Solid State, Applications in Chemistry, Materials, Biology, MRI, Instrumentation, Including a Public Lecture on the Role of MRI in Yoga

APNMR-2017

National Organising Committee Prof. Brar A.S, GNDU, Amritsar Prof. Chandrakumar N, IIT-Madras, Chennai Prof. Chary K.V.R, TIFR, Mumbai/Hyderabad Prof. Hosur R.V, CEBS and TIFR, Mumbai Dr. Jagadeesh B, IICT, Hyderabad Prof. Jagannathan N.R, AIIMS, New Delhi Prof. Khetrapal C.L, CBMR, Lucknow Dr. Kunwar A.C, IICT, Hyderabad Dr. Subhash Khushu, INMAS, New Delhi

Local Organising Committee Anil Kumar Atreya H.S. (Convener) Jayantha Chatterjee Mahavir Singh Raghothama S. Ramanathan K.V. Suryaprakash N. (Convener) Vasudevan S.

International Advisory Panel (APNMR) Prof. Byong-Seok Choi, Korea Advanced Institute of Science and Technology, Daejeon, South Korea Prof. Mitsu Ikura, Ontario Cancer Institute, University of Toronto, Ontario Canada Prof. Masatsune Kainosho, Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya, Japan Prof. Weontae Lee, Department of Biochemistry, Yonsei University, Seoul, Republic of Korea Prof. Maili Liu, Wuhan Institute of Physics and Mathematics, Wuhan, China Dr. Ping-Chiang Lyu, National Tsing Hua University, Taiwan Prof. Raymond Norton, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia Prof. Martin Scanlon, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia Prof. Ichio Shimada, The University of Tokyo, Tokyo, Japan Prof. Daiwen Yang, National University of Singapore, Singapore Prof. Mingjie Zhang, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

NATIONAL MAGNETIC RESONANCE SOCIETY, INDIA (REGD) INDIAN INSTITUTE OF SCIENCE BANGALORE – 560012, INDIA

Prof. N. Suryaprakash President

8th February, 2017 Message

I am extremely delighted to note that today we are organizing 7th Asia Pacific NMR Symposium in conjunction with the 23rd Annual Meeting of National Magnetic Resonance Society (NMRS) of India. I thank APNMR council for providing us an opportunity to take this academic initiative at Indian Institute of Science. NMR activity in India started within few years of its discovery. At IISc NMR activity dates back to early 50‘s when Prof. G. Suryan carried out his work on radiation damping and surface coil and NMR flow measurements. Significant number of notable contributions that are relevant to chemical, biological and medical sciences, have been made by large number of Indian NMR community. Prof. Dharmattis‘ contribution to discovery of NMR chemical shifts is also well known. Research activity progressively evolved in synchronous with technological advancements and diverse applications of magnetic resonance. To promote and accelerate the magnetic resonance activities in India, the National Magnetic Resonance Society was incorporated as a registered body in 1996 by a group of practicing Indian magnetic resonance spectroscopists, with its registered office at IISc. It is delighted to note that as of today, the society has nearly 1000 life members, 40 honorary members, and seven corporate members. Ever since its inception, NMRS has always been playing a pivotal role to create a harmonious scientific forum among the practitioners of magnetic resonance, not only to enhance the awareness but also to instill the creative interest on the developments and applications of NMR spectroscopy by frequent organization of symposia, conferences, workshops, summer schools, etc. Over the years the magnetic resonance activity in the country has experienced phenomenal growth in both scientific and technological fronts, and as a consequence several internationally recognized NMR research groups have emerged. I am happy to inform, presently the country has more than five hundred high-field NMR spectrometers, including number of 700 / 800 MHz spectrometers and more than three hundred MRI units. The NMRS holds a two-day annual meeting spread all across the country to reach the wider audience, and often prefixes a one or two-day special symposium to this meeting, to encourage NMR practitioners to discuss their latest findings. The previous meetings were held at Kharagpur, Amritsar, Tezpur, Lucknow, New Delhi, Mumbai, Bangalore, Hyderabad, Chennai, etc. This year 23rd meeting is being organized at Bangalore in conjunction with the 7th Asia pacific NMR Symposium. The participation of world renowned NMR scientists from all across the globe is growing year by year, giving the annual meetings an international character. Every year the society also elects distinguished overseas NMR scientists, as honorary members. The long list of honorary members contains internationally lauded scientists and also Nobel Laureates, such as, Richard Ernst, Kurt Wuthrich, A. Pines, E.D. Becker, P.C. Lauterbur, K. Akasaka, R. Kaptein, I.C.P.Smith, J. R. Pilbrow, J.H. Freed, Seiji Ogawa, Ad Bax, to mention a few. The NMRS also plays a significant role in nurturing the young talent, and has thus instituted number of awards given annually for students and younger faculty for their research contributions and also provides partial support for students to present their work in NMR related international conferences. The annual meetings of NMRS are unique for their depth, breadth, intellectual rigor and the highest level of the quality of the research that are discussed. In the next few days of deliberations, we have number of insightful presentations, such as, plenary lectures, invited talks, short oral and poster presentations. The discussion covers all aspects of NMR spectroscopy, such as, methodological developments in solution and solid state, applications in chemistry, materials, biology, MRI, Instrumentation, including a public lecture on the role of MRI in yoga. I am sure it will serve as an excellent academic feast for the scientific fraternity, a motivation for budding scientists, a platform to share the ideas, encouragement for establishing the long- range collaborations, in addition to paving the way towards accelerating the advanced research in the frontier and emerging areas of NMR spectroscopy. As a current president of NMRS, I would like to take this opportunity to cordially welcome all the delegates who have come from different parts of the globe and wish all of you a pleasant stay with the sumptuous academic feast to relish. I also would like to heartily congratulate all my faculty colleagues and the students of NMR Centre for taking this wonderful academic initiative and sincerely thank them for putting relentless efforts, day in and day out, in organizing this meeting.

President, National Magnetic Resonance Society

Dear Friends and Colleagues,

On behalf of the Asia-Pacific NMR community, I am very pleased to welcome all participants to excellent 7th APNMR symposium in this wonderful city ―Bengaluru‖.

I would like to take a chance to report the history of APNMR symposium briefly. Prof. Weontae Lee Very recently, since Asia-Pacific NMR community has grown Department of Biochemistry rapidly and all research environments set up very well, it is of Yonsei University, Korea importance to build human-network for efficient research collaboration including information exchange.

To make our idea in action, the 1st Pre-council meeting was held on August, 2004 at Pusan, Korea. The council members representing each countries were Professors Masatsune Kainosho (Japan), Tai-huang Huang (Taiwan), Mitsu Ikura (Canada), Ray Norton (Australia) , Mingjie Zhang (Hong-Kong) and Weontae Lee (Korea). The APNMR symposium has been successfully held during last 12 years; 1st APNMRS at Yokohama (2005), Japan, 2nd in Taiwan (2007), 3rd in Korea (2009), 4th in China (2011), 5th in Australia (2013) and 6th in Hong-Kong (2015).

The vision is to enhance the international recognition and visibility of the Asia-Pacific NMR community and to promote international scientific exchange between the participating countries as well as with other countries world-wide. The final goal in the APNMR symposium is to provide researchers and trainees in Asia and Pacific Rim excellent platform for having cutting-edge lectures, meetings with world prominent researchers, networking opportunities, information exchange and a relaxing environment. In particular, participation of young scientists in Asia-Pacific area should be strongly encouraged in the symposium.

SPONSORS

th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India February 16-19, Indian Institute of Science, Bangalore Scientific Program

February 16

09.00 - Registration

09.30 - 12.30 BRUKER Users Meeting (J. N. Tata Auditorium)

12.30 - 13.00 Lunch

13.00 - 16.30 JEOL Users Meeting (J. N. Tata Auditorium)

16.30 – 17.00 Tea Break

17.00 – 17.15 Inauguration (J. N. Tata Auditorium)

Inaugural Session

Chair: Anil Kumar

17.15 - 17.55 Lucio Frydman Principles and Applications of Weizmann Institute, Rehovot, Israel Ultrafast Imaging by Spatiotemporal Encoding

17.55 - 18.40 R. V. Hosur Tata Institute of Fundamental Some Recent Developments in Research & Centre for Excellence in NMR Methods and Applications Basic Sciences, Mumbai, India th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

February 17

Plenary Chair: Weontae Lee Lectures

09.00 - 09.40 Jiangfeng Du Single-molecule Electron Spin University of Science and Resonance Spectroscopy & Technology of China, Hefei, China Imaging by diamond sensor

Parallel Sessions

Session 1

J. N. Tata Auditorium: Biomolecular Structure and Dynamics

Chair: R. V. Hosur

9.40 - 10.10 Mitsu Ikura NMR Approaches to Dissect University of Toronto, Toronto, Oncogenic RAS Signaling At Ontario, Canada Biological Membrane 2+ 10.10 - 10.40 K. V. R. Chary Structure of Ca -binding Tata Institute of Fundamental protein-6 from Entamoeba Research, Mumbai & histolytica and its involvement in TIFR Center for Interdisciplinary trophozoite proliferation Sciences, Hyderabad, India regulation

10.40 - 11.10 Ho Sup Yoon FK506 Binding Proteins, Nanyang Technological University, Immunophilins with Functional Singapore Versatility

11.10 – 11.40 Tea Break

Chair: Gunnar Jeschke

11.40 - 12.10 Koh Takeuchi Dynamic multidrug recognition National Institute of Advanced and transcriptional regulation Industrial Science and Technology, by a multidrug resistant Japan transcriptional repressor, LmrR th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

12.10 - 12.30 Ranabir Das A complex interplay between National Centre for Biological post-translational modifications Sciences, Bengaluru, India observed during the Herpes infection

12.30- 12.50 Susmitha Ambadipudi Protein droplets: From Cellular German Center for Function to Neurodegeneration Neurodegenerative Diseases (DZNE), Göttingen, Germany

HALL A: Solid State NMR

Chair: T. Fujiwara

09.40 - 10.10 Matthias Ernst, Transient Compensation in ETH, Zurich, Switzerland Solid-State NMR

10.10 - 10.40 Neeraj Sinha, Atomic Level Structural Insight Centre of Biomedical Research, of Bones and Cartilage: Solid Lucknow, India State NMR Perspective

10.40 - 11.10 Yusuke Nishiyama Very fast MAS solid-state NMR JEOL RESONANCE Inc. & RIKEN and electron diffraction

11.10 - 11.40 Tea Break

Chair: N. Chandrakumar

11.40 - 12.10 Amir Goldbourt Magic-angle spinning NMR and Tel Aviv University, Israel SAXS reveal structural effects of single mutations in a filamentous viral capsid across multiple length scales

12.10 - 12.30 Alex Smirnov Solid-State NMR of Membrane North Carolina State University, Proteins Macroscopically North Carolina, USA Aligned by Nanopores

12.30 - 12.50 S. Jayanthi Deuterium Magic Angle Indian Institute of Space Science and Spinning NMR and Local Technology, Thiruvananthapuram, Molecular Dynamics Modelling India for the Study of Restricted Dynamics of Grafted and Adsorbed Molecules th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Hall B: New NMR Methods

Chair: AC Kunwar

09.40 - 10.10 Chojiro Kojima NMR structural analysis of Yokohama National University, unstable / partially unfolded Yokohama, Japan / aggregated proteins

15 10.10 - 10.40 Arthanari Haribabu N detected experiments – Harvard Medical School, Boston, Where being insensitive has USA its advantages

10.40 - 11.10 Eriks Kupce NMR Experiments for Two Bruker BioSpin, UK Receivers

11.10-11.40 Tea Break

Chair: P. Gooley

11.40 - 12.10 Patrick Giraudeau Recent developments, Université de Nantes, France applications and challenges in ultrafast multidimensional NMR

12.10 - 12.30 Paul Coote Rapid convergence of optimal Harvard Medical School, Boston, control in NMR using USA numerically-constructed toggling frames

12.30- 12.50 Jithender Reddy Rapid Structural Elucidation of Indian Institute of Chemical Proteins with 'All-in-one' NMR Technology, Hyderabad, India Spectroscopy

Hall C: MRI/MRS Chair: N. R. Jagannathan

09.40 - 10.10 S. Sendhil Valen Translational Metabolic Metabolic Imaging Group, Imaging Singapore Bioimaging Consortium, Singapore th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

10.10 - 10.40 Dennis W. Hwang MRI Contrast Enhancement by Department of Chemistry and Frequency Lock-in Suppression Biochemistry, National Chung Technique Cheng University

10.40 - 11.10 Shubash Khushu Functional MRI techniques: Indian Institute of Nuclear Medicine Application to Neuroscience and Allied Sciences, Lucknow, India

11.10 - 11.40 Tea Break

Chair: S. Khushu

11.40 - 12.00 Sairam Geethanath Tailored Magnetic Resonance Dayanand Sagar Institution, Fingerprinting Bangalore, India

Dinesh Kumar, Centre of 12.00 - 12.20 Biomedical Research, Lucknow Understanding the toxicity mechanism of Pyrazinamide as revealed by NMR based serum metabolomics and Biochemical analysis

12.20 - 12.40 Shivanand Pudakalakatti, Unique molecular signatures to UT M D Anderson Cancer Center, distinguish immunotherapy Houston, USA responding and resistant cell lines in melanoma by NMR spectroscopy and MR hyperpolarization

12.40 - 13.00 Shilpi Modi Functional, morphological and Indian Institute of Nuclear Medicine metabolic alterartions associated and Allied Sciences, Lucknow, India with trait anxiety

13.30 – 14.00 Lunch

14.00 – 16.00 Poster Session (ODD POSTER NUMBERS)

14.00 – 15.45 ACD LABS WORKSHOP th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

J. N. Tata Auditorium: Biomolecular Structure and Dynamics

Chair: S. Subramanian

16.00 - 16.30 Yangmee Kim Investigation of Thermostabilities and Dynamic Konkuk University, Seoul, Korea behaviors of Proteins from Thermophilic, Mesophilic, and Psychrophilic Bacteria

16.30 - 17.00 Paul Gooley The complex binding mode of University of Melbourne, Parkville, relaxin for its receptor RXFP1 Victoria, Australia

17.00 - 17.20 Krishna Mohan Poluri Mechanistic Insights into Indian Institute of Technology Chemokine Glycosaminoglycan Roorkee, India Interactions

HALL A: Solid State NMR

Chair: S. Vasudevan

16.00 - 16.30 Akira Naito Microwave Heating and Energy Yokohama National University, Flow Mechanisms of Liquid Yokohama, Japan Crystalline Systems as Revealed by Microwave Irradiation Solid- State NMR Spectroscopy 13 1 16.30 - 17.00 T. Narasimhaswamy C- H Dipolar Couplings and Polymer Science and Technology, Molecular Order of Mesogens: Central Leather Research Institute, Direct Evidence for Molecular Adyar, Chennai, India. Topology

17.00 - 17.20 Bholanath Pahari Multinuclear Solid-state NMR Goa University, Goa, India Investigations on Sr1-xNaxSiO3- 0.5x: A Family of Superior Ion Conductors th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Hall B: New NMR Methods

Chair: P. Giraudeau

16.00 - 16.30 Mehdi Mobli Structural characterisation of University of Queensland, Australia novel and engineered bioactive disulfide-rich peptides by advanced solution state NMR spectroscopy

16.30 - 17.00 Bharathwaj Sathyamoorthy Influence of aromatic side- Indian Institute of Science Education chains in biomolecular structure and Research, Bhopal, India and dynamics

1 17.00 - 17.20 Kalyan Chakrabarti High-power H CPD provides Max Planck Institute for Biophysical artifact free exchange-mediated Chemistry, Göttingen, Germany saturation transfer experiments

