002)

[10] A.O. Frank, J.C. Freudenberger, A.K. Shaytan, H. Kessler, B. Luy, Magn. Reson. Chem. 53, 213 (2015)

Poster Presentations

146

Small Molecules and Catalysis Poster Presentation P66

Interaction study of Brønsted acid catalyst / imine complex in solution by NMR.

M. Melikian1 , J. Greindl, J. Hioe, N. Sorgenfrei and R. M. Gschwind1 1Institut für organische Chemie, Regensburg, Germany E-mail: [email protected]

In the recent years, enantiomeric reactions mediated by Brønsted acid catalyst encountered a large success.[1] In particular, imine isomerization reaction allowed to partially converting E-imine into Z-imine. However, the Brønsted acid catalyst / imine complex structure in solution remains poorly understood.[2] In order to examine Brønsted acid catalyst / Imine complex, series of experiment were performed by NMR. The Mac Millan catalyst was selected. It consists of a symmetrical bi-naphthalene backbone supporting a phosphoric acid in the middle and two side chains composed each one by a Si(Ph)3 group. In absence of catalyst, E/Z Imine ratio is 99/1. The fraction of Z-imine varies in a 40-60 % range after catalyst addition. Splitting of Mac Millan catalyst NMR signals on HSQC spectrum indicated a specific interaction for each imine isomeric form. Selective 1D NOESY and Heteronuclear 1H 19F HOESY allowed identifying several intermolecular NOE. Finally, longitudinal T1 and transversal T2 relaxation experiment provided complementary information regarding the interaction between Mac Millan catalyst and the respective E and Z-imine forms. These results provided insights to further obtain a complex structure.

References [1] Storer, R. I.; Carrera, D. E.; Ni, Y.; MacMillan, D. W. C. J. Am. Chem. Soc., 128 (1), 84–86. (2006) [2] Rueping M., Sugiono E., Azap C., Theissmann T., Bolte M. Org Lett., 7 (17):3781-3. (2005)

Poster Presentations

147

Small Molecules and Catalysis Poster Presentation P67

Resolution enhanced 1H NMR method for separation of multiple spin system and mixture analysis N. Lokesh and R. Gschwind Universität Regensburg, Regensburg, Germany E-mail: [email protected]

NMR provides vital information about structure, dynamics, bonding and non-bonding interactions of the molecule in solution through chemical shifts, scalar couplings and NOEs. The presence of mixture of molecules or impurities complicates the system and becomes difficult to extract these structural parameters unambiguously. It is more common in organic reaction mixtures, natural extractions and metabolomics samples. 2D homonuclear and heteronuclear experiments partially solves the problems but fails often due to low resolution in indirect dimension and less natural abundance of 13C. Here, we present new 2D homonuclear NMR methods, which resolves different connected spin systems based on chemical shift difference, allowing mixture separation by retaining high resolution in both the dimensions. The high resolution in indirect dimension is achieved through homonuclear decoupling and less spectral width selection, while retaining the couplings and chemical shift information in direct dimension. The application of the methods were shown on system of mixture of molecules and organic reaction mixture.

References [1] D. Sinnaeve, M. Foroozandeh, M. Nilsson, G. A. Morris, Angew. Chem. Int. Ed., 55, (2016), 1090 – 1093 [2] Lokesh, S. R. Chaudhari, N. Suryaprakash, Org. Biomol. Chem., 12, (2014), 993-997.

Poster Presentations

148

Small Molecules and Catalysis Poster Presentation P68

IDNMR: Establishing a low-barrier online tool for pre-publication NMR shift assignment evaluation Stefan Kuhn,1 Johannes C. Liermann,2 and Nils E. Schlörer3 1 Ulidowski Group, Department of Computer Science, University of Leicester, UK 2 NMR facility, Institute of Organic Chemistry, Johannes Gutenberg University Mainz, Germany 3 NMR facility, Department of Chemistry, University of Cologne, Germany E-mail: [email protected]

While areas like X-ray crystallography or biomolecular NMR have long-established means of electronic research data submission and automatic evaluation prior to publication, the documentation and evaluation of NMR data in small molecule chemistry is still mostly stuck in the pre-digital age. Moreover, a large fraction of published NMR data has incorrect assignments or no assignments at all.[1] The consequence is a considerable number of incorrect structures in natural products chemistry[2] and assumedly many similar incidents in synthetic chemistry going unnoticed as long as there is no spectacular article retraction. Many of these cases could have been easily prevented by an automatic check of NMR shifts against a prediction before publication. CSEARCH is already offering an online “robot referee” system for several years for 13C evaluation.[3] However, this system requires prior registration and the report is sent by e-mail which does not allow an instant and anonymous evaluation. In addition, 1H chemical shifts are not evaluated. The project IDNMR (Initiative for Data Quality in NMR spectroscopy) addresses the insufficient evaluation of NMR data in scientific publications. In the course of this project, we are working on the development of a QuickCheck tool for 1H and 13C NMR assignments which is based on the open web-based NMR database NMRShiftDB.[4] Users can submit structures along with assigned or unassigned chemical shifts and will instantly receive a report on the agreement of their data with the proposed structure. The integration with the open database allows the use of its prediction and auto-assignment tools and encourages the subsequent submission of evaluated structures to the database. An even more streamlined workflow from acquired data to assigned structures is feasible by using the optional LIMS provided by the nmrshiftdb2 software.[5] References [1] W. Robien, TrAC, Trends Anal. Chem. 2009, 28, 914-922. [2] a) K. C. Nicolaou, S. A. Snyder, Angew. Chem., Int. Ed. 2005, 44, 1012-1044; b) M. Elyashberg, A. J. Williams, K. Blinov, Nat. Prod. Rep. 2010, 27, 1296-1328. [3] W. Robien, CSEARCH Robot Referee, http://nmrpredict.orc.univie.ac.at/c13robot/robot.php [4] NMRShiftDB, http://www.nmrshiftdb.org [5] S. Kuhn, N. E. Schlörer, Magn. Reson. Chem. 2015, 53, 582-589.

Poster Presentations

149

Small Molecules and Catalysis Poster Presentation P69

NMR Spectroscopic Investigations of Intermediate Dienamines and Iminium Ions in ACDC D. Schneider1 and R. Gschwind1 1University of Regensburg, Regensburg, Germany E-mail: [email protected]

Asymmetric counterion directed catalysis (ACDC) belongs to the field of organocatalysis and is described as induction of enantioselectivity by an ion pair consisting of an achiral cation and a chiral anion.[1] The concept was recently applied to the asymmetric transfer hydrogenation of α,β-unsaturated aldehydes with Hantzsch esters using salts that contain an achiral ammonium cation and a chiral phosphate anion as catalysts. With yields in the range of 63-90% and enantiomeric ratios of 95:5 and higher, these reactions seem to be very promising.[2] However, the question arises how such enantioselectivity is induced mechanistically. Therefore, structural investigations were carried out with morpholine as secondary amine. Chirality was provided to the system by (R)-3,3’-bis(2,4,6-triisopropylphenyl)- 1,1’-binaphthyl-2,2’-diyl hydrogen phosphate (TRIP), which combines the functionality of a phosphoric acid with a binaphthyl scaffold and triisopropylphenyl moieties as side chains. A variety of α,β-unsaturated aldehydes were used in attempt to synthesise the corresponding dienamines and iminium ions. For characterisation, NMR spectroscopic measurements were performed in different solvents and at several temperatures.

References [1] M. Mahlau, B. List, Angew. Chem. Int. Ed. 52, (2013) [2] S. Mayer, B. List, Angew. Chem. Int. Ed. 45, (2006)

Poster Presentations

150

Small Molecules and Catalysis Poster Presentation P70

STD NMR experiments identify fragments binding to a second site of West Nile Virus NS2B-NS3pro protease

T. Schöne,1 L. L. Grimm1, N. Sakai2, L. Zhang2, R. Hilgenfeld2 and T. Peters1 1Institute of Chemistry and Ratzeburger Allee 160, 23562 Lübeck, Germany 2Institute of Biochemistry, Ratzeburger Allee 160, 23562 Lübeck, Germany E-mail: [email protected]

West Nile Virus (WNV) belongs to the genus Flavivirus. Along with Zika and Dengue viruses, this mosquito-borne, human pathogenic RNA virus has been evolving into a global threat. Despite the potentially severe outcome of a WNV infection, no antiviral therapy has been developed yet. The viral protease NS2B-NS3pro is essential for viral replication. Therefore, NS2B-NS3pro has been a drug target for many years [1]. In order to identify second site binders for WNV NS2B-NS3pro protease (WNP) we screened a fragment library, the Maybridge Ro5 library, employing STD NMR as readout system [2] resulting in 126 fragments (30%) with binding affinity for NS2B-NS3pro. In a fluorophore-based assay 66% of these fragments showed inhibitory potency (µM range). Subsequent STD NMR competition experiments using a known active site binding inhibitor as reporter ligand [3] yielded 14 competitively binding fragments, and 22 fragments that did not compete with the known inhibitor. Of these non-competing binders 10 showed a significant increase of STD effect in the presence of the known inhibitor. The most attractive non-competitive compounds (IC50 ~500 µM) share a common structural motif that may be a starting point for the development of novel inhibitors aided by the identification of corresponding binding pockets using 1H,15N- TROSY-HSQC NMR.

References [1] Lim et al., Viruses, 5:2977-3006, (2013) [2] Meyer and Peters, Angew Chem Int Ed Engl, 42:864-90, (2003) [3] Ekonomiuk et al., PLoS Negl Trop Dis, 3:13, (2009)

Poster Presentations

151

Small Molecules and Catalysis Poster Presentation P71

Novel Experiments based on the ASAP-HSQC David Schulze-Sünninghausen2, Johanna Becker1,2, Martin R. M. Koos1,2 and Burkhard Luy1,2 1Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces 4 – Magnetic Resonance, 76344-Eggenstein-Leopoldshafen, Germany 2Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, 76131-Karlsruhe, Germany E-mail: [email protected]

Previously we introduced the ASAP-HSQC[1] as a fast method for the detection of heteronuclear single quantum coherence spectra of small molecules at natural abundance. On this poster we will present the latest developments of fast experiments based on the ASAP approach.

References [1] (a) Ē. Kupče, R. Freeman. Magn. Reson. Chem. 2007, 45, 2-4; (b) D. Schulze- Sünninghausen, J. Becker, B. Luy. J. Am. Chem. Soc. 2014, 136, 1242-1245.

Poster Presentations

152

Small Molecules and Catalysis Poster Presentation P72

Metabolic changes during cellular senescence investigated by NMR spectroscopy C. Windler1, T. Becker2, C. Gey3, D. H. Rapoport2, and K. Seeger1 1Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany 2 Fraunhofer EMB, Mönkhofer Weg 239a, 23562 Lübeck, Germany 3 Institute of Biology and Institute of Physiology, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany E-mail: [email protected]

Investigating cellular metabolism by NMR spectroscopy allows identification of altered metabolic pathways. Previously we found by an untargeted metabolomic approach using NMR spectroscopy that increased glycerophosphocholine (GPC) level seem to be a metabolic marker for cellular senescence in the human embryonic lung fibroblast cell line WI-38 [1]. The alterations in choline metabolism are opposed to the well-known changes in malignant cells underlining the importance of cellular senescence as tumour suppressor mechanism. Since cellular senescence is not only an important tumour suppressor mechanism but is also contributing to organismic ageing we wanted to investigate whether other cell types show the same metabolic changes. Primary human skin fibroblasts, and melanocytes have been cultured until they reached replicative senescence. Senescence has also been induced by addition of the cytostatic drug etoposide. Metabolic profiles of cell extract have been determined by NMR spectroscopy. Differences are observed not only between the different cell types but also between the two investigated senescence trigger. In accordance with our previous study GPC levels are elevated in senescent fibroblasts, in melanocytes changes in phosphocholine are more pronounced. We therefore conclude that the choline metabolism plays an important role in cell senescence and can be used to identify senescence.

References [1] Gey C, Seeger K. Mech Ageing Dev 134 (2013), 130-138.

Poster Presentations

153

Small Molecules and Catalysis Poster Presentation P73

Characterisation of self-assembled Pt-complexes: Insights into stacking and cooperative C-H....X hydrogen bonding from Solid State NMR P. Selter1, C. Rest2, A. Sampedro3, G. Fernandez3 and M. R. Hansen1 1Institute for Physical Chemistry, University of Münster, Germany 2Institute for Organic Chemistry and Center for Nanosystems Chemistry, University of Würzburg, Germany 3Institute for Organic Chemistry, University of Münster, Germany E-mail: [email protected]

Weak C-H…X interactions involving π-systems, electronegative atoms and anions are of high interest in many scientific fields from molecular biology to crystal engineering. Recently, a novel amphiphilic PtII-complex was described[1],which shows supramolecular polymerization and (hydro)gelation induced by cooperative π-π interactions as well as C-H…X hydrogen bonding. We performed a variety of 1H, 13C and 195Pt solid state NMR experiments on related complexes to further understand the driving forces behind the polymerization and the unconventional C-H…X interactions. Various 13C{1H} correlation spectra yield insight into the intricate stacking occurring due to π-π and π-halogen interactions, along with measurements of the 13C CSA and 1H-13C dipolar couplings. To explore the platinum coordination, 195Pt WURST-CPMG experiments were performed.