Hall C: MR Nucleic Acids-Protein Interactions

Chair: K. V. Ramanathan

16.00 - 16.30 Masato Katahira Interactions between non-coding Institute of Advanced Energy, Kyoto RNA and TLS/FUS, University, Japan and distinct difference in enzymatic behavior among APOBEC3 proteins as revealed by real-time NMR monitoring

16.30 - 17.00 Mandar Deshmukh Elucidating the small RNA Centre for Cellular and Molecular mediated gene silencing in A. Biology, Hyderabad, Telangana, thaliana India

17.00 - 17.20 Keisuke Kamba Sliding and intersegmental Kyoto University, Kyoto, Japan transfer along DNA enhance the enzymatic activity of APOBEC3G as revealed by real- time NMR monitoring methods

17.30 – 18.30 Special Lecture Integrating the Best of East Dr. H. R. Nagendra with the best of West in Medical S-Vyasa University, Bangalore Practice - Role of NMR /MRI th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

February 18

J. N. Tata Auditorium Chair: Akira Naito

09.00 - 09.40 Stanley Opella University of California, San Diego, USA

09.40 - 10.20 Weontae Lee New structural insight of Yonsei University, Seoul , Korea Proteoglycans, dimerization and molecular function related to melanoma cell migration and metastasis

10.20 - 10.40 Tea Break

Parallel Sessions:

J. N. Tata Auditorium: Biomolecular Structure and Dynamics

Chair: K. V. R. Chary

10.40 - 11.10 Chun Tang Preferred Conformational Wuhan Institute of Physics and States of Retroviral Capsid Mathematics, Wuhan, China Protein Visualized

11.10 - 11.40 Surajit Bhattacharya Analyses of Multi-Protein Nanyang Technological University, Complexes in Beta-2 Integrin Singapore Signaling

11.40 - 12.10 Ashutosh Kumar Motion, recognition and Indian Institute of Technology-Bombay, interaction of proteins Mumbai, India involved in the cell-cycle regulation

12.10 – 12.20 Short Break th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

J. N. Tata Auditorium Chair: S. Ganapathy

12.20 - 12.50 Jeong-Yong Suh Structural basis for the biased Seoul National University, Seoul, Korea interactions between auxin- response transcriptional machinery

12.50 - 13.10 Sujoy Mukherjee Is partial unfolding of native Indian Institute of Chemical Biology, proteins a necessary, Kolkata, India inaugural step in amyloidosis?

13.10 - 13.30 Vinesh Vijayan Determining the critical Indian Institute of Science Education & molecular interaction Research, Tiruvanthapuram, India responsible for tau fibril formation

HALL A: DNP/Solid State NMR

Chair: A. Goldbourt

10.40 - 11.10 Toshimichi Fujiwara Dynamic nuclear polarization Osaka University, Suita, Japan with double 460 GHz gyrotrons for the sensitivity enhancement of magic-angle- spinning NMR at 30 K

11.10 - 11.40 N. Chandrakumar Two To Tango, Three To Jive Indian Institute of Technology-Madras Chennai, India

11.40 - 12.00 Sachin Rama Chaudhari DNP Enhanced Solid-State University of Lyon, France NMR under Fast Magic Angle Spinning 12.10 - 12.10 Short break th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Hall B Chair: V. Venkataraman

12.10 - 12.40 Navin Khaneja Recoupling with phase change Indian Institute of Technology-Bombay, India

12.40 - 13.00 Krishnendu Kundu The Microwave Power and Weizmann Institute of Science, Rehovot, Time Dependence of Israel Static DNP induced NMR Signals

13.00 - 13.20 Soumya Roy A Cost-efficient and Fast University of York, Heslington, York, UK Technique of Achieving Sustainable Nuclear Spin Hyperpolarization

Hall B: Novel NMR Methods

Chair: S. V. Bhat

10.40 - 11.10 Petrik Galvosos Recent advances in NMR Victoria University of Wellington, diffusometry Wellington, New Zealand

11.10 - 11.40 Kavita Dorai Characterizing, detecting and Indian Institute of Science Education & preserving quantum Research, Mohali, India correlations on an NMR quantum information processor

11.40 - 12.10 T. S. Mahesh Quantum bits and their Indian Institute of Science Education & environment Research, Pune, India

12.10 - 12.20 Short break th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Hall B Chair: B. Jagadeesh

12.20 - 12.50 Scott Robson Towards Complete Sequence Harvard Medical School, Boston, USA Specific Assignment of Proteins from a Single Experiment: Applications of 13 C-Pyruvate labeling, Non- uniform sampling and Pattern Matching at Greater Than 42 kDa

12.50 - 13.10 B. V. N. Phani Kumar Fast Field Cycling NMR Central Leather Research Institute, Adyar, Investigations of Slow Chennai, India Dynamics in Soft Materials

13.10 - 13.30 Nilamoni Nath New methods for measuring Max Planck Institute for Biophysical RCSAs and RDCs for Small Chemistry, Göttingen, Germany Molecule Applications

Hall C: NMR and Drug discovery

Chair: T. N. Guru Row

10.40 - 11.10 Martin Scanlon The application of NMR Monash Institute of Pharmaceutical spectroscopy in fragment- Sciences, Parkville, Australia based drug design

11.10 - 11.40 TysrYu Transthyretin-related Academia Sinica, Taipei, Taiwan familiar amyloid polyneuropathy

11.40 - 12.10 Koichi Kato NMR views of functional Nagoya City University, Japan roles of glycoconjugates of biological and pharmaceutical interest

12.10 - 12.20 Short break th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Hall C Chair: M. Scanlon

12.20 - 12.40 Ke Ruan NMR Characterization of University of Science and Technology of Weak Interactions between China, Hefei, Anhui, China RhoGDI2 and Fragment Screening Hits

12.40 - 13.00 Biswaranjan Mohanty Methyl NMR as a tool for Monash Institute of Pharmaceutical determining the ligand Sciences, Parkville, Australia binding modes in weak protein–ligand complexes

13.00 - 13.30 Praful Gupta Optimized frameworks for Daiichi Sankyo India Pharma Private Ltd synthetic chemistry

13.30 – 14.00 Lunch

14.00 – 16.00 Poster Session (EVEN POSTER NUMBERS)

14.00 – 15.45 ACD LABS WORKSHOP

J. N. Tata Auditorium: Metabolomics/Small Molecules

Chair: N. Jayaraman

16.00 - 16.30 Sunghyouk Park Real-time monitoring of p53’s Seoul National University, Korea effects on pyruvate metabolism in live mitochondria using in- organelle NMR metabolomics

16.30 - 17.00 Anant Bahadur Patel Exploring Brain Energy Metabolism in Alzheimer's Cellular and Molecular Biology, Disease Hyderabad, India

17.00 - 17.20 Palwindar Singh Design of Small Peptides: Guru Nank Dev University, Amritsar, India Synthesis and Biological Studies for the Development of Anti-inflammatory Agents

th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

HALL A : Biomolecular Structure and Dynamics-1

Chair: M. Ikura

16.00 - 16.30 Christian Griesinger NMR Based Structural Max-Planck Institute for Biophysical Biology In Immunity And Chemistry, Göttingen, Germany Neurodegeneration/Protectoin

16.30 - 17.00 Junfeng Wang Structural study of fibroblast High Magnetic Field Laboratory, China growth factor 21 (FGF21)-a potential anti-diabetic drug

17.00 - 17.30 Viji Sarojini, University of Auckland, Structural and Functional Auckland, New Zealand Studies on Synthetic Peptides

HALL B : Biomolecular Structure and Dynamics-2

Chair: S. Sarma 16.00 - 16.30 Gunnar Jeschke, ETH Zürich, Zürich, Combining EPR and NMR Switzerland restraints in structure determination of biological systems

16.30 - 17.00 Shinichi Tate, Hiroshima University, Inter-domain communication Hiroshima, Japan in protein controlled by intrinsically disordered region (IDR) – a functional role of intramolecular ‘fly- casting’ mechanism in ligand binding

17.00 - 17.20 Tatyana Smirnov, North Carolina State Nanoparticle/biological University, Raleigh NC, USA interface: effect of silica support on membrane surface potential, dynamics of transmembrane peptide, and effective pKa of ionisable side chain

17.20 - 17.40 H. Kadavath, Max Planck Institute for NMR Investigation of the Biophysical Chemistry, 37077 Göttingen, Molecular Basis of Germany Microtubule Regulation by Neuronal Microtubule- Associated Proteins th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

HALL C: Nucleic Acids-Protein Interactions

Chair: Koichi Kato

16.00 - 16.30 Joon Hwa Lee NMR study of the left-handed Gyeongsang National University, Z-form conformation of Gyeongnam, Korea. nucleic acids induced by Z- DNA binding proteins

16.30 - 17.00 Neel Sarovar Bhavesh Towards a code for single International Centre for Genetic stranded RNA recognition Engineering and Biotechnology, New Delhi, India

17.00 - 17.20 Sukakshana Mukherjee NMR Characterization of 107 Indian Institute of Technology kDa Ternary Complex Roorkee, India Involved in Termination of NF-kappaB Signaling

J. N. Tata Auditorium Chair: N. Suryaprakash

17.45 – 18.15 Subramanian Award Lecture Structure, Dynamics, Anirban Bhunia, Topology, Membrane- disrupting Mechanism and Bose Institute, Kolkata Function of Antimicrobial, Amyloid and Cell-Penetrating Peptides 18.15 – 18.30 CBMR NMRS Award

Veera Mohana Rao Kakita New Methods for NMR of Centre for Excellence in Basic Sciences, Complex Mixtures Mumbai, India

18.30 – 18.45 Jharana Rani Samal Award Lecture Structure-activity- relationship of designed Rajiv K. Kar peptides using low-resolution spectroscopy and high- Bose Institute, Kolkata resolution NMR technique: Case studies of antifreeze and anti-amyloid peptides th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

February 19

J. N. Tata Auditorium: Biomolecular Structure and Dynamics-1

Chair: R. Varadarajan

09.00 - 09.30 Marc Antoni Sani Solid-state NMR study of live Univeristy of Melbourne, Victoria, bacteria in the presence of Australia antimicrobial agents

09.30 - 10.00 Jierong Huang Intrinsically disordered regions of National Yang-Ming University, Taipei, proteins mediate their self- Taiwan association

10.00 - 10.30 Jooyoung Lee Protein Structure Determination Korea Institute for Advanced Study, Seoul, Korea By Multi-Tier Global Optimization Using Early-Stage NMR Data

10.30 - 11.00 Tea Break

Chair: Kavita Dorai

11.00 - 11.30 Chunyang Cao NMR studies on the mechanism of Shanghai Institute of Organic Chemistry, epigenetic modification of the Shanghai, China biological molecules

11.30 - 11.50 Ravi Ampapathi, Structure function studies of SIX3 Central Drug Research Institute, and its mutants in the disease Lucknow, India Holoprosencephaly (HPE)

11.50 - 12.10 Amit Srivastava Korea Institute for Advanced Study, Exploring the conformational Seoul, Korea transition of leucine transporter using an all atom structure based Lorentzian potential model 12.10 - 12.30 K. Asakura JEOL, Japan th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

HALL A: Solid State NMR

Chair: Matthias Ernst

09.00 - 09.30 Hartmut Oshckinat Structural Investigations on Outer FMP, Berlin Membrane Protein G (OmpG), Channelrhodopsin and Phytochrome Cph1 by Very Fast MAS and Dynamic Nuclear Polarisation (DNP)

09.30 - 10.00 Oc Hee Han Water Confined in Nano-meter Korea Basic Science Institute, Seoul, Scale Space of Polymer Korea Electrolyte Membranes

10.00 - 10.30 T. G. Ajithkumar Insights into the mechanical National Chemical Laboratories, Pune, properties of polymers by probing India their functional group, and segmental motions using solid-state NMR 10.30 - 11.00 Tea Break

Chair: Y. Nishiyama

11.00 - 11.30 Shenlin Wang Structure of membrane proteins Peking University, China by solid-state NMR 1 1 11.30 - 11.50 Vipin Agarwal H- H Distance Restraints in fully TIFR Centre for Interdisciplinary protonated proteins at 111 kHz Sciences, Hyderabad, India and above MAS frequencies 13 11.50 - 12.10 Nitin Prakash Lobo Solid State c NMR Central Leather Research Institute, Investigations Of Molecular Chennai, India Order And Topology Of Thiophene Based Mesogens th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

Biomolecular Structure

and Dynamics -2

HALL B: Chair: C. Griesinger

09.00 - 09.30 Ashish Arora Structure and Dynamics of Central Drug Research Institute, Bacterial Peptidyl-tRNA Lucknow, India Hydrolase

09.30 - 10.00 Bong-Jin Lee Two distinct mechanisms of Seoul National University, Seoul, Korea transcriptional regulation by redox sensor YodB

10.00 - 10.30 Bikash Bhaisya F1 Bilinear Rotation Decoupled Center of Biomedical Research, ultra-high resolution diagonal Lucknow, India suppressed Total Correlation Spectroscopy

10.30 - 11.00 Tea Break

Chair: S. Raghothama

11.00 - 11.20 Chinar Pathak Toxins from TA system of Seoul National University, Seou, Korea Helicobacter pylori and insight into mRNase activity

11.20 - 11.40 Jayasubba Reddy NMR Studies of Hierarchical Ecole Polytechnique Fèdèrale de Protein Dynamics Lausanne, Lausanne, Switzerland

11.40 - 12.00 Bipul Sarma Nonmonotonic sweet taste and Tezpur University, Napaam, India molecular conformation of the natural high potency sweetener Rebaudioside A

12.00 - 12.20 Garima Jaipuria Cholesterol-mediated allosteric Deutsches Zentrum für regulation of the structure of Neurodegenerative Erkrankungen, membrane-embedded Göttingen, Germany translocator protein from solid- state NMR

12.30 – 14.00 Lunch th rd 7 Asia Pacific NMR Symposium and the 23 Annual Meeting of the National Magnetic Resonance Society, India

February 16-19, Indian Institute of Science, Bangalore Scientific Program

STUDENT ORAL PRESENTATIONS (14.00-16.00) J. N. Tata Auditorium

Jitendra Das: Mapping Conformational Dynamics of Transthyretin by Solution NMR

Sandeep K. Mishra: Complete eradication of axial peaks and Falls couplings with Ultrahigh resolution in an NMR correlation experiment

Upasana Rai: Structural-functional aspects of DRBs in gene regulation of Arabidopsis thaliana.

Amandeep Singh: Characterizing Quantum Correlations, Beyond Entanglement, on an NMR Quantum Information Processor

Phani Babu: Design and Solid-state NMR/DFT Characterization of Self-assembled 13C, 15N-labelled Small Cyclic-Peptides as Mimics of Amyloid type Aggregation /Inhibition.