References [1] C. Rest et. al., Angew. Chem. Int. Ed. 53, 700-705 (2014)

Poster Presentations

154

Small Molecules and Catalysis Poster Presentation P74

Study of the counter-ion effect in strychnine by 1D and 2D spectroscopy with compact NMR Kawarpal Singh and Bernhard Blümich

Institut für Technische und Makromolekulare Chemie, RWTH Aachen University, Germany Email: [email protected]

The new generation of compact NMR instruments [1] provides novel possibilities for the use of NMR spectroscopy on the chemical workbench to elucidate chemical structures of illicit and toxic drugs and to monitor chemical reactions. Strychnine has 22 protons and 6 prochiral carbon atoms. It is a comparatively complex yet small molecule and is popular for reference in high-field NMR spectroscopy. The origin of these illicit chemicals and its counter ions such as strychnine sulphate and strychnine chloride can be identified, or two or more distinct samples obtained from the same chemical synthesis procedure can be matched by identifying the counter-ion [2] with analytical techniques such as gas chromatography. In this work, we demonstrate that the chemical- shift changes due to the different counter-ions of strychnine can be followed 1D and 2D NMR spectroscopy and that peaks can be assigned with the help of 13C, HETCOR and COSY experiments with a compact NMR spectrometer [3].

References [1] B. Blümich, S. Haber-Pohlmeier, W. Zia, Compact NMR, de Gruyter, Berlin, 2011. [2] A. E. Metaxas, J. R. Cort, Magn. Reson. Chem. 51 (2013) 292-298. [3] K. Singh, B. Blümich, Trends. Anal. Chem., doi:10.1016/j.trac.2016.02.014

Poster Presentations

155

Small Molecules and Catalysis Poster Presentation P75

Organocatalytic Enyne Formation – NMR Spectroscopic Investigations and Reaction Optimization Verena Streitferdt, Michael Haindl, Markus B. Schmid, Fabio Morana, Polyssena Renzi, Felicitas von Rekowski, Alexander Zimmermann, Kirsten Zeitler and Ruth M. Gschwind University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany E-mail: [email protected]

The conjugated enyne moiety can be found in several natural products and pharmaceuticals[1], which draws a high interest on the synthesis of such enynes. Common methods for the synthesis of conjugated enynes are mainly metal catalytic cross-couplings[2], whereas metal free approaches are still rare so far.[3] To the best of our knowledge, organocatalytic procedures haven’t been reported yet. In our group, a completely new, organocatalytic procedure for the synthesis of conjugated enynes was designed and mechanistic studies were carried out by NMR. This new procedure allows the enyne formation via a nitroalkene dimerization followed by a base induced fragmentation. The latter was conducted in presence of benzoic acid and several bases were tested. Triethylamine proved to be the best choice with highest, but still moderate NMR yields. It is assumed, that beside the enyne formation also polymerization of the nitroalkene dimer can occur.[3] In order to optimize the reaction from the dimer to the enyne, attempts to reduce the polymerization were made. Therefore, this reaction was carried out with varying concentrations regarding the nitroalkene dimer, benzoic acid and triethylamine. Furthermore, different acids were tested and the reaction was conducted at elevated temperatures. The mentioned reactions were monitored by NMR. Also a further attempt by a dropwise addition of the dimer to acid and base was performed.

References [1] a) Konishi, et al, J. Antibiot. 42, (1989), b) Rudisill, D. E.; Castonguay, L. A.; Stille, J. K., Tetrahedron Lett. 29, (1988), c) Stutz, A.; Petranyi, G., J. Med. Chem. 27, (1984), d) Huang, et al., J. Med. Chem. 53, (2010), e) Masaoka, T.; et al. H. Eur. J. Cancer. 12, (1976) [2] a) Hatanaka, Y.; Hiyama, T., J. Org. Chem. 53, (1988), b) Hatanaka, Y.; Matsui, K.; Hiyama, T., Tetrahedron Lett. 30, (1989), c) Andreini, et al., Tetrahedron. 45, (1989), d) Shao, L. X.; Shi, M., J. Org. Chem. 70, (2005), e) Saha, D.; Chatterjee, T.; Mukherjee, M.; Ranu, B. C., J. Org. Chem. 77, (2012), f) Miyaura, N.; Yamada, K.; Suzuki, A., Tetrahedron Lett. 20, (1979) [3] Li, P. F.; Wang, H. L.; Qu, J., J. Org. Chem. 79, (2014) [4] M. Haindl, Dissertation: “NMR Spectroscopic Investigations On Small Organic Molecules: Catalyst Stabilities, Low-Abundance Conformers, Intermediates And Reaction Mechanisms”, (2016)

Poster Presentations

156

Methods & Theory Poster Presentation P76

EPR-Based Localization of Metal Ions in Biomolecules Dinar Abdullin, Nicole Florin, Fraser Duthie, Gregor Hagelueken, and Olav Schiemann Institute of Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany E-mail: [email protected]

Metal ions are present in many proteins and nucleic acids and are involved in numerous essential biological functions. The knowledge of the location of metal ions in biomolecules is important for elucidation of the biological functions of metal ions. Here, an EPR-based trilateration approach for localization of paramagnetic metal ions in biomolecules will be presented. The idea of the approach is that the position of a metal ion can be determined via distance constraints measured between this ion and a number of spin labels attached at selected positions of a biomolecule. The approach was successfully tested on the Cu(II) ion of the protein azurin [1]. The determined precision of the approach of 0.26 nm was shown to be limited mostly by the precision of the estimated locations of the spin labels in the azurin structure. Therefore, the detailed study of these locations was carried out by means of EPR and X-ray crystallography [2]. In addition, the possibility to extend the trilateration approach towards other metal ions, such as Fe(III), and other biomolecules, such as RNA, was considered. This includes the comparison of different pulsed EPR techniques for distance measurements [3], as well as the synthesis and detailed characterization of a new spin label for RNA.

References [1] D. Abdullin et al., Angew. Chem. Int. Ed. 54, 1827 (2015). [2] D. Abdullin et al., Phys. Chem. Chem. Phys. 18, 10428 (2016). [3] D. Abdullin et al., J. Phys. Chem. B 119, 13534 (2015).