Vinay Ganapathy: Description of Multi Quantum (MQ) excitation in Half Integer Quadrupolar nuclei using Effective Floquet Hamiltonians

D. Adilakshmi: Noise Suppression in Spin Noise NMR using Wavelet Transform Analysis Dilip Senapati: Amyloid betaA peptide fragments analysis by NMR

Hall A

Akhila Viswan: Acute Respiratory Distress Syndrome (ARDS) Clinical Spectrum: From The Standpoint Of NMR Based Metabolomics

Tesmine Martin: Interaction of a dimeric Cyclophilin A with Myb1 transcription factor in Trichomonas vaginalis

Nancy Jaiswal: Structural investigations on Helicobacter pylori-Hup and Designing its inhibitors as starting precursors for developing Next Generation Antibiotics

Indrani Pal: Mechanistic insights into the action of nano-conjugates

Garima Verma: Pyrimidine favored recognition by RRM1 of PfSR1

Deepshikha Verma; An Unusual Calcium Binding Protein From Entamoeba histolytica That Exhibits GTPase Activity

Sadhana Kumari: Altered metabolism in Parkinson‘s disease based on serum metabolomics

Richa Dubey: Recombinant hIAPP (rhIAPP) forms smaller aggregates and cytotoxic than synthetic hIAPP- leads obtained from biophysical and cellular studies 16.00 – 16.30 Tea Break 16.30 – 17.00 NMRS General Body Meeting and Oral/poster Prize Announcements

LECTURES

Principles and Applications of Ultrafast Imaging by Spatiotemporal Encoding

Lucio Frydman, Weizmann Institute, 76100 Rehovot, Israel

Over the last decade we and others have introduced and perfected a so-called spatiotemporal encoding (SPEN) methodology to collect multidimensional NMR spectra and images in a single scan. This talk will focus on introducing this technique and showing its potential to deliver superior imaging information, particularly in comparison with established methods such as spin-echo EPI. The figure below illustrates some of our ongoing achievements in this area including (a) Breast-cancer diffusion-based studies executed on a patient with both cysts and a lesion. In the brain cases the SPEN/PE axes run along the vertical (RO horizontal), whereas in the breast scan SPEN/PE was imparted horizontally. (b) Single-shot diffusion brain characterizations. (c) Brain images collected at 7T. (d) Single-shot 3T data collected on a volunteer‘s head with non-ferromagnetic dentures. Notice in (a) the difficulties to deal with fat-derived replicas that complicate the identification of cysts and lesions, in (b) the much better resolution, in (c) the weaker distortions, and in (d) EPI‘s complication to deal with the implant-related field inhomogeneities.

(a) 3T bilateral breast of cancer patient with cysts revealed by ADC maps (d) Head of volunteer with metallic dentures 40mm

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(b) 3T brain apparent diffusion coefficient maps c) Brain images @7T

Single-shot SPEN Single-shot SE-EPI Res: 3x3mm Res = 0.9x1mm 3

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Some Recent Developments in NMR Methods and Applications

Ramakrishna V. Hosur1,2,*

1Department of Chemical Sciences, Tata Institute of Fundamental Research, 1-Homi Bhabha Road, Colaba, Mumbai-400005, India 2UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Santa Cruz, Mumbai- 400098, India

Nuclear Magnetic Resonance (NMR) spectroscopy has come a long way since its discovery seven decades ago and continues to evolve unabated with new applications emerging in many areas of biology and chemistry, such as, structural biology, metabolomics, proteomics, drug discovery, to name a few prominent ones. Methodological developments continue to occur for stretching the limits of existing NMR applications, and for generating new ones. Enhancing the speed of structure determination, pushing the size limits to higher and higher molecular weights, elucidation of structure-function relationships, investigation of large molecular assemblies, intrinsically disordered proteins, folding intermediates, molten globules, drug-protein interactions, protein fibrillation etc are some of the goals in such endeavours. Encompassing the above, this talk will summarize some of the accomplishments and the recent developments in our laboratory to deal with NMR of complex biological systems, on one hand, and NMR of complex mixtures as in metabolomics and herbal drug preparations on the other.

Single-molecule Electron Spin Resonance Spectroscopy & Imaging by diamond sensor

Jiangfeng Du

Hefei National Laboratory for Physical Sciences at the Microscale and Department of Modern Physics, University of Science and Technology of China, Hefei 230026, China

E-mail: [email protected]

Single-molecule magnetic resonance spectroscopy and imaging is one of the ultimate goals in magnetic resonance and will has great applications in a broad range of scientific areas, from life science to physics and chemistry. We and co-workers have successfully obtained the first single-protein spin resonance spectroscopy under ambient conditions [1], realized atomic-scale structure analysis of single nuclear-spin clusters in diamond [3], detected nuclear magnetic resonance spectroscopy with single spin sensitivity [4], and succeeded in detection of (5nm)3 hydrogen nuclear spin magnetic resonance spectroscopy [6].

Among these works, I will specially introduce the ―single-protein magnetic resonance spectroscopy under ambient conditions‖ [1]. The spin of a single nitrogen vacancy (NV) center in diamond is a highly sensitive magnetic-field sensor. We used the NV center to detect a nitroxide labeled protein and gained the world‘s first magnetic resonance spectrum of single protein through electron spin resonance under ambient conditions. We not only revealed the position and orientation of the spin label relative to the NV center, but also elucidate the dynamical motions of the protein on the diamond surface. Now, we are trying to detect the coupling signal of electron spin pairs on DNA duplex and image single ferritins in situ on nanoscale resolution. References:

[1] Fazhan Shi, Qi Zhang, Pengfei Wang, Hongbin Sun, Jiarong Wang, Xing Rong, Ming Chen, Chenyong Ju, Friedemann Reinhard, Hongwei Chen, Joerg Wrachtrup, Junfeng Wang, and Jiangfeng Du. Single-protein spin resonance spectroscopy under ambient conditions, Science, 347, 1135 (2015) [2] Pengfei Wang, Zhenheng Yuan, Pu Huang, Xing Rong, Mengqi Wang, Xiangkun Xu, Changkui Duan, Chenyong Ju, Fazhan Shi, and Jiangfeng Du. High-resolution vector microwave magnetometry based on solid-state spins in diamond, Nature Communication,6, 6631 (2015) [3] Fazhan Shi, Xi Kong, Pengfei Wang, Fei Kong, Nan Zhao, Renbao Liu, and Jiangfeng Du. Sensing and atomic-scale structure analysis of single nuclear spin clusters in diamond, Nature Physics, 10, 21 (2014) [4] C. Mueller, X. Kong, J. M. Cai, K. Melentijevic, A. Stacey, M. Markham, J. Isoya, S. Pezzagna, J. Meijer, J. F. Du, M. B. Plenio, B. Naydenov, L. P. McGuinness, and F. Jelezko. Nuclear magnetic resonance with single spin sensitivity. Nature Communications, 5:4703 (2014)

[5] Fazhan Shi, Qi Zhang, Boris Naydenov, Fedor Jelezko, Jiangfeng Du, Friedemann Reinhard, and Joerg Wrachtrup. Quantum logic readout and cooling of a single dark electron spin, Phys. Rev. B, 87, 195414 (2013) [6] Tobias Staudacher, Fazhan Shi, S. Pezzagna, Jan Meijer, Jiangfeng Du, Carlos A. Meriles, Friedemann Reinhard, Joerg Wrachtrup. Nuclear magnetic resonance spectroscopy on a (5nm)3 volume of liquid and solid samples, Science, 339, 561 (2013)

NMR Approaches to Dissect Oncogenic RAS Signaling at Biological Membrane

Zhenhao Feng, Teklab Gebregiworgis, Ki-Young Lee, Le Zheng, Mohammad Mazhab-Jafari, Mathew J. Smith, Christopher B. Marshall, Mitsu Ikura

Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, M5G 1L7

RAS proteins are frequently mutated in cancer (~30% of all human tumours). Among three major isoforms of RAS, K-RAS is a well-established ‗driver‘ in pancreatic, colorectal and lung cancers where the protein is mutated at three hotspots: G12, G13, and Q61. Despite of enormous efforts in the RAS research over three decades, there is no clinically approved RAS inhibitor and the RAS protein remains to be a challenging target for cancer therapy development. This is largely owing to the lack of understanding of how mutant RAS functions and alters related signaling pathways in the RAS-driven tumours. Fully-processed RAS is prenylated and methylated at the carboxy-terminus, which enables RAS to anchor to the plasma membrane where it functions. There is a large gap in understanding how the lipidated RAS protein functions at the surface of the plasma membrane. To overcome those outstanding issues in the RAS field, we sought to develop better conformational and functional assays for RAS both in isolation and on lipid bilayers. We are particularly interested in elucidating how oncogenic mutations alter the RAS structure and function especially when RAS is at the membrane. We combine isotope-aided, time-resolved, multi-dimensional NMR spectroscopy with the nanodisc technology (developed by Sligar and coworkers) to study membrane- anchored RAS. I will discuss how the biological membrane affects the structure and function of K-RAS in both wild-type and mutants. These structural and functional characterizations of KRAS by NMR may help to develop therapeutic strategies with novel anti-cancer agents targeted to K-RAS driven cancers (Supported by CCSRI, CRS, CFI; Special thanks to the Princess Margaret Cancer Foundation).

Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation

Kandala V. R. Chary1, Deepshikha Verma1, Aruna Murmu2 and Alok Bhattacharya2

1Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India and TIFR Center for Interdisciplinary Sciences, Hyderabad, India 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that the localization of this protein in the nucleus is dependent on its ability to bind Ca2+. Our results, including structural inferences, characterize EhCaBP6 as an unusual CaBP that, as a functional equivalent of Calmodulin in E. histolytica, is involved in regulating cell proliferation.

FK506 Binding Proteins, Immunophilins with Functional Versatility

Ho Sup Yoon

School of Biological Sciences, Nanyang Technological University, Singapore

Immunophilins consist of a family of highly conserved proteins binding with immunosuppressive drugs such as FK506, Rapamycin and Cyclosporin A. Canonical immunophilins such as FKBPs and cyclophilins show peptidylprolyl cis/trans isomerase (PPIase) activity, where the catalytic activity is modulated by cognate ligands. Small-size immunophilins contain only ligand-binding domains, whereas immunophilins with large molecular weights possess extra domains such as tetratricopeptide repeat domain for protein- protein interaction, calmodulin binding domain for sensing calcium and transmembrane domain for anchoring at biological membranes. While the immunosuppressive roles of immunophilin-ligand binary/ternary complexes in the T-cell activation pathways have been extensively studied, their involvements in other biochemical processes including protein folding, receptor signaling, protein trafficking, apoptosis, autophagy, and transcription currently remain poorly understood. To this end, we have been studying the high resolution structural portraits and underlying molecular basis of several canonical and noncanonical FKBPs involved in apoptosis, conformational disorders, malaria, and nucleic acid recognition. The structural information not only provides insights into molecular mechanisms of the emerging FKBP family members in such cellular processes, but also aids in designing novel and selective non-immunosuppressive immunophilin ligands with pharmacological efficacies. In this talk, structures, mechanisms of the multi-domain FKBP family members, design strategy of new immunophilin ligands, and opportunity of their therapeutic potentials will be discussed.

Dynamic multidrug recognition and transcriptional regulation by a multidrug resistant transcriptional repressor, LmrR

Koh Takeuchi†, ‡, Misaki Imai§, and Ichio Shimada†, ¶

†Biomedicinal Information Research Center & Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Japan. ‡PRESTO, JST, Japan. §Research and Development Department, Japan Biological Informatics Consortium, Japan. ¶Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.

LmrR is a multidrug resistance transcriptional repressor that controls the expression of a major multidrug resistance transporter, LmrCD, in Lactococcus lactis. Although the X-ray structures of LmrR are available, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region to induce the expression of LmrCD cannot be explained from the structural snapshots. Here, we structurally and dynamically characterized LmrR in the apo, compound- bound, and operator-bound states by using solution NMR. The compound-binding site of LmrR exhibits ps–μs dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition [1]. Meanwhile, the compound binding induces redistribution of ps–ns dynamics to the allosteric sites, which entropically favors the high-affinity compound recognition [1]. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound- and promoter-bound states, which reduces the affinity of LmrR to the lmrCD operator by several fold. We further found that the transcription regulation of LmrR is achieved through an equilibrium between the lmrCD operator-bound and the non-specific DNA-adsorption states in the L. lactis genome. Together with the 2:1 binding stoichiometry between LmrR dimer and the operator, the effective lmrCD operator with the non-specific DNA- adsorption is close to the endogenous LmrR concentration, which allows a substantial reduction of the LmrR occupancy at the operator site upon ligation to the compound. Taken together, LmrR represents a dynamic multidrug recognition and transcriptional regulation in prokaryotic multidrug resistance systems, where the conformational change induced by the promiscuous compound ligations is coupled to the relocalization of the repressor in the genomic DNA.

References

[1] Koh Takeuchi, Yuji Tokunaga, Misaki Imai, Hideo Takahashi, and Ichio Shimada, Scientific Reports 4, 6922 (2014).

A complex interplay between post-translational modifications observed during the Herpes infection

DSS Hembram, Hitendra Negi, Divya MV and Ranabir Das*

NCBS, TIFR, Bangalore

Post-translational modification of proteins by chains of ubiquitin (Ub) molecules has long been known to play several functions in the inducible and reversible control of signaling pathways. Ubiquitylation is a multistep process where several classes of enzymes function in a sequential regulated manner. First, Ub is activated by an activating enzyme (E1). The activated ubiquitin is then conjugated to the conjugating enzymes (E2s). The E2s interacts with another class of proteins known as ubiquitin ligase (E3s), which function to transfer ubiquitin to the targeted protein. Repeated cycles of ubiquitylation can assemble a poly- ubiquitin chain on a substrate protein. A substrate tagged with a particular form of poly- ubiquitin chains (K48-linked chains) are destined to be degraded by the macro-molecular machinery known as the 26S proteasome. It has been reported earlier that viruses can hijack the Ubiquitin-26S proteasome pathway to suppress anti-viral responses. In this talk, we will report recent results that provide molecular mechanism of how the herpes simplex virus may modulate this pathway for an effective viral replication.

Protein droplets: From Cellular Function to Neurodegeneration

1 3 3 1,2 Susmitha Ambadipudi , Jacek Biernat , Eckhard Mandelkow and Markus Zweckstetter

1. German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany, 37077 2. University Medical Center Göttingen, University of Göttingen, Germany, 37073 3. German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany, 53175

Abnormal aggregation of amyloid proteins (Aβ, Tau and α-synuclein), is the hallmark of neurodegeneration and tauopathies. Neurotoxicity is often attributed to conformational conversion of monomeric proteins into insoluble amyloid aggregates; however, the mechanism underlying this conversion remains unclear. Recent studies propose that proteins undergo phase separation and condense into droplets before aggregating. Proteins comprising low complexity sequences exhibit reversible phase behavior in presence of RNA to form stress granules, speckles and foci with liquid-like properties1. These protein-RNA droplets facilitate cellular functions such as transcription and stabilization of nucleoli2. Aberrant phase separation results in further condensation of protein droplets into solid amyloid fibers. For example, RNA-binding proteins, Fused in Sarcoma (FUS) and heterogeneous nuclear Ribonucleoprotein A1 (hnRNPA1) have been shown to cause neurotoxicity due to irreversible droplet formation in vivo3,4. In this context, we present an integrative framework for uncovering sequence-encoded phase separation, liquid-gel properties and fibrillization of Alzheimer-related protein, Tau.

References:

1. Quiroz, F.G; Chilkoti A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 2015, 14(11): 1164-1171. 2. Nott, T.J; Craggs, T.D; Baldwin, A.J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 2016, 8(6): 569-575. 3. Patel, A; Lee, H.O; et al. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 2015, 162(5): 1066-1077. 4. Molliex, A; et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 2015, 163(1): 123-133.

Transient Compensation in Solid-State NMR

Johannes Hellwagnera, Johannes J. Wittmanna, Nino Willia, Kazuyuki Takedab Beat Meiera, Matthias Ernsta aLaboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland bDivision of Chemistry, Graduate School of Science, Kyoto University, 606-8502 Kyoto, Japan

Pulse sequences in NMR spectroscopy are usually designed under the assumption that amplitude and phase of the radio frequency are identical in experiments, analytical calculations and numerical simulations. In reality, however, the experimental B1-field the nuclear spins experience in the sample can deviate significantly from this ideal behavior.1,2 Depending on various properties of the rf circuit an exponential build-up or decay of the in-phase rf-field amplitude as well as quadrature components are observed at any discontinuity during the pulse sequence (Figure 1B). The resulting differences from the intended ideal spin trajectories can lead to a significant decrease in the performance of pulse sequences.3,4 Therefore, one of the selection criteria for Figure 1. Comparison of input shape and probe successful NMR pulse sequences is their response for rectangular excitation (A and B) stability against pulse transients. Linear and a transient-compensated pulse (C and D). response theory offers a way to calculate pulse shapes (Figure 1C) which counteract the formation of pulse transients and generate the desired pulse shape (Figure 1D) in the NMR coil.5

In this contribution, we will discuss the sensitivity of different pulse sequences in solid-state NMR to phase transients. We have shown recently that in symmetry-based sequences (e.g., POST-C7) a stable and reproducible recoupling can be achieved by using transient- compensated pulses.6,7 We have started analyzing other types of pulse sequences, in order to analyze which sequences profit in which way from using transient-compensated pulses. So far we have looked at π-pulse sequences (RFDR and REDOR) and some heteronuclear decoupling sequences with unexpected results. RFDR shows a strong sensitivity to transient effects while REDOR sequences are not sensitive at all. Such differences can be understood when analyzing the sequences using triple-mode Floquet theory to calculate the effective Hamiltonians.

References (1) Mehring, M.; Waugh, J. S. Rev. Sci. Instrum. 1972, 43 (4), 649. (2) Barbara, T. M.; Martin, J. F.; Wurl, J. G. J. Magn. Reson. 1991, 93 (3), 497.

(3) Rhim, W. K.; Ellemann, D. D.; Vaughan, R. W. J. Chem. Phys. 1973, 59 (7), 3740. (4) Rhim, W. K.; Ellemann, D. D.; Schreiber, L. B.; Vaughan, R. W. J. Chem. Phys. 1974, 60 (11), 4595. (5) Tabuchi, Y.; Negoro, M.; Takeda, K.; Kitagawa, M. J. Magn. Reson. 2010, 204 (2), 327. (6) Wittmann, J. J.; Takeda, K.; Meier, B. H.; Ernst, M. Angew. Chem. Int. Ed. Engl. 2015, 54 (43), 12592. (7) Wittmann, J. J.; Mertens, V.; Takeda, K.; Meier, B. H.; Ernst, M. J. Magn. Reson. 2016, 263 (C), 7.

Atomic Level Structural Insight of Bones and Cartilage: Solid State NMR Perspective

Neeraj Sinha

Centre of Biomedical Research SGPGIMS Campus, Raebarelly Road Lucknow – 226014 INDIA

Bone and cartilage are important class of biomaterials in which solid state nuclear magnetic resonance (ssNMR) play a pivotal role in providing atomic level structural insight. Bone is a mineralized tissue consisting of inorganic minerals (calcium hydroxyapatite), organic proteins (mainly triple helical collagen proteins) and water molecules. Atomic level structural details help in understanding the bone architecture and associated changes due to disease condition such as osteoporosis. While X-ray and other forms of spectroscopy has given significant understanding of bone architecture, ssNMR has unique advantage of providing atomic resolution structural details in its absolute native state. This talk summarizes ssNMR methods, recently developed in our laboratory, which gives structural details of bone and cartilage. Methods such as 1H detected experiments, sensitivity enhancement of collagen backbone natural abundance 15N resonance due to paramagnetic doping and sensitivity enhancement of natural abundance 13C resonance by employing heteronuclear Overhauser enhancement with equilibration of magnetization by low power irradiation, will be presented.

Very fast MAS solid-state NMR and electron diffraction

JEOL RESONANCE Inc. & RIKEN, Yusuke Nishiyama

Very fast MAS paves the new way for analyzing natural abundance samples in solid-state NMR. The advantages1 are a) narrowing of 1H resonances, b) sensitivity enhancement by 1H indirect detection, c) very small sample amount, d) wide spectral range in rotor-synchronous 1 acquisition, e) elongated H T2‗ decay, f) strong rf irradiation by micro-sized sample coil, g) use of low power 1H heteronuclear decoupling, etc. The combination of these benefits allows us to measure multi-dimensional correlation experiments even in natural abundance. Here we will discuss the following examples.

1) Observation of low-gamma nuclei including integer nuclei by 1H indirect detection.2-6, 2) 14N/14N homonuclear correlation experiments to elucidate the intermolecular packings 7,8 3) Use of 1H chemical shift to elucidate the atomic resolution structures.9-13 4) Use of 1H/X dipolar interactions for precise determination of internuclear distances.14,15 5) HRuMAS for monitoring tissue samples less than 1 uL.16,17 In addition that, we will introduce the combined approach of solid-state NMR with electron diffraction to distinguish crystalline form in micro-crystalline organic samples.18

References:

1. Y. Nishiyama, Solid State Nucl. Magn. Reson. 78 (2016) 24-36. 2. N.T. Duong, Y. Nishiyama, submitted. 3. Y. Nishiyama et al., J. Magn. Reson. 208 (2011) 44-48. 4. Y. Nishiyama et al., J. Magn. Reson. 230 (2013) 160-164. 5. M.K. Pandey, et al., PhysChemChemPhys 18 (2016) 6209-6216. 6. G.N. Manjunatha et al., Anal. Chem. In press. 7. M.K. Pandey, Y. Nishiyama, J. Magn. Reson. 258 (2015) 96-101. 8. M.K. Pandey, et al., PhysChemChemPhys 18 (2016) 22583-22589. 9. M.K. Pandey, et al., J. Magn. Reson. 250 (2015) 45-54. 10. M.K. Pandey, Y. Nishiyama, Solid State Nucl. Magn. Reson. 70 (2015) 15-20. 11. M.K. Pandey, et al., J. Magn. Reson. 261 (2015) 1-5. 12. M.K. Pandey, Y. Nishiyama, J. Magn. Reson. 261 (2015) 133-140. 13. M.K. Pandey, et al., Solid State Nucl. Magn. Reson. 76-77 (2016) 1-6. 14. P. Paluch, et al., J. Magn. Reson. 252 (2015) 67-77. 15. Y. Nishiyama et al., Solid State Nucl. Magn. Reson. 73 (2016) 15-21. 16. Y. Nishiyama et al., Solid State Analyst 140 (2015) 8097-8100. 17. N.T. Duong, et al., Analytical Methods 8 (2016) 6815-6820. 18. T. Oikawa et al., submitted.

Magic-angle spinning NMR and SAXS reveal structural effects of single mutations in a filamentous viral capsid across multiple length scales

Gili Abramov1, Rona Shaharabani1, Omry Morag1, Ram Avinery2, Anat Haimovich1, Inbal Oz1, Roy Beck2, and Amir Goldbourt1

1School of Chemistry and 2School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel.

Viral mutations are a main source of resistance to medical treatment. By combining SAXS and magic-angle spinning (MAS) NMR we relate single-site mutations in the filamentous bacteriophage virus fd to structural changes over several length scales. The atomic-scale was examined by solving the quaternary structure of the phage[1], and by comparing NMR chemical shifts of three forms: wild-type, D12N, and Y21M. Inter-particle interactions experienced by different mutants on the nano-scale were studied by the SAXS osmotic pressure technique and the micron-scale, i.e. liquid crystal formation and phage alignment, was examined from SAXS and from optical birefringence. We show that a single-site charge mutation (D12N – M13 virus) on the surface of the capsid, has a negligible effect on its structure however, a mutation in the hydrophobic packing region (Y21M) has a significant impact: it changes the symmetry, the subunit structure and the packing of the phage, and on the micron-scale it significantly alter the pitch of cholesteric liquid-crystals formed by the virus. Despite the fact that changes occurring in the atomic- scale propagate to the macroscopic scale, those changes skip the nano-scale; SAXS measurements we employed on the three forms of the virus (wt-fd, D12N, fd-Y21M) at different salt and osmotic pressure conditions suggest that inter-particle interactions are not significantly affected by the hydrophobic Y21M mutation. On the other hand, these interactions are significantly affected by the D12N charge mutation. Modelling of the SAXS data also allowed us to re-estimate the effective particle charge and show that the non- stoichiometric nucleotide-to-subunit ratio is the source of the net charge on the virus. Since such mutation affect differently the organization of phage particles, our studies can direct smart design of phage-inspired bio- and nano-technology.

[1] Morag O, Sgourakis NG, Baker D, Goldbourt A, PNAS 27, 971-976 (2015).

Solid-State NMR of Membrane Proteins Macroscopically Aligned by Nanopores Alex I. Smirnov, AlexanderA. Nevzorov

Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, North Carolina 27695-8204, USA

Solid-state NMR of macroscopically aligned samples has emerged as a powerful tool for studying membrane proteins under their native-like conditions mimicking cellular membtranes. Further progress in this method could be achieved by development of novel experimental methods and membrane mimetics for the structure-function studies of these important biological constituents. Here we describe experimental methods for forming lipid nanotubural bilayers by self-assembly inside cylindrical nanopores of anodic aluminum oxide (AAO). Such hybrid nanostructures, which we named lipid nanotube arrays, represent a new type of substrate-supported and macroscopically-aligned lipid bilayers that have many attractive features for both biotechnology as well as structure-function studies by magnetic resonance methods. We also describe a recent progress in reconstructing both small pore- forming and transmembrane peptides1,2 as well as large membrane protein complexes, such as bacterial reaction center (RC) protein from Rhodobacter sphaeroides. Cylindrical geometry of lipid nanotubes was verified and further studied by spin-labelling EPR and solid- state 31P NMR. Moreover, a wide range of temperature and pH stability of lipid bilayers within the nanopores enabled solid-state NMR experiments under a wider range of conditions than ever before. Uniaxial motional averaging by rotational diffusion of membrane proteins ensures the line narrowing of experimental NMR linewidths, thus, providing structural and dynamic information for the lipid-embedded proteins without the need for crystallization.

(1) Marek, A.; Tang, W.; Milikisiyants, S.; Nevzorov, A A.; Smirnov, A I. Biophysical J., 108, 5. (2) Nevzorov, A. A.; Smirnov, A. I. In Protein NMR: Modern Techniques and Biomedical Applications; Berliner, L., Ed. 2015; Vol. 32, p 159.

Deuterium Magic Angle Spinning NMR and Local Molecular Dynamics Modelling for the Study of Restricted Dynamics of Grafted and Adsorbed Molecules

S. Jayanthi1, Shifi Kababya2, Asher Schmidt2 and Shimon Vega3

1Department of Physics, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala, India. 2Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion, Israel Institute of Technology, Haifa, Israel. 3Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel

Dynamics of molecules located at organic and inorganic surfaces has always been an area of interest due to their extended applicability in surface chemistry, catalysis, slow release and others. Over the past decade silica based organic-inorganic hybrid materials have been investigated for the characterization of catalytic reactions. Identifying the structure-function relationship of these materials enhances the knowledge of these systems together with the possibility of using them for catalysis, storage, etc. The present work reports on solid state Nuclear Magnetic Resonance investigations carried out on similar systems to probe the mobility of free adsorbates and covalently bound linkers close to the surface. Dynamics of molecules has been reported time and again in solid state NMR, by exploiting the orientational dependence of the deuterium quadrupole tensor, mostly under static conditions. Instead, we have employed deuterium Magic Angle Spinning (MAS) NMR to study kinetics of grafted linkers and adsorbed molecules at the inner surface of mesoporous silica materials.

Dynamic deuterium MAS NMR spectra of the grafted molecules in various mesopores revealed the existence of non-uniaxial tensors under various levels of water adsorption indicating molecular mobility On the other hand similar spectra implied complex dynamic properties in the case of adsorbed molecules between molecules undergoing exchange between surface bound and free states, also dependent on the presence of water molecules. Challenges associated in retrieving dynamic parameters utilizing and modifying standard dynamic Floquet theory for computational affability, are addressed.

In the present studies, Molecular Dynamic (MD) tools were employed to visualize the local dynamics. No a-priori assumptions of dynamic models were made in these studies. MD observations corroborate the NMR results thereby providing molecular conformations associated with dynamics in the case of grafted molecules whereas the dynamics of adsorbed molecules were interpreted in terms of an exchange process that is gated by the diffusion of water molecules to and from surface adsorbing sites.

Further studies in systems where dynamics and its associated molecular conformations correlated to catalytic activity are under investigation.

NMR structural analysis of unstable / partially unfolded / aggregated proteins

Chojiro Kojima

Graduate School of Engineering, Yokohama National University Institute for Protein Research, Osaka University

For high-resolution NMR structure determinations, a sufficient number of NOE peaks with nearly complete and accurate assignments are required. In the case of CYANA, more than 8.4 NOE restraints per residue and more than 90% of chemical shift assignments are necessary for a successful NMR structure determination using the automated NOE assignment procedure. These requirements cannot always be met, and depend on the nature of the target protein being examined. Especially for membrane proteins and unstable / partially unfolded / aggregated proteins, these requirements are hardly met.

In an effort to overcome these difficulties, we have developed some new methods to analyze the high-resolution NMR structure. (1) Simultaneous use of multiple PRE/PCS-derived distance restraints: this approach is useful when the number of NOE constraints is insufficient. In fact, the high-resolution structure of an unstable protein could be determined using multiple PRE-derived distance restraints [1,2]. (2) Fully automated structure determination: this approach was useful when the number of NOE constraints is sufficient in part. We have developed a fully automated structure determination platform, MagRO-FLYA, and successfully determined the high-resolution structure of a partially unfolded protein. (3) Non-linear sampling: this approach was useful when S/N is insufficient. At very low concentration, 17 µM, triple-resonance NMR spectra of a protein possessing aggregation propensity were successfully measured, and 99% of the backbone chemical shifts were successfully assigned.

References

[1] Furuita K, Kataoka S, Sugiki T, Hattori Y, Kobayashi N, Ikegami T, Shiozaki K, Fujiwara T, Kojima C. J Biomol NMR, 2015, 61, 55-64. [2] Tatebe H, Murayama S, Yonekura T, Hatano T, Richter D, Furuya T, Kataoka S, Furuita K, Kojima C, Shiozaki K. eLife.

15N detected experiments – Where being insensitive has its advantages

Arthanari Haribabu

15N detection never had the limelight in biomolecular NMR due to the inherent low sensitivity compared to 1H/13C-detected experiments. The low gyromagnetic ratio of 15N results in favorable relaxation properties and it is the slowest relaxing nucleus in protein NMR, which results in narrow linewidths. With the advancement in data collection using Non-Uniform Sampling and improvement in cryoprobe technology, sensitivity should no longer be seen as a number, rather the ability to see the weakest resonance of interest. This talk will introduce a suite of 15N-detected 2D and 3D experiments, discuss their advantages and application in studying Intrinsically Disordered Proteins (IDPs) and large systems.

NMR Experiments for Two Receivers

Helena Kovacs,1 and Ēriks Kupče,2

1Bruker BioSpin AG, Industriestrasse 26, CH-8117, Fällanden, Switzerland. 2Bruker BioSpin, Banner Lane, Coventry CV4 9GH, UK.