Poster Presentations

157

Methods & Theory Poster Presentation P77

Methyl induced cross-relaxation during dynamic nuclear polarization C. Bengs1, J. Heiliger1, D. Daube1 and B. Corzilius1 1Institute of Physical and Theoretical Chemistry, Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany E-mail: [email protected]

Nuclear Magnetic Resonance (NMR) Spectroscopy is one of the most developed spectroscopic techniques due to easy manipulation of the present quantum system. One of the major downsides of NMR is its inherently low sensitivity. To overcome this problem considerable effort has been put into the development of hyperpolarization techniques. One of the most promising hyperpolarization techniques, dynamic nuclear polarization (DNP), has enabled investigation of otherwise challenging systems. DNP is used to transfer the high thermal polarization of electrons onto the proton bath. A second polarization transfer onto a hetero-nucleus allows for detection of a substantially enhanced signal.

Recent results from our group have shown that incoherent dynamics under DNP conditions can facilitate the polarization transfer from protons to carbon. Experimentally we could observe spectral line inversion in specific parts of solid-state carbon spectra of proteins and amino acids. Necessities for this line inversion are spatial proximity of a reorienting methyl group to the carbon and significant hyperpolarization of the protons. This process is highly reminiscent of the classic Nuclear Overhauser Effect (NOE) observed in solution-state NMR. Analysis based on Redfield-Relaxation theory allows for basic description of this observation. Additionally, numerical simulations were employed to further explore influence of several control parameters.

Poster Presentations

158

Methods & Theory Poster Presentation P78

Site-directed spin labeling of a protein with Gd(III) for MAS NMR dynamic nuclear polarization Jörg Heiliger1, Diane Daube1, Thorsten Bahrenberg1, Robert Silvers2, Harald Schwalbe2, Björn Corzilius1 1Institute of Physical and Theoretical Chemistry, Institute of Biophysical Chemistry, and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue- Str. 7-9, 60438 Frankfurt am Main, Germany. 2Institute of Organic Chemistry and Chemical Biology, and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Str. 7-9, 60438 Frankfurt am Main, Germany. E-mail: [email protected]

Dynamic nuclear polarization (DNP) is a method to overcome the inherent low sensitivity of NMR by transferring the higher polarization of unpaired electron spins to the nuclei by irradiation with microwaves during low temperature MAS NMR experiments. Nitroxides such as AMUPol or paramagnetic high-spin transition metal ions such as Gd(III) can be used as polarizing agents (PA). Ubiquitin is a small, easy to handle, robust protein. The absence of cysteines easily allows for specific mutations for site-directed spin labeling. Therefore, ubiquitin is well suited as a biological model system. In this work we present recent results of site directed spin labeling on a protein to show the effect of the transition metal Gadolinium on a biological system. The influence of different metal chelators are compared and we investigate the influence of parameters such as metal- protein distance or EPR linewidth on DNP. Furthermore, the impact of such a high-spin PA on features such as paramagnetic relaxation resulting in line broadening and reduced DNP enhancement factors will be reported and compared to standard biradicals like AMUPol. Additionally, heteronuclear cross relaxation of the enhanced proton spins with 13C leads to resonance inversion and enhancement for specific functional groups. The results presented will help to design improved metal chelate complexes and are a first step to site-specific DNP. Further exploitation of the selective peak inversion may lead to new DNP applications.

Poster Presentations

159

Methods & Theory Poster Presentation P79

Comparison of Calculated Chemical Shifts (DFT) with Experiment and Spectrum Prediction B. Kirste1 1Institut für Chemie und Biochemie der Freien Universität, Berlin, Germany E-mail: [email protected]

Chemical shifts for a series of natural products have been calculated by routine DFT hybrid methods (B3LYP/6-311G(d,p), Gaussian 09) focusing on 13C NMR. The results are compared with experimental data and with data from spectrum prediction. Roughly, the precision of the quantum-chemical calculations is similar to that of spectrum prediction, i.e., the agreement with experimental data is usually within a few ppm, but occasionally deviations exceed 5 ppm (for 13C NMR) [1]. The quantum-chemical calculations refer to the three- dimensional structure of the molecules, whereas in spectrum prediction only connectivities are taken into account (increment methods using the HOSE code approach).

While it is clear that approximate MO methods will only yield approximate results, the interesting question here is the influence of the geometry. The calculations begin with geometry optimization, but only in the case of rigid geometries a clear-cut result can be expected. Otherwise there might be complications due to the flexibility of the molecule, i.e. problems of finding the global energy minimum, or equilibria between different conformers. Calculations are usually performed for the gas phase, but they may be extended to solutions.

References [1] B. Kirste, Chemical Sciences Journal, 2016, 7:127.

Poster Presentations

160

Methods & Theory Poster Presentation P80

Ligand Binding to the Histamine H2 Receptor Investigated by NMR Spectroscopy P. Nitschke1, M. Melikian1, A. Buschauer1 and R. Gschwind1 1University of Regensburg ,Universitätsstraße 31, 93053 Regensburg, Bavaria, Germany E-mail: [email protected]

G-Protein coupled receptors (GPCRs) make up over 40 % of all drug targets.[1] To develop new GPRC drug candidates by a structure based design, a further understanding of ligand binding to the protein binding site is mandatory. Besides crystallization of the GPCRs, nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful tool to characterize the binding of a ligand to the active site of proteins.[2] Especially the method of saturation transfer difference (STD) NMR has become a prominent tool in drug discovery.[3] As its name suggests the receptor of interest is saturated by a low- power pulse irradiating a spectral region of the receptor that doesn’t contain ligand signals. By the process of spin diffusion the magnetization spreads rapidly throughout the protein thereby saturating all protein 1H-NMR signals. Ligands binding to the protein get saturated via non-scalar magnetization transfer resulting in decreased 1H-NMR ligand signals. A second experiment with the low power pulse irradiating a region far away from the ligand and the protein signals is executed making sure no saturation takes place. The difference spectrum of the two yields the STD-spectrum, which indicates the binding of the ligand.[4] The proportion of the STD signals to each other can further be used to establish the ligand conformation within the active site of the protein.[5] Although STD NMR is well established investigating ligand binding to purified proteins, only a few examples of GPCRs incorporated in natural membranes have been done yet, to our [2,4] knowledge. Hereby we present STD-NMR studies on the human H2 receptor (hH2R-GsaS) in a natural membrane preparation, as it poses a prominent member of the GPCR family. The hH2R-GsaS containing membranes were obtained from SF9 insect cells overexpressing the human H2-receptor as a fusion protein with GsaS. As ligands we used commercially available common histamine H2 antagonists such as Famotidine and Ranitidine to gain further insight on how to optimise STD NMR for membrane preparations.

References [1] J. Drews, Science. 287, (2000) [2] S. Bartoschek, T. Carlomagno, C. Griesinger, Angew. Chem. Int. Ed. 49, (2010) [3] A. Bhunia, S. Chatterjee, Drug Discovery Today. 17, (2012)

[4] R. Venkitakrishnan, J. L. Markley, Methods Mol Biol. 914, (2012) [5] M. Mayer, B. Meyer, J. Am. Chem. Soc. 123, (2001)

Poster Presentations

161

Methods & Theory Poster Presentation P81

Measurement of Spatially Selective CLIP-HSQC M. Reller1, M.R.M Koos2, S. Wesp3, M.Reggelin3 and B. Luy1,2 1Institute for Organic Chemistry, Karlsruhe Institute for Technology, Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany) 2Institute for Biological Interfaces 4 – Magnetic Resonance, Karlsruhe Institute for Technology, Postfach 3640, 76201 Karlsruhe, Germany 3Institute für Organische und Biochemie, Technische Universität Darmstadt, Alarich-Weiss- Str. 4, 64287 Darmstadt, Germany E-mail: [email protected]

Inhomogeneously aligned samples are a challenge for the accurate measurement of residual dipolar couplings due to the arising line broadening. To overcome this problem, two methods are introduced to measure spatially selective CLIP-HSQCs [1]. The first pulse sequence works with slice selective excitation, the second approach is based on the introduction of an additional spatial dimension [2].

References [1] A. Enthart, J.C. Freudenberger, J. Furrer, H. Kessler, B. Luy, J. Magn. Reson. 2008, 192, 314-322. [2] P. Trigo-Mourino, C. Merle, M. R. M. Koos, B. Luy, R. R. Gil, Chem. Eur. J. 2013, 19, 7013- 7019.

Poster Presentations

162

Methods & Theory Poster Presentation P82

2+1 artifact suppression in DEER traces using Gaussian pulses M. Teucher1 and E. Bordignon1 1Ruhr University Bochum/ Department of Chemistry and Biochemistry, Bochum, Germany E-mail: [email protected]

Double Electron-Electron Resonance (DEER) is a versatile technique for obtaining high precision distance information in proteins. The sensitivity of this technique is strongly dependent on the achievable excitation bandwidth of the microwave pulses with respect to the available spectral width of the spin probes. However, in optimized X-band, as well as in high power Q-band [1] DEER setups, the excitation bandwidths of rectangular pump and observer pulses slightly overlap due to the large side bands of rectangular pulses in Fourier space. This causes an artifact at the end of each time trace since the spins within the overlap are subjected to a single frequency experiment called “2+1” pulse train ESE [2]. For proper data analysis, it is necessary to exclude the last part of the time domain data from background fitting. Otherwise, the artifact causes problems in background correction and can lead to artificially narrower widths in distance distributions, especially for long distances. Here, we present an optimized DEER setup for AWG-equipped spectrometers that exclusively uses Gaussian pulses. This allows almost complete suppression of the 2+1 artifact without significant sensitivity losses, which makes the entire length of the DEER time trace accessible for data evaluation.

References [1] Polyhach et al., Phys. Chem. Chem. Phys. 14:10762, (2012) [2] Kurshev et al., J. Magn. Res. 81:441, (1989)

Poster Presentations

163

Methods & Theory Poster Presentation P83

UTOPIA NMR: Activating unexploited magnetization using interleaved low-gamma detection A. Viegas1, T. Viennet1, T.-Y. Yu3, F. Schumann4, W. Bermel4, G. Wagner3, M. Etzkorn1,2 1Institute of Physical Biology, Heinrich-Heine-University Düsseldorf, Germany 2Instititue of Complex Systems, Forschungszentrum Jülich, Germany 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA 4Bruker BioSpin GmbH, Rheinstetten/Fällanden, Germany/Switzerland E-mail: [email protected]

A growing number of nuclear magnetic resonance (NMR) spectroscopic studies are limited by the information content provided by the standard set of experiments conventionally recorded. This is particularly true for studies of challenging biological systems like large, unstructured, membrane-embedded and/or paramagnetic proteins. Here we introduce the concept of unified time-optimized interleaved acquisition NMR (UTOPIA-NMR) [1] for the acquisition of standard proton-detected and low-gamma-detected experiments using a single receiver. Our aim is to activate the high level of polarization and information content distributed on low-gamma nuclei without disturbing conventional magnetization transfer pathways. We show that using UTOPIA-NMR we are able to recover nearly all of the normally non-used magnetization without disturbing the standard experiments. In other words, additional spectra, that significantly increase the NMR insights, are obtained for free. We anticipate a broad range of possible applications and demonstrate for the soluble protein Bcl-xL (ca. 21 kDa) and OmpX in nanodiscs (ca. 160 kDa) that, unlike other techniques, UTOPIA-NMR is particularly useful for challenging protein systems including perdeuterated proteins. Moreover, we also demonstrate that UTOPIA-NMR setup can be useful for screening and/or structural characterization of small compounds.

References [1] A. Viegas, T. Viennet, T.-Y. Yu, F. Schumann, W. Bermel, G. Wagner, M. Etzkorn, Biomol NMR 64, (2016)

Poster Presentations

164

Hardware, Instrumentation, Methodology & Imaging Poster Presentation P84

Removal of digital artefacts from time-domain signals G. Hempel Martin-Luther-Universität, Institut für Physik, Halle, Germany E-mail: [email protected]

Analysis of time-domain signals with short decay times requires accuracy of the first data points (for example: proton FID shape of rigid-amorphous or crystalline components of polymers). However, modern NMR spectrometers process signals digitally. This includes oversampling followed by decimation of data points which leads to strong ripple-like artefacts at the first data points in the time-domain signal. The decimation procedure can be regarded as multiplication of the original spectrum with a narrower rectangular function corresponding to a convolution of the FID with inverse Fourier transform of the rectangular function. Use of filters reduces such artefacts but does not suppress them completely. Here a method is introduced by which the original signal can be retrieved via deconvolution of the recorded signal from the device function of the digital signal processor. The device function is estimated by differentiation of a digitally processed, originally step-function-like signal. For obtaining such a signal the FID of a sample with very slow transversal relaxation was recorded with very short aquisition time. The shape of the device function depends on kind and bandwidth of filter, sweep width and other receiver characteristics as expected. The method was applied to “de-ripple” a number of short FID’s. The resulting signals are free of ripple artefacts and correspond to analogously processed signals with virtually infinite bandwidth.