NMR experiments involving several receivers provide a unique way of increasing the sensitivity and information content of data recorded in a given period of time [1-3]. We present a comprehensive series of such experiments designed for simultaneous detection of abundant nuclei, such as 1H, 19F and 31P, as well as samples enriched with magnetically active isotopes including 13C and 15N. The multiple receiver experiments are categorized into three main types – (a) parallel acquisition, (b) sequential acquisition and (c) interleaved experiments. The optimum implementation is shown to depend on the relaxation properties of the involved nuclei as well as the intrinsic sensitivity of the directly observed nuclei. We particularly focus on the routine NMR experiments involving 1H and 19F nuclei not least because of the particularly important role that 19F plays in drug discovery and pharmaceutical industry [3]. We show that essentially any of the basic 2D NMR experiments, such as COSY, NOESY, TOCSY, DOSY, HSQC, HMQC, HMBC, HETCOR or relaxation measurements that are routinely used in small molecule NMR can be easily adapted for and more efficiently recorded on systems equipped with multiple receivers.

Figure 1. Two-dimensional 1H-1H and 1H-19F COSY experiments recorded in parallel with direct detection of 1H (receiver 1) and 19F (receiver 2) nuclei on a 700 MHz AVIII HD NMR system equipped with a QCIF CryoProbe.

Many of these experiments are amenable to further reduction of experiment time by combining them with other fast NMR techniques, such as Hadamard NMR, non-uniform sampling, spatial encoding or rapid pulsing methods. We believe that the multi-receiver technology will boost the development of new NMR experiments as well as NMR research in general, making the NMR instruments more efficient and making the NMR spectroscopy even more unique in the universe of analytical tools and experimental techniques.

References.

[1] R. Freeman and Ē. Kupče, in Modern NMR Approaches To The Structure Elucidation of Natural Products,(Eds. A. J. Williams, G. E. Martin and D. Rovnyak), vol. 1, pp 117-145, Royal Soc. Chem. (2016). [2] Ē. Kupče, eMagRes, 2015, 4, 721–732. DOI 10.1002/9780470034590.emrstm1404. [3] H. Kovacs and Ē. Kupče, Magn. Reson. Chem., 2016, DOI 10.1002/mrc.4428.

Recent developments, applications and challenges in ultrafast multidimensional NMR

Patrick Giraudeau 1,2

1. CEISAM, Université de Nantes, France 2. Institut Universitaire de France, Paris, France

Ultrafast (UF) multidimensional NMR spectroscopy is a generic approach that makes it possible to record nD spectra in a single scan [1-2]. UF NMR has recently evolved into a powerful analytical tool, with particular interest in situations where the experiment duration is critical such as the monitoring of fast kinetics or dynamics, or the coupling with other techniques [3]. UF NMR is also recognized for its high precision in quantitative analysis [4]. In this presentation, we will describe some of the most recent developments and applications of UF 2D NMR in our group. These include the development of double- quantum experiments for the quantitative analysis of complex mixtures [5], as well as new experiments for UF 2D NMR in oriented media [6] or in the solid-state [7]. We will also present the successful implementation of UF 2D NMR on a benchtop spectrometer to monitor organic syntheses [8] or for the fast screening of food products [9]. We will also discuss the perspectives arising from the application of UF 2D NMR for high-throughput metabolomics [10], as well as the promising combination of UF 2D NMR with dissolution DNP applied to biological samples [11].

References

1. L. Frydman, T. Scherf, and A. Lupulescu, Prod. Natl. Acad. Sci. USA, 2002, 99, 15858-15862. 2. B. Gouilleux, L. Rouger, and P. Giraudeau, eMagRes, 2017, 5, 913-922. 3. P. Giraudeau, and L. Frydman, Annu. Rev. Anal. Chem., 2014, 7, 129-161. 4. S. Akoka, and P. Giraudeau, Magn. Reson. Chem., 2015, 53, 986-994. 5. L. Rouger, B. Gouilleux, M. Pourchet-Gellez, J.-N. Dumez, and P. Giraudeau, Analyst, 2016, 141, 1686-1692. 6. P. Lesot, P. Berdague, and P. Giraudeau, Chem. Commun., 2016, 52, 2122-2125. 7. L. Rouger, M. Yon, V. Sarou-Kanian, F. Fayon, J.-N. Dumez, and P. Giraudeau, submitted for publication, 2017. 8. B. Gouilleux, B. Charrier, E. Danieli, J.-N. Dumez, S. Akoka, F.-X. Felpin, M. Rodriguez-Zubiri, and P. Giraudeau, Analyst, 2015, 140, 7854-7858. 9. B. Gouilleux, B. Charrier, S. Akoka, F.-X. Felpin, M. Rodriguez-Zubiri, and P. Giraudeau, Trends Anal. Chem., 2016, 83, 65-75. 10. T. Jézéquel, C. Deborde, M. Maucourt, V. Zhendre, A. Moing, and P. Giraudeau, Metabolomics, 2015, 11, 1231-1242. 11. 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, and P. Giraudeau, Analyst, 2015, 140, 5860-5863.

Rapid convergence of optimal control in NMR using numerically- constructed toggling frames

Paul Coote, Harvard Medical School

We present a numerical method for rapidly solving the Bloch equation for an arbitrary time- varying spin-1/2 Hamiltonian. The method relies on fast computations such as summation and quaternion multiplication, rather than slow computations such as matrix exponentiation. A toggling frame is constructed in which the Hamiltonian is time-invariant, and therefore has a simple analytical solution. The key insight is that constructing this frame is faster than solving the system dynamics in the original frame. Rapidly solving the Bloch equations for an arbitrary Hamiltonian is particularly useful in the context of NMR optimal control. Optimal control theory can be used to design pulse shapes for NMR spectroscopy. However, it requires multiple simulations of the Bloch equations at each stage of the algorithm, and for each relevant set of parameters (e.g. chemical shift frequencies). This is typically very time consuming and therefore limits the application of optimal control in NMR. We demonstrate that by working in an appropriate toggling frame, optimal control pulses can be generated much faster.

We present an adjustment to the well-known GRAPE (Gradient Ascent Pulse Engineering) algorithm to continuously update the toggling-frame as the optimal pulse is generated.

The procedure is as follows: The time-varying part of the Hamiltonian is approximately removed using a numerically-constructed toggling frame. This construction requires an integration (i.e. cumulative sum of a digital signal), a subtraction, and a rotation of the Hamiltonian. All of these are very fast numerical operations. With iteration, this quickly converges to a time-invariant Hamiltonian, i.e. an average or equivalent Hamiltonian. Since the average Hamiltonian is time-invariant, it has a simple analytical solution, and the corresponding density matrix trajectory is easily extracted. The toggling frame is encoded by a quaternion rotation vector, so that switching between the original frame and toggling frame is achieved by a fast computation. In contrast, solving the Bloch equations in the original frame of reference, in which the Hamiltonian is time-varying, requires repeated matrix exponentiation which is computationally slow.

We demonstrate that the toggling-frame method of generating the trajectories is about two orders of magnitude faster than working in the original frame of reference. This extends the applicability of optimal control pulse sequences in a range of applications. Figure 1: Systematic removal of time-dependence by toggling frames. A.-C: A time- dependent Hamiltonian expressed in successive toggling frames after 0, 3, and 6 iterations of 23 the toggling frame algorithm. By C, the time variation is removed. D: The magnetization trajectory expressed in the original frame of reference. This method of solving the Bloch equation is much faster than the standard approach using repeated matrix exponentiation.

Rapid Structural Elucidation of Proteins with 'All-in-one' NMR Spectroscopy

Jithender Reddy Gurrala1 and Hanudatta S Atreya2

1Indian Institute of Chemical Technology, Hyderabad, India 2Indian Institute of Science, Bangalore, India

Nuclear magnetic resonance (NMR) spectroscopy is routinely used technique for structural characterization of biomolecules. For de novo structure determination of proteins, it is desirable to acquire rapidly several data sets which are required for backbone sequential correlations, side-chain assignments and nuclear Overhausser effect (NOE) based distance constraints. We present a three-dimensional (3D) "all-in-one" experiment (named 3D ANSY) that provides all this information in a single experimental data set. The method involves simultaneous acquisition of multiple 1H and 13C data using the dual receiver system. Four 3D data set comprising two 3D (13C and 15N edited) 1H-1H NOESY for distance constraints and two 3D spectra each for backbone sequential walk and side-chain resonance assignments are acquired at the same time with high resolution. The sensitivity of this experiment is optimized by channelizing cosine (real) and sine (imaginary) modulations of chemical shifts into four different magnetization pathways which are finally detected as four different 3D data sets. Utility of this experiment for structural characterization of proteins in solution is demonstrated. The data collection can be further accelerated by combining the technique with other fast data collection approaches such as non-uniform sampling and reduced dimensionality approaches. The method presented here demonstrates for the first time that four or more high dimensional NMR data can be serially or parallely acquired with multiple receivers for rapid protein structural studies. Thus, our study opens up new avenues for high-throughput applications such as monitoring conformational changes during protein aggregation, unfolding, protein-ligand interactions.

Translational MetabolTranslational Metabolic Imaging S. Sendhil Velan Metabolic Imaging Group, Singapore Bioimaging Consortium, Singapore Our lab is focused on developing and implementing advanced Magnetic Resonance Imaging techniques suitable for investigating metabolic diseases. The study of fat distribution is important to understand the pathophysiology of obesity related disorders, diabetes mellitus and cardiovascular diseases. In this talk I will highlight some of our ongoing effort in imaging white and brown adipose tissue function, fat partitioning during obesity, fat mobilization and metabolic interventions. Pre-clinical longitudinal studies allow identification of imaging biomarkers during the evolution of disease which can be translated to clinic. In this talk I will highlight some of our findings from both rodents and human studies. We have also translated our methodology to clinical platform to investigate the Asian ethnicities (Indian, Chinese and Malay) within the Singapore Adult Metabolism Study (SAMS). South Asians are more insulin resistant than Caucasians despite similar or lower BMI. We investigated whether the propensity to accumulate fat in specific depots with increasing adiposity mediates the interactions between ethnicity, adiposity, and insulin sensitivity. References: 1. Pola A, et al., Prog. Nucl Magn Reson Spectrosc. 2012, 65, 66-76. 2. Nagarajan V, et al., Am J Physiol Heart Circ Physiol. 2013. 304 , H1495-504. 3. Agarwal, N et al., Magn Reson Imaging, 2014, 32, 379-384. 4. Khoo CM et al., Diabetes, 2014, 63, 1093-1102. 5. Prakash KN et al., MAGMA, 2016, 29, 277-86.

MRI Contrast Enhancement by Frequency Lock-in Suppression Technique

Dennis W. Hwang Department of Chemistry and Biochemistry, National Chung Cheng University

MR molecular imaging is non-invasive and enables the visualization of molecular process in living organisms. However, contrast in MRI may be very weak due to the small difference of MR properties between the lesion and normal regime. Frequency lock-in technique by using a electronic feedback device generates a specific narrow frequency bandwidth RF field is presented. The effects of this RF field on magnetic resonance images are assessed both theoretically and experimentally. Spectroscopy and imaging experiments were performed. Frequency tuning allowed the selected spectral peak to be suppressed. Phantom tests using methanol, ethanol, and water showed different contrasts using different feedback RF field frequencies. The frequency lock-in was also found to help differentiate among the small structural variations in biological tissues. The contrast achieved in in vivo mouse brain imaging using the lock-in suppressed technique indicated a better spatial discrimination when compared with that achieved using conventional imaging methods, especially in the hippocampus region. Selective lock-in suppressed imaging is a new approach to provide frequency information in magnetic resonance imaging; rather than determining an object‘s evolution over time, this approach allows small susceptibility variations to be distinguished by tuning the frequency of the narrow bandwidth lock-in RF field. A new and enhanced contrast can be achieved using this technique. In addition, we also take of advantage of frequency lock-in suppressed technique to obtain the positive contrast of iron-contain region. It can distinguish signal induced by paramagnetic ions from other artifacts, such as inhomogeneous spin distribution.

Functional MRI techniques: Application to Neuroscience

Dr Subash Khushu Institute of Nuclear Medicine & Allied Sciences (INMAS), Lucknow Road, Timar Pur, Delhi-110054 email: [email protected]

Magnetic Resonance Imaging (MRI) has shown a great potential for in-vivo assessment of morphology, function and metabolism in living systems. The availability of high field human MRI systems with parallel imaging capabilities has resulted in enormous improvements in the sensitivity and resolution facilitating development of advanced techniques. Advanced MR techniques based on the multi-parametric contrast of MRI have opened up a new era of applications in neuroscience for studying the brain in health and disease1.

The developments of functional MRI, resting state fMRI based on BOLD contrast2,3 and Diffusion Tensor Imaging4 have become most popular approaches for brain mapping and possibly as a surrogate marker for neural function in cognitive and clinical neuroscience. There has also been a great interest in using these techniques to assist in understanding, clinical diagnosis and management of Neuro-developmental, neuro-endocrine and psychiatric disorders.

In this presentation the applications of these techniques together with Voxel Based Morphometry and Magnetic Resonance Spectroscopy in various endocrine5 psychiatric6, and stress related neurocognitive studies carried out at our center will be highlighted.

References

1. Tianzi Jiang & Yong Liu & Feng Shi & Ni Shu & Bing Liu & Jiefeng Jiang & Yuan Zhou Multimodal Magnetic Resonance Imaging for Brain Disorders: Advances and Perspectives, Brain Imaging and Behavior, (2008); 2: 249–257 2 Ogawa S, Lee T M, Kay A R, and Tank D W. Brain magnetic resonance imaging with contrast dependent on blood oxygenation. Proc Natl Acad Sci U S A. (1990); Dec; 87(24): 9868–9872. 3 Biswal B, Yetkin FZ, Haughton VM, et al. Functional connectivity in the motor cortex of resting human brain using echo-planar MRI. Magn Reson Med (1995); 34:537–41. 4 Mori, S. & Zhang, J.. Principles of Diffusion Tensor Primer Imaging and Its Applications to Basic Neuroscience Research. Neuron (2006); 51, 527–539. 5 Singh S, Kumar M, Modi S, Kaur P, Shankar LR, Khushu S Alterations of Functional Connectivity Among Resting-State Networks in Hypothyroidism., J Neuroendocrinol. (2015); 27(7):609-15 6 Singh S, Modi S, Goyal S, Kaur P, Singh N, Bhatia T, Deshpande SN, Khushu S. Functional and structural abnormalities associated with empathy in patients with schizophrenia: An fMRI and VBM study. J Biosci. (2015); Jun;40(2):355-64.

Tailored Magnetic Resonance Fingerprinting

Shaik Imam Ahmed1, Shivaprasad Ashok Chikop1, Amaresh Shridhar Konar1, 2, Vimal Chandran3, Ramesh Venkatesan2, Mauricio Reyes3, Sairam Geethanath1*

1Medical Imaging Research Centre, Dayananda Sagar Institutions, India

2Wipro-GE Healthcare, India

3University of Bern, Switzerland

Speaker: Sairam Geethanath, Ph.D.,

Magnetic Resonance Fingerprinting (MRF) is an accelerated acquisition-reconstruction strategy to simultaneously generate multi-parametric MR maps (1). In this work, we present a tailored design for MRF that optimizes acquisition parameters to enhance desired contrasts in sequence of ‘blocks’. The method also allows direct access to signal intensity images of multiple contrasts of acceptable quality. This allows accurate determination of long T2 components, which has been grossly underestimated previously (1). The work also involves development of in vitro breast, cardiac and carbomer phantoms, to demonstrate the utility of the proposed method prospectively. These phantoms are user interface driven and mimic the structural and functional characteristics of the particular anatomy. Conventional MR imaging of these phantoms at 1.5T on multiple venodors has been performed and compared with in vivo data. Reconstruction of the MRF data has been performed using sliding window and random forests methods, which allow for dictionary-less matching. This is compared with conventional matching methods such as vector product and partial least squares. These findings on in vivo brain data in addition to phantom validation will be reported in detail.