Poster Presentations

165

Hardware, Instrumentation, Methodology & Imaging Poster Presentation P85

Monitoring disorders in glucocorticoid receptor-deficient zebrafish using μMRI and NMR U. Roy1, M. Schaaf2, J. Matysik1, A. Alia3,4 1Institut für Analytische Chemie, Leipzig, Germany 2Institute of Biology Leiden, Leiden, The Netherlands 3Institut für Medizinische Physik und Biophysik, Leipzig, Germany 4Leiden Institute of Chemistry, Leiden, The Netherlands Email: [email protected]

Dysregulation in the hypothalamus-pituitary-adrenal (HPA) axis is a hallmark of major depression in human patients. It is characterized by diminished cortisol signalling through GC receptor (GR). GR controls several biological processes including metabolism, immune response and brain function. However, it is not clear whether the GR deficiency can cause any specific structural and chemical changes in the brain. In this study we have optimized and applied high resolution µMRI and NMR methods to examine anatomical and neurochemical details in the brain of GR mutant zebrafish which displays a hyperactive HPA axis and increased depression-like behaviour in response to stress1. μMRI methods were optimized at to obtain anatomical details in adult zebrafish brains. Interestingly, the size of the telencephalon was bigger than mesencephalon in GR fish indicating development of megalencephaly. Also ventricles appeared larger in GR than in controls. Fat mapping with CHESS sequence shows that hyper-intensities corresponding to fat signal were higher in GR. 1H HRMAS NMR methods3 on intact brain tissue shows that GR has reduction in NAA but increase in brain lipids than controls. These results suggest that lack of glucocorticoid signalling is responsible for hypertrophy in the brain structures and neurochemical profile which might play key role in depression.

Reference: (1) Ziv et al., Mol Psychiatry. 18, (2013) (2) Kabli et al., Zebrafish. 7, (2010) (3) Berry et al., Zebrafish. accepted

Poster Presentations

166

Hardware, Instrumentation, Methodology & Imaging Poster Presentation P86

Distances and orientations with DEER at 263 GHz frequencies Igor Tkach1, Karin Halbmair1 and Marina Bennati1,2

1 Research Group EPR Spectroscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen , Germany 2 Institute of Organic and Biomolecular Chemistry, Georg-August University of Göttingen, Tammannstr. 2, Göttingen, Germany E-mail: [email protected]

The low field (0.3T/9GHz) pulsed electron-electron resonance (PELDOR/DEER) is a widely used technique for distance measurements in biomacromolecules. If applied at higher fields (3T/95GHz and 9T/263GHz), the method possesses higher absolute sensitivity and allows performing experiments on limited sample volumes. Furthermore, orientation selectivity is enhanced to a degree where it becomes an advantage since it permits to go beyond spin- spin distance measurements and to determine spatial arrangements of paramagnetic centers. To perform high-field/orientation-selective DEER we have installed a high power (100 mW) 263 GHz DEER setup. Our experiments with double-labelled RNAs and model simulations indicate that the available spectral resolution at 263 GHz should reflect clear alteration of a DEER signal as a function of a relative label configuration. However, a probability of forbidden transitions remains high, which makes the analysis of the experimental traces demanding. To enhance the constraints improving orientation measurements, we apply a 2D-DEER concept, which is only feasible with a variable pump-detect frequency separation. We are going to discuss this concept with the examples showing how the relative label configuration depends on the helical pitch of peptides or nucleic acids and how in turn it affects the modulation depth and dipolar frequencies of a DEER signal. Other advantages of high-field DEER measurements will be discussed as well.

Poster Presentations

167

Hardware, Instrumentation, Methodology & Imaging Poster Presentation P87

Reaction Monitoring with Compact NMR D. Weidener1, K. Singh1 and B. Blümich1 1Institut für Technische und Makromolekulare Chemie RWTH Aachen University, Worringerweg 2, 52074 Aachen, Germany E-mail: [email protected]

Compact NMR spectroscopy is playing a significant role for quality control and reaction monitoring [1]. Compact NMR spectroscopy at low field gives better time resolution than conventional NMR spectroscopy at high field due to shorter feed lines apart from cost effectiveness and differences in operating skills. Chemical reaction kinetics can easily be followed by using in-line and online monitoring under the fume hood. Recent developments in low field NMR give the opportunity for quantitative and qualitative analysis by using different nuclei such as 1H, 19F and 13C, which can be studied with basic 1D and 2D pulse sequences. In the present study, we report the monitoring of the production of α-fluoro-α, β- unsaturated esters via deacylation of α-fluoro-β-keto esters [2]. These esters are key compounds in the synthesis of various pharmaceutically relevant substances e.g. antitumor or anti-inflammatory agents [3]. The reaction was monitored using 1D 1H and 19F and 2D 1H-1H COSY experiments. The real time measurements confirm that the reaction consist of two steps first the deprotonation of the α-fluoro-β-keto ester and second the reaction of deprotonated compound with the respective aldehyde. The influence of the reaction temperature was investigated from 296 K to 333 K and reaction rate constants for both reaction steps were calculated, compared and the respective percentage yields were calculated.

References [1] K. Singh and B. Blümich, TrAC, (2016) [2] V. Sans, L. Porwol, V. Dragone and L. Cronin, Chem. Sci., 6(2), (2015) [3] J. Qian, W. Yi, M. Lv and C. Cai, Synlett, 26(1), (2015)

Poster Presentations

9. Index of Contributors

169

Abdullin, D. 47, 156 Bordignon, E. 38, 48, 108, 162 Achilles, A. 122 Borggräfe, J. 84 Adams, A. 131, 136 Bothe, S. 126 Adjei-Acheamfour, M. 110 Boutis, G. S. 117, 118 Adler, J. 12 Bruns, C. 100 Ahmadian, R. 88, 103 Büll, A. K. 109 Ahrem, L. 135 Buntkowsky, G. 126, 137 Akbey, Ü. 71 Buschauer, A. 160 Akdogan, Y. 42 Cadalbert, R. 15, 54 Akgül, S. 81 Cantrelle F.-X. 26 Albrecht, B. 86 Carbery, D. R. 139 Alia, A. 130, 165 Cattaneo, G. 58 Altmayer, S. 114 Celebre, G. 143 Amininasab, M. 101 Chang, Y.-N. 94 Anders, K. 48 Chrysina, M. 46 Arens, S. 84, 87, 88, 128 Classen, T. 104 Argyropoulos, D. 139 Clauser, J. 58 Artero, V. 106 Colas-Debled, E. 25, 69 Assafa, T. 48 Corzilius, B. 7, 73, 157, 158 Ayala, I. 89 Cox, N. 46, 74 Bachmann, R. 140 Creutznacher, R. 85, 98 Bahrenberg, T. 158 Crublet, E. 25, 69, 89 Balbach, J. 96 Dames, S. A. 24 Bannister, S. 119 Daube, D. 157, 158 Bärenwald, R. 122 de Groot, B. 53 Barth, K. 82 De Luca, G. 143 Becker, S. 53 Decaneto, E. 116 Becker, J. 141, 151 Del Federico, E. 113 Becker, T. 152 Delaforge, E. 64 Beerwerth, J. 110 Delcourt, M. P. 123 Bengs, C. 157 Dettmer, K. 20 Bennati, M. 76, 166 Deupi, X. 22 Berg, N. 142 Di Pietro, M. E. 143 Berger, S. 127 Díaz, M. D. 86 Bermel, W. 163 Dick, M. 104 Bernarding, J. 100 Dietrich, C. 127 Berndhäuser, A. 115 Dimitrova, L. 20 Bernhard, F. 39 Ding, Y. 119 Bertram, R. 135 Dingley, A. 66 Bessi, I. 83 Dötsch, V. 39 Beumer, C. 116 Drochner, A. 137 Bhowmik, D. 12 Drögemüller, J. 65 Biernat, J. 95 Duthie, F. 156 Bilici, K. 117, 118 Eisenmann, P. 144 Bill, E. 74 Elter, S. 87, 93 Binas, O. 83 Engelmann, J. C. 20 Birrell, J. 74 Ernst, M. 54 Blackledge, M. 64 Essen, L 48 Blommers, M. 17 Essen, L.O. 125 Blümich, B. 58, 113, 131, 136, 138, 154 Esters, B. 121 Blümler, P. 56 Etzkorn, M. 4, 84, 87, 88, 93, 103, 109, Böckmann, A. 15 128, 129, 163 Boeske, A. 81 Euchner, F. 100 Böhmer, R. 110 Falke, M. 84, 88, 103 Boisbouvier, J. 25, 69, 89 Farès, C. 75 Bommerich, U. 100 Feist, M. 20