Qualitative and quantitative error quantification has been performed. The error for the long T2 components is less than 5%. In summary, this design is expected to automate combination of multiple sequences of a desired protocol, into a single sequence based on tailored choices of MR contrast.

References:

(1) Dan Ma et. al., Nature, 2013

Urinary metabolomic profiling in idiopathic Parkinson’s disease

S. Senthil Kumaran, Sadhana Kumari

Department of NMR, All India Institute of Medical Sciences, New Delhi

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder, caused by progressive death of dopaminergic neurons in the substantia nigra parscompacta. The present study used 1H NMR technique to investigate urine samples to find possible biomarkers in PD patients with respect to healthy controls (HC).

Method: Urine samples were collected from PD patients (n= 28; mean age: 56.1 ± 6.78 years) and HC (n=10; mean age: 51.5 ± 5.57 years) after 12 hours fasting. 1D spectrum with water suppression was acquired using a single 90 pulse with 128 scans and 14 s relaxation delay. Binning data were estimated using MestReNova software (version 10.0, Mestrelab Research, Spain) and Multivariate partial least square discriminate analysis (PLS-DA) using MetaboAnalyst, a web-based metabolomics data processing tool.

Figure 1. Representative one dimensional (1D) 700MHz 1H NMR spectrum of urine sample acquired at 25 C in D2O in a patient of Parkinson ‘s disease (red color) and a Healthy Control (black color).

Results: The concentration of lactate, glutamine, phenylalanine, tryptophan, glycine and alanine were observed to be significantly higher, while that of citrate, leucine were lower in PD patients. (representative 1H NMR spectra shown in Figure 1). PLS-DA depicts clear separation between PD and HC.

Discussion: Lower levels of leucine, isoleucine and citrate indicate abnormalities in protein synthesis and energy production in PD patients1. Altered glycine and tryptophan levels in PD patients may be attributed to mitochondrial disturbances1 and impairment of brain energy metabolism2. Elevated lactate may ascribe mitochondrial dysfunction to reduced bioenergetics efficiency in patients with respect to HC3. The study indicates metabolic acidosis and hypokalemia, which may be the effect of poor renal handling in PD patients.

References

1. Luan, Hemi, et al. Scientific Reports 2015, 5:13888. 2. Moroni, Flavio. European Journal of Pharmacology 1999, 375: 87-100. 3. Ozelius, Laurie J., et al. New England Journal of Medicine 2006, 354: 424-425.

Unique molecular signatures to distinguish immunotherapy responding and resistant cell lines in melanoma by NMR spectroscopy and MR hyperpolarization

Shivanand Pudakalakattia, Ashvin Jaiswalb, Prasanta Duttaa, Michael Curranb, Pratip Bhattacharyaa*

Department of Cancer Systems Imaging, UT M D Anderson Cancer Center, Houston, Texasa Department of Immunology, UT M D Anderson Cancer Center, Houston, Texasb

Background: Cancer immunotherapy is utilizing body‘s own immune system to destroy cancer cells, which is achieved by blocking the two negative regulatory proteins of T-cell activation known as cytotoxic T-lymphocyte associated protein-4 (CTLA4) and programmed death-1 (PD1). The therapy is successful in treating more than 50-60% of patients with melanoma. However, about 40-50% of patients doesn‘t respond to immunotherapy. So, understanding the response of immunotherapy in individuals at metabolic level and predicting the therapy response in advance is the overarching goal of this investigation. The mouse derived melanoma cancer cell lines are used to study the role of metabolism in immunotherapy response. Methods: The standard one dimensional (1D) 1H NMR with water suppression sequence was used to acquire the data on cell line B16/BL6 TMT (responsive to immunotherapy) and B16/BL6 3I F4 (completely resistant). The data was processed in Topspin 3.1 and resonances are identified using Chenomx, human metabolic database (HMDB), 2D [1H-1H] TOCSY and 2D [1H-13C] HSQC. The patient derived xenograft models are employed to study real time dynamics using hyperpolarized 1-13C pyruvate by magnetic resonance spectroscopy to distinguish immunotherapy responding vs resistant‘s[1][2]. Results: The analysis of 1D 1H NMR revealed distinct difference in metabolic activity in two different cell lines. The metabolites lactate, alanine and phosphocholine (PC) were altered significantly in the two individual cell lines (Figure 1). Conclusion: This study validates our observation that altered concentration of lactate, alanine and phosphocholine (PC) are correlated to the immunotherapy resistant individuals. The in vivo study in murine derived tumors from these two individual cell lines to identify metabolic biomarkers that can potentially be employed for MR imaging to classify patients responsive to immunotherapy is in progress.

Figure 1: Comparative bar plots of metabolites showing marked difference across two cell lines: I) immunoresponsive, B16/BL6 TMT and II) immunoresistant, B16/BL6 3I F4.

Reference:

[1] M. J. Albers, R. Bok, A. P. Chen, C. H. Cunningham, M. L. Zierhut, V. Y. Zhang, S. J. Kohler, J. Tropp, R. E. Hurd, Y.-F. Yen, S. J. Nelson, D. B. Vigneron, J. Kurhanewicz, Cancer research 2008, 68, 8607-8615. [2] Salzillo TC, Hu J, Nguyen L, Pudakalakatti S, et al. (2016) Interrogating metabolism in brain cancer (Book Chapter). Mag Reson Imag Clin in North America 2016 4:687-703.

Functional, morphological and metabolic alterartions associated with trait anxiety Shilpi Modi, Mukesh Kumar, Sanjeev Nara, Divesh Thaploo and Subash Khushu NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. S. K. Mazumdar Marg, Delhi-54, India Anxiety is an aversive emotional and motivational state that can be characterized by harm avoidance behaviour such as worrying, irritability, difficulty to relax, or by predisposition to interpret ambiguous situations as threatening. Anxiety has been found to be associated with many functional consequences such as increased distractibility, attentional bias in favor of threat-related information and hyper-responsive amygdala even for unattended threat-related stimuli. These effects are seen not only in clinical anxiety but also within the normal population as a personality dimension which is generally assessed by measures of trait anxiety such as the Spielberger‘s State-Trait Anxiety Inventory (STAI)1. The STAI consists of two scales – Y2, which measures the participants‘ predisposition to respond anxiously (trait), and Y1, which measures how anxious the subject/participant feels at the moment (state). The literature largely suggests that high levels of anxiety, whether trait or state anxiety, can negatively affect cognitive performance. There are strong evidences that suggest that high anxiety trait as an important risk factor for the development (onset, severity and outcome) of not only depression but also anxiety disorders. Therefore, in order to identify individuals that are at risk for the development of clinical anxiety disorders and depression, identifying hallmarks of anxiety becomes important so that timely preventive interventions (such as exercise training, stress management training, cognitive and behavioural interventions to prevent anxiety and improve self esteem) may be given to them.

Nuclear Magnetic Resonance (NMR) imaging has emerged as a powerful tool in the field of diagnostic and biomedical research in the last few decades and is routinely and widely used as diagnostic imaging techniques under the banner of Magnetic Resonance Imaging (MRI). With the development of faster electronics and other hardware, newer techniques emerged within MRI, namely, blood oxygenated level dependent (BOLD) fMRI, resting state fMRI, diffusion tensor imaging (DTI), magnetic resonance spectroscopy (MRS), Voxel based morphometry (VBM) etc. which have wide applications in neuroscience and other functional systems. Neuroimaging using MRI offers considerable potential for understanding the neuro- pathophysiological mechanisms and biological underpinnings associated with the cognitive deficits associated with sub-clinical trait anxiety2,3,4. The presentation will cover the leads from the preliminary work that has been done to assess the functional, morphological, and metabolic alterations associated with sub-clinical trait anxiety through multi-parametric MR approach. References: 1. Eysenck, M.W., Derakshan, N., Santos, R., Calvo, M.G., 2007. Anxiety and cognitive performance: attentional control theory. Emotion 7 (2), 336–353. 2. Bishop, S.J., 2008. Trait anxiety and impoverished prefrontal control of attention. Nature Neuroscience 12 (1), 92-98. 3. Donzuso G, Cerasa A, Gioia MC, Caracciolo M and Quattrone A. The neuroanatomical correlates of anxiety in a healthy population: differences between the State-Trait Anxiety Inventory and the Hamilton Anxiety Rating Scale. Brain and Behavior 2014; 4(4): 504–514.

4. Grachev, I.D., Apkarian, A.V., 2000. Anxiety in healthy humans is associated with orbital frontal chemistry. Molecular Psychiatry 5, 482–488.

Investigation of Thermostabilities and Dynamic behaviors of Proteins from Thermophilic, Mesophilic, and Psychrophilic Bacteria

Min-Cheol Jeong, Yeongjun Lee, Dasom Cheon, Geun Sang Yang, Daeseop Guem, and Yangmee Kim Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea Flexibilities in protein conformations are essential for their functions, playing key roles in molecular recognition, rate-limiting conformational transitions, and catalysis. Motion in protein is essential for its ability to interact with functionally different proteins or nucleic acids. Acyl carrier protein (ACP) ACP has to interact with functionally different enzyme partners in the rapid delivery of acyl chain from one partner to another in fatty acid synthase (FAS). The overall structure of ACPs shows the common ACP folding pattern consisting of four α-helices connected by three loops. However, the thermostabilities of ACPs from thermophilic, mesophilic, and psychrophilic ACP are very different. Cold-shock proteins (Csps)—proteins expressed when ambient temperature drops below growth-supporting temperature—bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share high primary sequence homology and a high degree of three-dimensional structural similarity. In this study, we will discuss the key factors in the thermostabilities of these proteins from thermophilic, mesophilic, and psychrophilic bacteria. Molecular details of dynamics in these proteins provide insights into the communication with protein partners and nucleic acids for their functions.

The complex binding mode of relaxin for its receptor RXFP1

Ashish Sethi1,2, Shoni Bruell1,2,3, Samantha Lagaida1,2, Nitin Patil3,4, Akhter Hossain3,4, Daniel Scott1,3, Emma Petrie1,2, Ross Bathgate1,3, Paul Gooley1,2

1Department of Biochemistry & Molecular Biology, 2Bio21 Molecular Science & Biotechnology Institute, 3Florey Institute of Neuroscience & Mental Health, 4School of Chemistry, the University of Melbourne, Parkville, Victoria, Australia 3010.

The ectodomain of the relaxin GPCR receptor, RXFP1, comprises an N-terminal LDLa module, essential for activation (1), tethered to a leucine-rich repeat (LRR) domain by a 32- residue linker. Activation is proposed to proceed by relaxin binding with strong affinity to the LRR domain and then, through an unknown process, enable the LDLa module to bind and activate the transmembrane domain. However, we have found mutations within a conserved region of the linker immediately C-terminal to the LDLa module (GDNNGW, residues 41- 46) that significantly weaken relaxin affinity, suggesting an additional binding site. Using NMR spectroscopy and titrations of 15N-labelled LDLa-linker with relaxin or a paramagnetic (Mn2+) labelled relaxin we have elucidated a discrete relaxin-binding site (residues 46-63) on the linker (2). Additional NMR experiments show residues 49-52 of the linker have a weak propensity for helix which on relaxin titration stabilizes. Mutations within GDNNGW show loss of relaxin-binding and an increase in structural disorder around this helical region of the linker, suggesting an indirect role in binding relaxin. We engineered a soluble protein scaffold (3) to include the transmembrane domain exoloop-2 and show that the LDLa-linker interacts specifically with this loop. We propose two binding sites for relaxin are required: one on the LRR domain, the other on the linker. Mechanistically, the LDLa-linker may weakly interact with the transmembrane exoloops; this is intensified by relaxin binding which stabilizes a helical conformation within the linker. Stabilization of this helix positions the LDLa module to interact and activate the transmembrane domain of RXFP1. 1. Scott, D.J., Layfield, S., Yan, Y., Sudo, S., Hseuh, A.J., Tregear, G.W. and Bathgate, R.A.D. Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J. Biol. Chem. 2006 281, 34942-34954. 2. Sethi, A., Bruell, S., Patil, N., Hossain, M.A., Scott, D.J., Petrie, E.J., Bathgate, R.A.D. and Gooley, P.R. ―The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1. Nature Communications 2016 7:11344, 1-12, 3. Diepenhorst, N.A., Petrie, E.J., Chen, C.Z., Wang, A., Hossain, M.A., Bathgate, R.A.D. and Gooley, P.R. ―Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)‖ J. Biol. Chem. 2014, 289, 34938-52.

Mechanistic Insights into Chemokine Glycosaminoglycan Interactions

*Krishna Mohan Poluri Department of Biotechnology and Center for Nanotechnology, Indian Institute of Technology Roorkee Roorkee 247667, Uttarakhand, India *Email: [email protected] / [email protected]

Chemokines are a group of chemotactic cytokines that are involved in regulating leucocyte migration to the infected tissue. They do so by binding to glycosaminoglycans (GAGs) on the endothelial cell surface and thus activating the G-protein coupled receptors (GPCR) present on the leucocytes. Glycosaminoglycans (GAGs) such as heparan sulfate are highly negatively charged linear polysaccharides. They are ubiquitously expressed on cell surfaces, and mediate a wide variety of biological functions. Neutrophil activating chemokines (NACs) are one of the major classes of CXC chemokines recognized by their highly conserved ―ELR‖ motif that recruit neutrophils at the site of infection. Growth related Oncogene (Gro) chemokines, subfamily NACs, consisting of three members CXCL1 (GRO-), CXCL2 (GRO-) and CXCL3 (GRO-). In the current study, a variety of Protein NMR spectroscopy and biophysical techniques were employed to decipher the oligomerization potencies of GRO chemokines in their native form and in the presence of synthetic GAGs/GAG-mimetics. These studies indicated that the charge of sulfation alone is not sufficient for shifting the Gro chemokine oligomerization equilibrium. The positioning of sulfates, chain length and backbone saccharide conformation etc, of GAGs significantly contributes to their binding to GRO proteins. Mutational Studies indicated that the dynamic side chain interactions of the unstructured polypeptide chain of GRO proteins can potentially regulate the GAG induced oligomerization. These data together suggest that GAG-bound oligomers regulate in-vivo neutrophil trafficking by multiple mechanisms including, defining the gradient formation and increasing the life time of ‗active‘ chemokines via homo/hetero oligomerization on the endothelial cell surface for sustained recruitment. Further, this study promises to aid in creation of chemokine and GAG based decoys to circumvent the endogenous GAG based chemokine recruitment in numerous infectious/inflammatory diseases.

References:

1. Gulati K and Poluri KM* ―Mechanistic and therapeutic overview of glycosaminoglycans: the unsung heroes of biomolecular signaling‖, Glycoconjugate Journal 33(1), 1-17, 2016. 2. Poluri KM, Joseph PR, Sawant KR and Rajarathnam K, ―Molecular Basis for Glycosaminoglycan binding to Chemokine CXCL1 Dimer‖. ‖, J Biol Chem., 278(22), 19980-19985, 2013. 3. Gulati K and Poluri KM* ―Deciphering the in vitro homo and hetero oligomerization characteristics of CXCL1/CXCL2 chemokines‖, RSC Advances 6, 28213-18, 2016.

Microwave Heating and Energy Flow Mechanisms of Liquid Crystalline Systems as Revealed by Microwave Irradiation Solid-State NMR Spectroscopy

Akira Naito1, Yugo Tasei, Fumikazu Tanigawa1, Izuru Kawamura1, Teruaki Fujito2, Motoyasu Sato3

1Graduate School of Engineering, Yokohama National University, Yokohama, Japan. 2Probe Laboratory Inc., Tokyo, Japan. 3Faculty of Engineering, Chubu University, Kasugai, Japan

Microwave (MW) heating effects are widely used in the acceleration of organic and enzymatic reactions. These effects are primarily caused by the dielectric loss induced by MW irradiation. However, the detailed molecular mechanisms associated with MW heating effects on the chemical reaction have not yet been well understood. This study investigated the MW heating effect of N-(4-methoxybenzyliden)-4-butylaniline (MBBA) in liquid crystalline and isotropic phases by analyzing 1H NMR spectra using in-situ MW irradiation solid-state NMR.

A microwave irradiation solid-state NMR spectrometer was equipped with a MW generator transmitting 1.3 kW pulsed or continuous MW at a frequency of 2.45 GHz. MWs were transmitted from the MW generator to the magnet through the MW wave guide, and finally to the resonance circuit at the probe head [1].

When MW of 130 W was irradiated for 90 s at 20 ºC, a small amount of isotropic phase appeared within the bulk liquid crystal at 35 ºC which is lower than the phase transition temperature of 41 ºC (Fig. 1) [2] The partial transition to the isotropic phase can be attributed to a non-equilibrium local heating state induced by the MW irradiation. The MW at 195 W was irradiated for 5 min to isotropic MBBA at 50 ºC and the sample temperature raised to

160 ºC. However, CH3-O, and CH=N protons showed the temperatures at 220 and 350 ºC, respectively. The different protons showed significantly different temperatures in the same molecule (Fig. 1) [2].

Fig. 1. MW heating processes of liquid crystalline and the molecular MBBA [2]. A nematic-isotropic phase-correlated 2D NMR spectra were successfully obtained in 4‘- ethoxybenzylidene-4-n-buthylaniline (EBBA) using MW irradiation solid state NMR and local dipolar fields of 11 magnetically different protons in EBBA in the nematic phase were 39 separately observed in the F1 dimension by means of well-resolved signals in the isotropic phase [3].

References

[1] Y. Tasei et al. J. Magn. Reson. (2015) 254, 27-34. [2] Y. Tasei et al. Phys. Chem. Chem. Phys. (2015) 17, 9082-9089. [3] A. Naito and Y. Tasei Materials Science and Technology (2010) 2886-2894.

13C-1H Dipolar Couplings and Molecular Order of Mesogens: Direct Evidence for Molecular Topology

T. NARASIMHASWAMY

Polymer Science and Technology, Central Leather Research Institute, Adyar, Chennai 600 020, India.

Thermotropic Liquid Crystals are important class of molecular materials. An increasing interest in these materials is witnessed due to their applications in many avenues. One of the most important design parameters of mesogens is the molecular topology which greatly influences the packing in the liquid crystalline phase leading to various mesophase morphologies. Accordingly, rod-like, polycatenar, bent-core, -shape, U-shape etc. topologies of mesogens lead to nematic, smectic, columnar, banana phases with interesting characteristics. We have been actively involved in the molecular design and synthesis of topologically variant mesogens and the study of their structure-property relationship. Solid state 13C NMR studies of these materials led to atomistic information of the mesogens in their mesophase contributing to an understanding of the orientational constraints of the core as well as the terminal chains. The 2D SLF NMR measurements particularly provided the 13C- 1H dipolar couplings that are directly correlated to local order from which the information about molecular topology is deduced. Further, the replacement of phenyl rings in the mesogenic core with thiophene led to dramatic changes in mesophase properties like phase transition temperatures. Also the 13C-1H dipolar couplings of thiophene rings led to crucial information about the molecular shape of mesogens in liquid crystalline phase. The talk will focus on the importance of 13C-1H dipolar couplings in the study of structurally different novel mesogens and the kind of information deduced from such studies. The presentation will highlight the advantages of 13C NMR spectroscopy over other tools that are routinely used for structural as well as mesophase characterization of thermotropic liquid crystals.

Multinuclear Solid-state NMR Investigations on Sr1-xNaxSiO3-0.5x: A Family of Superior Ion Conductors

Bholanath Pahari,§ P. Lokeswara Rao,†,₶ Shivanand M,¶ Tukaram Shet, ¶ and K. V. Ramanathan†

§Department of Physics, Goa University, Taleigao Plateau, Goa-403206, India †NMR Research Center & ₶Physics Department, Indian Institute of Science, Bangalore- 560012, India ¶Materials Research Centre, Indian Institute of Science, Bangalore-560012, India email – [email protected]

Sr1-xNaxSiO3-0.5x family of material is recently been reported as superior ion conducting materials. However, there is a considerable amount of uncertainty and debates about the values, nature, and origin of the ion conductivity in these materials. We intend to demonstrate the unique capability of solid-state NMR to address those unsolved issues.

References: 1. P. Singh and J. B. Goodenough, J. Am. Chem. Soc.,2013, 135, 10149. 2. I. R. Evans, J. S. OEvans, H. G. Davies, A. R. Haworth, M. L. Tate, Chem. Mater., 2014, 26, 5187. 3. C. Tealdi, L. Malavasi, I. Uda, C. Ferrara, V. Berbenni, P. Mustarelli, Chem. Commun., 2014, 50, 14732. 4. Y. Jee, X. Zhao, K. Huang, Chem. Commun., 2015, 51, 9640. 5. J. R. Peet, C. M. Widdifield, D. C. Apperley, P. Hodgkinson, M. R. Johnson, I. R. Evans, Chem. Commun., 2015, 51, 17163.

Structural characterisation of novel and engineered bioactive disulfide-rich peptides by advanced solution state NMR spectroscopy

M. Mobli

Centre for Advanced Imaging, The University of Queensland, St. Lucia, QLD 4072, Australia

Disulfide-rich peptides (DRPs), including hormones, toxins and antimicrobial peptides, are short polypeptide chains (peptides) that fold into defined three-dimensional structures.1 They are secreted molecules that have naturally evolved high potency and selectivity, to regulate biological processes while remaining stable in the harsh extracellular milieu. DRPs owe their remarkable stability to formation of multiple, covalent, disulfide bonds between the side chains of different cysteine residues that act to ―tie‖ the molecule into well-defined ―scaffolds‖.

Here I will present work on (1) the production and isotope labeling of disulfide rich peptides, including the production of segmentally labelled two domain peptides, (2) application of non- uniform sampling in structural characterisation of these peptides2 and finally (3) the interaction of these peptides with both their receptors and to membrane bilayers.3

References

1. Klint, J. K.; Chin, Y. K. Y.; Mobli, M., Rational engineering defines a molecular switch that is essential for activity of spider-venom peptides against the analgesics target NaV1.7. Molecular Pharmacology 2015, 88 (6), 1002-1010.

2. Mobli, M., Reducing seed dependent variability of non-uniformly sampled multidimensional NMR data. Journal of Magnetic Resonance 2015, 256, 60-69.

3. Lau, C. H. Y.; King, G. F.; Mobli, M., Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels. Scientific Reports 2016, 6, 34333.

Influence of aromatic side-chains in biomolecular structure and dynamics Bharathwaj Sathyamoorthy Indian Institute of Science Education and Research, Bhopal

Aromatic side-chains in biomolecules are active participants of diverse interactions, the most prominent being pi-pi networks in proteins, stacking and H-bonding in nucleic acids. Early studies of aromatic ring flips in hydrophobic core of bovine pancreatic trypsin inhibitor (BPTI) unraveled the molecular plasticity of proteins1. More recently, purine bases in DNA, thought previously as rigid entities within the Watson-Crick double helical framework, were observed to undergo ring flips that result in an alternate "Hoogsteen" H-bonding pattern2. This presentation will illustrate application of aromatic chemical shifts and RDCs towards characterization of biomolecules. In particular, aromatic RDCs help in assessing protein core modeling in structures and characterize high-energy conformational states accessed during ring-flip excursions in BPTI3. Also, structural and dynamic preferences of a modified duplex DNA that comprises of a single Hoogsteen base pair will be presented4.

References:

[1] NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor. Wüthrich K, Wagner G. FEBS Lett, 1975, 50(2):265-8. [2] Transient Hoogsteen base pairs in canonical duplex DNA. Nikolova EN, Kim E, Wise AA, O'Brien PJ, Andricioaei I, Al-Hashimi HM. Nature, 2011, 470(7335):498-502. [3] Protein conformational space populated in solution probed with aromatic residual dipolar 13C-1H couplings. Sathyamoorthy B, Singarapu KK, Garcia AE, Szyperski T. Chembiochem, 2013,14(6):684-8. [4] Characterizing sequence-dependent structural and dynamic perturbations in duplex DNA induced by m1A•T Hoogsteen base pairs using solution NMR. Bharathwaj Sathyamoorthy, Huiqing Zhou, Honglue Shi, Yi Xue, and Hashim M. Al-Hashimi. Manuscript under preparation

Abstract category:

First preference: Biomolecular Structure and Dynamics (solution or solid state)

Second preference: Nucleic Acids/Protein-Nucleic Acid Interactions

High-power 1H CPD provides artifact free exchange-mediated saturation

transfer experiments

Kalyan S. Chakrabarti1, David Ban2, Supriya Pratihar1, Jithender G. Reddy1, Stefan Becker1,

Christian Griesinger1, Donghan Lee1,2

1Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

2James Graham Brown Cancer Center, Department of Medicine, University of Louisville, 505 S. Hancock St., Louisville, KY 40202, USA

Email: [email protected]

Abstract Exchange-mediated saturation transfer (EST) provides critical information regarding dynamics of molecules. In typical applications EST is studied by either scanning a wide range of 15N chemical shift offsets where the applied 15N irradiation field strength is on the order of hundreds of hertz [1], or scanning a narrow range of 15N chemical shift offsets where the applied 15N irradiation field-strength is on the order of tens of hertz [2] during the EST period. The 1H decoupling during the EST delay is critical as incomplete decoupling causes broadening of the EST profile, which can result in inaccuracies of the extracted kinetic parameters and transverse relaxation rates. Currently two different 1H decoupling schemes have been employed, intermittently applied 180 pulses [1] and composite-pulse decoupling (CPD) [2], for situations where a wide range or narrow range of 15N chemical shift offsets are scanned, respectively. We show that high-power CPD provides artifact free EST experiments, which can be universally implemented regardless of the offset range and irradiation field- strengths [3]. References: [1] N.L. Fawzi, J. Ying, R. Ghirlando, D.A. Torchia, G.M. Clore (2011) Nature 480, 268- 272. [2] P. Vallurupalli, G. Bouvignies, L.E. Kay (2012) J. Am. Chem. Soc. 134, 8148-8168. [3] K.S. Chakrabarti, D. Ban, S. Pratihar, J.G. Reddy, S. Becker, C. Griesinger, D. Lee (2016) J. Magn. Reson. 269, 65-69.

Interactions between non-coding RNA and TLS/FUS, and distinct difference in enzymatic behavior among APOBEC3 proteins as revealed by real-time NMR monitoring

Masato Katahira

Institute of Advanced Energy, Kyoto University, Japan

Translocated in liposarcoma protein (TLS/FUS) plays an important role in the regulation of cyclin D1 (CCND1) transcription. TLS is recruited to the CCND1 promoter by the interaction with the promoter-associated non-coding RNA (pncRNA). We determined the sequence of pncRNA. The secondary structure of pncRNA was determined by NMR. It was revealed that the third RGG motif (RGG3) of TLS preferably interacts with the 5′-region than the 3′-region of pncRNA (1). The mode of the interaction of TLS with pncRNA was deduced. TLS also works as a key regulator of telomere lengthening via the specific binding with G- quadruplexes of telomeric DNA (teloDNA) and telomeric repeat-containing RNA (TERRA). Formation of the ternary complex results in recruitment of histone methyl transferases to histone, which leads to the repression of telomere elongation. RGG3 was indicated to play the central role in binding with G-quadruplexes. The mode of the interaction of TLS RGG3 with teloDNA and TERRA quadruplexes was deduced.

We developed the method to monitor the deamination reaction by APOBEC3G protein with real-time NMR monitoring (2-4). This time we applied this method to the other APOBEC3 family protein. Unexpectedly, distinct difference in enzymatic behavior was found among APOBEC3 proteins. Rational interpretation of the newly found enzymatic behavior is given.

(1) Yoneda, R. et al. Cell Biosci. 2016, 6, 4. (2) Furukawa, A. et al. Angewandte Chem, 2014, 53, 2349-2352. (3) Kamba, K. et al. PLoS One 2015, 10, e0124142. (4) Kamba, K. et al. Front. Microbiol. 2016, 7, 587.

Elucidating the small RNA mediated gene silencing in A. thaliana

C. Sai Chaitanya, Upasana Rai, Ramdas Aute, Mandar V Deshmukh

CSIR – Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India

Plants exhibit a very elaborate small RNA mediated gene regulation mechanism compared to other higher eukaryotes such as nematodes (C. elegans), insects (D. melanogaster) and primates (H. sapiens). For example, A. thaliana relies on four Dicer-like RNase III enzymes (DCL1-4) that pairs with unique dsRBPs (DRB1-4) and the trigger dsRNA. The Dcr:DRB:dsRNA pair is indispensable for specificity in the mRNA biogenesis as these genes do not complement each other. The lack of adaptive immunity and the polyploidy have perhaps allowed such a diverse small RNA pathway in plants. Here, we provide structural and mechanistic details for the divergence in the plant RNAi pathway.

The studies on individual and concatenated DRB4 dsRBDs demonstrate that the DRB4- dsRBD1 bind to dsRNA with higher affinity than DRB4-dsRBD2 because of favorable structural orientation and ms- -domain orientation determined using PREs and validated by SAXS, suggests that the eight amino acid long linker restrict the mobility of dsRBD leading in further reduction in its dsRNA binding activity. Our study deciphers functional roles of DRB4 domains by showing that dsRBD1 drives the tasiRNA/siRNA pathway. Studies on DRB1, DRB2 and DRB3 suggest that only one dsRBD is recruited for dsRNA binding implying that the non-functional domain may mediate protein:protein interactions. Results elaborate on the divergence in seemingly conserved and highly homologous proteins implying a fine balance in which subtle changes can ―make or break‖ the small RNA mediated gene silencing in plants.

Sliding and intersegmental transfer along DNA enhance the enzymatic activity of APOBEC3G as revealed by real-time NMR monitoring methods

Keisuke Kamba, Takashi Nagata, Masato Katahira

Institute of Advanced Energy, Kyoto University

Human enzyme APOBEC3G (A3G) modifies retroviral genome by converting cytosine (C) into uracil (U) within single-stranded DNA (ssDNA). This A3G-mediated hypermutation inactivates HIV-11. Deamination activity of A3G is highly specific; A3G favors the third position of a triplet cytosine residues (CCC, underlined), and A3G possesses a deamination bias called 3'→5' polarity; A3G edits CCCs located closer to the 5'-end more effectively than one less close to the 5'-end2. We have been characterizing deamination mechanism of A3G involving NMR based enzymatic assay, so called ―real-time NMR monitoring method‖. Using this method we previously found that the 3'→5' polarity is caused by sliding2. Here we analyzed the deamination activity of A3G against ssDNA substrates, containing one (or two) CCC(s), in several buffers with different salt and pH conditions3, 4. Firstly, we found that the electrostatic interaction between positively charged residues of A3G and phosphate backbone of ssDNA is dominant over other interactions such as those involving base and sugar moieties for sliding3, 4. Then it turned out that the addition of ssDNA lacking CCCs to the ssDNA substrates increases the deamination efficiency by A3G. These results indicate that intersegmental transfer as well as sliding enhance the deamination activity of A3G. We will also provide the possible application of our real-time NMR monitoring method to different systems in our presentation. Figure The picture of sliding and intersegmental transfer

1. Harris, R. S.; Liddament, M. T. Nat. Rev. Immunol., 2004, 4, 868–877. 2. Furukawa, A.; Sugase, K.; Morishita, R.; Nagata, T.; Kodaki, T.; Takaori-Kondo, A.; Katahira, M. Angew. Chem. Int. Ed. Engl., 2014, 53, 2349–2352. 3. Kamba, K.; Nagata, T.; Katahira, M. PLoS One, 2015, 10, e0124142. 4. Kamba, K.; Nagata, T.; Katahira, M. Front. Microbiol., 2016, 7, 587.