Index of Contributors

170

Fernandez, G. 153 Heckel, A. 83 Feuerstein, S. 27 Heid, L. 93 Fischer, M. 140 Heil, W. 56 Flohr, J. 58 Heiliger, J. 157,158 Florin, N. 156 Hein, C. 39 Frey, H. 133 Heise, H. 3, 14, 92, 116, 120, 121, Fritz, M. 74 124, 128, 129 Gans, P. 25, 69, 89 Hempel, G. 111, 112, 164 Gardiennet, C. 15 Henrich, E. 39 Gärtner, W. 114, 125 Herold, D. 43 Geertsma, E. R. 94 Herrmann, T. 100 Gelev, V. 87, 128 Hilgenfeld, R. 150 Gemmecker, G. 90 Himmelsbach, M. 144 Gey, C. 152 Hinderberger, D. 42, 133 Giller, K. 53 Hintze, C. 9 Giraudeau, P. 41 Hioe, J. 146 Glaenzer, J. 91 Hofele, R. V. 95 Godt, A. 73 Hoffman, B. 39 Goldbourt, A. 98 Hoffmann, S. 81 Goncalves, D. P. N. 83 Hoffmann, M. M. 126 Görges, A. 131 Högborn, M. 74 Goss, R. 130 Höger, S. 132 Grahl, A. 22 Hoyer, W. 14, 27, 69, 92, 109, 129 Granwehr, J. 138 Huber, M. 13 Greindl, J. 146 Hughes, J. 125 Gremer, L. 14, 27, 69, 120, 124 Hummel, M. 20 Griese, J. J. 74 Huster, D. 12 Grimm, L. L. 150 Hutter, C. A. J. 108 Gronwald, W. 20 Huvent, I. 26 Grossmann, T. N. 128 Immel, S. 134 Grzesiek, S. 22 Isogai, S. 22 Gschwind, R. 79,142, 146, 147, 149, 155 Ivanov, K. L. 72 Guan, J.-Y. 25 Jacob-Dubuisson, F. 123 Guan, J. Y. 69 Jaeger, K.-E. 104 Guérin, J. 123 Jähnigen, S. 114 Gutmann, T. 126 Jaremko Ł. 95 Gutmann, T. 137 Jaremko, M. 95 Haarmann, F. 32 Jassoy, J. J. 115 Hackl, T. 140 Jaumann, E. A. 94 Hadjiali, S. 126 Jbeily, M. 122 Hagelueken, G. 91, 156 Jensen, M. R. 64 Hagelüken, G. 47 Jeschke G. 15 Haindl, M. 155 Johann, T. 133 Haist, C. 104 John, M. 145 Halbmair, K. 166 Jonker, H. 83 Haller, J. D. 44 Joseph, B. 67, 82, 94 Hamelin, O. 89 Kadavath, H. 95 Han, S. 62, 138 Kaila, V. R. I 24 Hanoulle, X. 123 Kaila, V. 90 Hansen, M. R. 153 Kaminker, I. 62 Hansman, G. S. 97 Kansy, M. 130 Hansmann, S. 43 Kant-Das, A. 12 Härd, T. 27 Kasanmascheff, M. 76 Hasecke, F. 92 Kattnig, D. R. 42 Hauck, C. 99 Kaushik, M. 73 Haumann, M. 74 Kehlet, C. 113

Index of Contributors

171

Keller, K. 15 Luy, B. 44, 141, 143, 144, 151, 161 Kemnitz, E. 135 Ma, P. 81 Kentgens, A. 10 Macek, P. 25, 69 Kerfah, R. 89 Madhu, P. K. 12 Khazaei, S. 127 Maiti, S. 12 Kirste, B. 159 Mallagaray, A. 85, 97 Kiryution, A. S. 72 Mandelkow, E. 95 Klapper, W. 20 Märker, K. 9 Kleo, K. 20 Mas, G. 25, 69, 89 Klingler, R. J. 19 Matsuoka, H. 132 Klockmann, S. 140 Mattea, C. 57 Knapp, A. 104 Matysik, J. 72, 114, 119, 125, 127, 130 Knauer, S. H. 65 Maul, A. 56 Knipp, M. 116 Meier, B. H. 15, 54 Knoche, S. 137 Meister, A. 133 Köberle, M. 43 Meldrum, T. 113 Köhler, L. 130 Melikian, M. 146, 160 König, A. 120, 124 Meng, L. 72 Koos, M. R. M. 44, 141, 151, 161 Meyer, A. 47 Korn, A. 12 Michaelis, M. 98 Kositzki, R. 74 Mirecka, E. A. 14, 27 Kottke, T. 119 Möller, H. M. 98, 99, 107 Kragelj, J. 64 Morana, F. 155 Kressler, J. 122 Morgan, S. W. 117, 118 Krushelnitsky, A. 96 Moriscot, C. 69 Kube, D. 20 Moser, A. 139 Kuhn, S. 148 Mroginski, M. A. 125 Kumar, S. 95, 101 Muhle-Goll, C. 144 Küppers, M. 58 Münnemann, K. 18 Küppers, A. 128 Munt, M. 100 Kutin, Y. 46, 74 Nanao, M. 64 Lacabanne, D. 5 Nedielkov, R. 98 Laguerre, A. 39 Neese, F. 132 Lakomek, N.-A. 54 Neudecker, P. 2, 66, 92, 104 Lang, C. 125 Neudert, O. 138 Lappan, U. 59 Nick, T. U. 76 Laskay, T. 102 Niklas, T. 145 Leavesley, A. 62 Nitschke, P. 160 Lecher, J. 66 Nuhn, L. 133 Lee, W. 76 Oefner, P. J. 20 Lends, A. 54 Opitz, C. 22 Lewandowski,J. R. 78 Ordikhani-Seyedlar, A. 57 Li, W. 29 Otten, E. 56 Liermann, J. C. 148 Palencia, A. 64 Link, S. 96 Pantazis, D. A. 46 Linser, R. 53 Panwalkar, V. 66, 105 Lippens, G. 26 Paone, C. 99 Lockhauserbäumer, J. 97 Parr, M. K. 86 Löhr, F. 39 Parra, F. 97 Lokesh, N. 147 Paul, S. 72 Lopez, J. 26 Penzel, S. 54 Löser, L. 96 Peter, M. 91 Lowe, J. P. 139 Peters, T. 85, 97, 150 Lubitz, W. 46, 49, 74, 75, 106 Peters, O. 86 Lund, A. 62 Petrov, O. 128 Luo, Q. 24 Petzold, K. 52

Index of Contributors

172

Piechatzek, T. 121 Schmidts, V. 43, 134 Pietruszka, J. 104 Schmitt, L. 132 Pintacuda, G. 37 Schmitz, M. 66 Plaumann, M. 100 Schneider, R. 26, 123 Pohl, C. 121 Schneider, D. 149 Prestel, A. 99 Schoehn, G. 25, 69 Prisner, T. F. 67, 82, 94 Scholz, G. 135 Qi, M. 73 Schölzel, D. 92, 120, 124 Rademacher, C. 97 Schönborn, J. 124 Rapoport, D. H. 152 Schöne, T. 150 Rawat, A. 12 Schriek, S. 103 Reggelin, M. 161 Schröder, G. F. 27, 34 Rehahn, M. 134 Schulte, M. 66, 104, 105 Rehorn, C. 113 Schulze-Sünninghausen, 141, 151 Reichenwallner, J. 133 D. Reijerse, E. J. 49, 75, 106 Schumann, F. 163 Reinartz, E. 14, 92 Schwalbe, H. 83, 158 Reinders, J. 20 Schwarten, M. 81 Reller, M. 161 Schwarzfischer, P. 20 Renzi, P. 155 Schweimer, K. 65 Rest, C. 153 Sebastiani, D. 114, 127 Retegan, M. 132 Seeger, K. 102, 152 Rezaei-Ghaleh, N. 101 Seeger, M. A. 108 Richer, B. 102 Seegerer, A. 142 Richter, C. 83 Seidel, C. A. M. 35 Ringleb, R. 100 Seifert, U. 42 Risse, T. 30 Selter, P. 153 Rittscher, V. 134 Shafaat, H. S. 74 Roos, A. 42 Shaykhalishahi, H. 27, 109, 129 Roos, M. 112 Shenderovich, I. G. 31 Rösch, P. 65 Siaw, T. A. 62 Rösener, N. 120 Sidabras, J. W. 49 Roth, F. 43 Silvers, R. 158 Rovo, P. 53 Silverstein, M. C. 117, 118 Roy, S. 106 Singh, K. 154, 167 Roy, U. 165 Skoczinski, P. 104 Rumpel, S. 75, 106 Slavov, C. 83 Saalwächter, K. 96, 112, 122 Smith, C. 53 Sakai, N. 150 Sommer, C. 106 Salei, N. 102 Song, C. 114, 125 Sampedro, A. 153 Sood, A. 85 Sattler, M. 90 Sorgenfrei, N. 146 Sauskojus, W. 29 Sounier, R. 89 Savitsky, A. 5, 46, 49 Spang, R. 20 Scarbath-Evers, L. 114 Srinivas, V. 74 Schaaf, M. 165 Stalke, D. 145 Schade, M. 68 Stapf, S. 57, 138 Schanda, P. 25, 51, 69 Steinhoff, H.-J. 61 Scheidt, H. A. 12 Stoldt, M. 27, 81 Scheler, U. 59 Storek, M. 110 Schertler, G. 22 Strauß, M. 65 Schiemann, O. 47, 115, 132, 156 Streitferdt, V. 155 Schipke, C. 86 Stubbe, J. 76 Schlörer, N. E. 86, 148 Sun, H. J. 118 Schmedt auf der Günne, J. 29 Supekar, S. 90 Schmid, M. B. 155 Suter, D. 49

Index of Contributors

173

Yurkovskaya, A. V. 72 Szczepanowski, M. 20 Zeitler, K. 155 Tampé R. 82 Zhang, Y. 117, 118 Taruttis, F. 20 Zhang, J. 131 Tepper, K. 95 Zhang, L. 150 Terradot, L. 15 Zia, W. 113 Teucher, M. 162 Zill, J. 130 Teymouri, Y. 131, 136 Zimmermann, A. 155 Thankamony, A. S. L. 137 Zolofo, G. 113 Thevarpadam, J. 83 Zweckstetter, M. 95, 101 Thiele, C. M. 43, 134 Thomas, G. H. 91 Thomas, S. 107 Thomas, A. 133 Thompson, J. 113 Tilly, J. 110 Timachi, M. H. 108 Tkach, I. 166 Trettin, R. 29

Tripsianes, K. 90 Überrück, T. 138 Uetrecht, C. 97 Uluca, B. 129

Urlaub, H. 95 Varghese, S. 116 Veprintsev, D. 22 Viegas, A. 84, 87, 93, 128, 163 Viennet, T. 93, 109, 128, 129, 163 Viess, J. 58 Vogel, H. 137 von Rekowski, F. 155 Wachtveitl, J. 83

Wagemann, M. 131 Wagner, G. 87, 163 Walter, J. 101 Wang, X. 72 Wang, Y. 117, 118 Weber, J. 29 Weidener, D. 167 Weirich, F. 14, 124 Wesp, S. 161

Westhoff, J. 144 Wiegand, T. 15 Wilhelm, C. 130 Willbold, D. 27, 66, 69, 81, 104, 105, 120 Windler, C. 152 Wittmann, J. 9 Wittwer, M. 24 Woelk, K. 19 Wöhrl, M. 65

Woods, R. J. 85 Wördehoff, M. M. W. 27, 109, 129 Wurl, A. 112 Yu, J. J. 103

Yu, T.-Y. 163 Yulikov, M. 15

Index of Contributors