Solid-state NMR of membrane proteins: Application to the chemokine IL-8 and its receptor CXCR1

Stanley J. Opella

Department of Chemistry and Biochemistry

University of California, San Diego

La Jolla, California 92037 U.S.A.

Both magic angle spinning (MAS) and oriented sample (OS) solid-state NMR methods enable the study of membrane proteins in the near-native environment of phospholipid bilayers under physiological conditions. The human genome encodes 800 G-protein coupled receptors (GPCRs), making this the largest class of membrane proteins. They are involved in a wide range of biological signaling, resulting in their being the targets of one-third or more of all drugs used in medicine. 50 chemokines have been identified; they are small, globular proteins involved in the innate immune system. IL-8 is the first one to have been characterized, and the structure of a monomeric form in aqueous solution will be described. IL-8 binds to CXCR1, the first chemokine receptor to have been identified, and its structure in phospholipid bilayers will be described. Recent results on complexes that include IL-8, CXCR1 and G-proteins provide a step towards understanding the molecular basis for the recognition and activation of the receptor, which triggers intracellular events that result in directed chemotaxis of neutrophils to sites of infection and injury.

New structural insight of Proteoglycans, dimerization and molecular function related to melanoma cell migration and metastasis Ji-Hye Yun1, Youngsil Choi2, John R. Couchman3, Eok-Soo Oh2, and Weontae Lee1*

1Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Korea

2Department of Life Sciences, Division of Life and Pharmaceutical Sciences and the Research Center for Cellular Homeostasis, Ewha Womans University, Korea

3Department of Biomedical Sciences, University of Copenhagen, Biocenter, 2200 Copenhagen, Denmark

Syndecans are type I transmembrane heparan sulfate proteoglycans found on most eukaryotic cell surfaces, which comprising a large extracellular domain, a single transmembrane domain, and a short cytoplasmic domain. The extracellular domain is heavily glycosylated with heparan sulfate chains and cytoplasmic domain functionally interacts with numerous soluble ligands/proteins for its optimum function. The syndecan cytoplasmic domain forms a compact intertwined dimer with a symmetrical clamp shape and two antiparallel strands are twisted with a cavity. NMR data also revealed the detailed inter-molecular interactions between transmembrane domains of syndecans in solution. Since syndecans lack any intrinsic signaling capacity, the interactions of their cytoplasmic domains with various adaptor proteins can play essential regulatory roles. These adaptor proteins include the postsynaptic density protein, disc large, and the zonula occludens (PDZ) domain-containing scaffold protein, syntenin-1, which can link the cytoplasmic domain of certain syndecans to the cytoskeleton. Syndecan/syntenin-1 complex shows an unusual symmetrical pair of dimers anchored by a syndecan-4 dimer, inhibiting the functions of syndecan-4. We have shown that multi-merization of both syndecan and syntenin-1 is crucial for the negative regulation of proteglycan signaling. I will also discuss structure and dynamics of homo- and hetero- oligomerization of syndecan mediated by transmembrane domains in solution, lighting molecular insight into the cancer signaling mechanisms by syndecan oligomerization. Our findings propose that three-dimensional structure of syndecan proteoglycan mainly governs its cellular function such as cytoskeletal organization and cell migration.

1. Choi Y. et al., Scientific Reports (2016) In Press 2. Kwon M. et al., J. Biol. Chem. (2015), 290, 9, pp5772 3. Choi Y. el al., J. Biol. Chem. (2015), 290, 27, pp16943

Preferred Conformational States of Retroviral Capsid Protein Visualized

Chun Tang, Wen-Xue Jiang, Xu Dong, Zhou Gong, Zhu Liu

CAS Key Laboratory of Magnetic Resonance in Biological Systems, National Center of Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences

Discovered in 1901, the Rous sarcoma virus (RSV) is a prototypical retrovirus. Several thousand copies of the RSV capsid (CA) protein interact and form a nanoparticle in various morphologies, encapsidating the viral genome. A RSV CA comprises two domains, namely the N-terminal domain (NTD) and the C-terminal domain CTD, connected by a short linker. The CA assembly has been characterized with cryoEM, which reveals specific interactions between NTD and CTD and between CTD and CTD from adjacent CA proteins. In solution, NMR studies have indicated the two domains of an individual CA protein tumble independently. Nevertheless, the pathway from CA protein to CA assembly is not clear. Here I will present our recent work depicting the structure and dynamics of RSV CA protein, with conjoined use of fluorescence resonance energy transfer (smFRET), NMR lanthanide pseudo- contact shift (PCS), and other solution-based biophysical techniques. We found that an RSV CA protein dynamically interconvert among three conformational states with different arrangements for the two domains; they are open, compact and closed states. The compact state is responsible for the formation of spherical and tubular CA assembly, while the closed state can be dissipated upon the addition of unlabeled RSV CA protein through intermolecular NTD-CTD interactions. Thus the quaternary arrangement of each building block dictates the morphology of the final assembly product.

Analyses of Multi-Protein Complexes in Beta-2 Integrin Signaling

Surajit Bhattacharyya

School of Biological Sciences, Nanyang Technological University, Singapore 637551

Integrins, hetero-dimeric (alpha and beta subunits) signal-transducer proteins, are essential for cell adhesion and migration. Beta cytosolic tails (beta-CTs) of integrins interact with a number of cytosolic proteins including talin, Dok1, 14-3-3z and filamin. The formation of multi-protein complexes with beta-CTs is involved in the activation and regulation of integrins. However, the sequence of event of interactions remains unclear. The leukocyte- specific beta2 integrins are essential for leukocyte trafficking, phagocytosis, antigen- presentation and proliferation. We are examining and characterizing molecular interactions of beta2-CT and alpha CTs with cytosolic proteins, in binary and ternary complexes by NMR and biophysical methods. We find novel interactions that may have strong implications in integrin signalling mechanism. Our current results demonstrated (1) talin head domain and 14-3-3z form a stable ternary complex with phosphorylated beta2-CT, (2) Dok1, a negative regulator of activation, binds to a non-canonical motif in beta2-CT, (3) direct interactions between 14-3-3z and Dok1 or 14-3-3z/filamin modulate recruitment of positive and negative regulator to the b2-CT. These observations help in building new models of regulation of integrins.

Motion, recognition and interaction of proteins involved in the cell-cycle regulation

Ashutosh Kumar Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India Precisely regulated protein function is vital for most of the cellular processes. Such fidelity in regulation is required for prevention of premature and adverse interactions among various signaling cascades in cellular compartments to ensure that binding is spatially and temporally appropriate. In real scenario, proteins do not adopt a unique structure, but are more aptly described by an equilibrating set of time-dependent structures. Distinct conformations of proteins are continuously sampled within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and fem to seconds to hours. Understanding the structural bases of various dynamic and regulatory processes is important for realizing the full spectrum of macromolecular function.

NMR spectroscopy is one of the elegant tools for characterization of biological macromolecules with its unique ability to provide a coherent insight into the structure and intrinsic dynamics. In this presentation, I will talk about the dynamic regulation of proteins involved in the cell cycle control, Skp1and its interacting partners F-box proteins, at residue- level using NMR methods and molecular dynamics simulations.

Subsequently, I will discuss about the recognition and regulation that could take place in the SUMO machinery upon plasmodium infection to human.

Structural basis for the biased interactions between auxin-response transcriptional machinery

Mookyoung Han, Youngim Kim, Migyeong Jeong, and Jeong-Yong Suh

Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea

The plant hormone auxin regulates various developmental processes in plants, including morphogenesis, organogenesis, vascular differentiation, and tropic responses to environmental stimuli such as light and gravity. Transcriptional regulation by auxin is mediated by the auxin response transcription factor (ARF) and the transcriptional repressor (Aux/IAA). Aux/IAA associates with ARF via conserved domains III‒IV for transcriptional repression that is reversed by auxin-induced Aux/IAA degradation. Given that ARFs per se are classified into transcription activators and repressors, it has been intriguing whether Aux/IAA would interact with ARF repressors as well as ARF activators. Recent structures of domain III‒IV revealed a PB1 -grasp fold that is well conserved among different ARF activators and Aux/IAAs. Here we show that ARF repressors also adopt a similar PB1 fold of domain III‒IV, but present much weaker affinity to Aux/IAA. Differences in the affinity originates from a unique strategy that modulates the binding interface without perturbing the backbone fold. Our finding suggests that Aux/IAA preferentially associates with ARF activators and avoid futile association with ARF repressors, which would help prompt response to cellular Aux/IAA accumulation when auxin is depleted.

Han, M., Park, Y., Kim, I., Kim, E. H., Yu, T. K., Rhee, S., and Suh, J. Y. (2014) Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17. Proc. Natl. Acad. Sci. USA 111, 1861318618.

Is partial unfolding of native proteins a necessary, inaugural step in amyloidosis?

Jitendra K. Das, Aritra Bej, Shyam S Mall and Sujoy Mukherjee

Structural Biology and Bioinformatics division. CSIR - Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Kolkata, West Bengal 700 032 The conversion of soluble protein into insoluble amyloid fibrils is the hallmark of a large variety of protein aggregation diseases. Among the important factors responsible for initiating this process, protein‘s conformational dynamics is believed to play a key role in the conversion of native proteins via formation of partially unfolded non-native intermediates.1 In recent work,2 we have probed the role of backbone conformational flexibility in the amyloid forming protein, transthyretin (TTR). The wild type as well as mutant forms of TTR are implicated in neuropathies, cardiomyopathy, ocular, leptomeningeal and other forms of amyloidosis.3 We have investigated motions from fast (picoseconds-nanosecond) to slower timescales (~milliseconds) using NMR and microsecond-scale, atomistic MD simulations and found the existence of non-native intermediates in exchange with the native TTR. Our results show that these non-native conformers are formed more efficiently for mutant, pathogenic TTRs due to lowering of activation energy (i.e. kinetic barrier) as well as the free energy difference between the native and non-native states (i.e. thermodynamic barriers) in comparison to wild type protein. This suggests a correlation between the known pathogenicity of TTR mutants with their formations of non-native intermediates. This results in reshaping of energy landscape in the amyloid forming pathway with unique features not observed in other protein aggregation diseases. Finally, we obtain the change in heat capacities between native and non-native states to show that for certain but not all pathogenic mutants, the initial, non-native state is partially unfolded in nature.

References.

1. Chiti, F.; Dobson, C. M., Amyloid formation by globular proteins under native conditions. Nat Chem Biol 2009, 5, (1), 15-22.

2. Das, J. K.; Mall, S. S.; Bej, A.; Mukherjee, S., Conformational flexibility tunes the propensity of transthyretin to form fibrils through non-native intermediate States. Angew Chem Int Ed Engl 2014, 53, (47), 12781-4.

3. Saraiva, M. J., Transthyretin mutations in health and disease. Hum. Mutat. 1995, 5, 191-196.

Determining the critical molecular interaction responsible for tau fibril formation

Jiji A.C and Vinesh Vijayan

School of Chemistry, IISER Thiruvananthapuram, Trivandrum 695016, India

Abnormal aggregation of tau protein forming the paired helical filaments (PHFs) is found to be major components of neurofibrillary tangles (NFTs) in nerve cell that is considered a pathological marker for Alzheimers disease. The R2 and R3 repeats of tau protein that houses the hexapeptide region are known to initiate the tau fibril formation. Despite recent progress in understanding the structure of tau fibrils through solid-state NMR1,2, the high- resolution structure of the tau fibril is still not available. Hence the key structural interactions that modulate the tau fibril formation remain mostly unknown.

Using different tau repeat construct, we show that in R3, addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. The time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide reveals a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. We identify S316 as the key residue in the turn that provides the templating capacity to R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results3 and studies on the inhibitory interaction existing in longer construct of Tau will also be discussed.

REFERENCE

[1] O. C. Andronesi, M. von Bergen, J. Biernat, K. Seidel, C. Griesinger, E. Mandelkow, M. Baldus, J. Am. Chem. Soc. 130 5922 (2008). [2] V. Daebel, S. Chinnathambi, J. Biernat, M. Schwalbe, B. Habenstein, A. Loquet, E. Akoury, K. Tepper, H. Muller, M. Baldus, C. Griesinger, M. Zweckstetter, E. Mandelkow, V. Vijayan, A. Lange, J. Am. Chem. Soc. 134 13982 (2012). [3] A.C. Jiji, A. Shine and V. Vijayan, Angew Chem. 55 11562 (2016).

Dynamic nuclear polarization with double 460 GHz gyrotrons for the sensitivity enhancement of magic-angle-spinning NMR at 30 K

Toshimichi Fujiwara and Yoh Matsuki

Institute for Protein Research, Osaka University, Suita 565-0871, Japan NMR sensitivity is the crucial factor for studying the structures of large biomolecules and material sufaces. We are improving the instrumentation for high-field dynamic nuclear polarization (DNP) to increase the sensitivity of high-resolution solid-state 13C NMR. Our previous experiments showed that DNP at about 30 K provided much higher polarization than those at about 90 K. At first, these magic-angle-spinning DNP experiments were performed by boiling liquid helium. Thus the large liquid He consumption made the routine DNP experiments difficult. To minimize the He consumption, we have developed a closed He-gas circulation system with an electrical gas chiller for MAS experiments at about 30 K. This circulation system not only reduced the operation cost but also stabilized the gas flow over an experimental time of a week for multidimensional NMR experiments. The large Boltzmann polarization and reduced thermal noise at the low temperature increased the NMR sensitivity. We developed a high-power submillimeter wave irradiation system with two gyrotrons to saturate electron spin resonances of paramagnetic compounds efficiently at 16.4 T. One second-harmonics gyrotron generates a high-intensity 460-GHz wave with a continuously tunable range of about 1 GHz. The other gyrotron provides the submillimeter wave with frequency modulation at the rate about 10 kHz. The submillimeter waves are transmitted to a sample rotor through a diplexer connected to corrugated waveguides. This system was used for double resonance experiments in a submillimeter wave range which should improve the high-field DNP experiments. We will present the experimental results obtained with our DNP-NMR spectrometer.

Acknowledgements Instrumentation for DNP was developed with Research Center for Far-Infrared Region in Fukui University and JEOL RESONANCE Ltd. under the support by JST Sentan and NMR platform, Japan.

Reference

Matsuki, Y.; Idehara, T.; Fukazawa, J.; Fujiwara, T. Advanced instrumentation for DNP- enhanced MAS NMR for higher magnetic fields and lower temperatures, J. Magn. Reson., 2016, 264, 107-115.

TWO TO TANGO, THREE TO JIVE

N. Chandrakumar

MRI – MRS Centre & Department of Chemistry Indian Institute of Technology Madras Chennai – 600036, Tamil Nadu, India

Some results from recent Overhauser Dynamic Nuclear Polarization (DNP-OE, or ODNP) studies performed in our Laboratory at moderately low fields are presented.

DNP-OE has long been known in solution state, and has been investigated on a number of nuclear spins. Of these, 1H and 19F are among the most significant since they lie at the molecular periphery, and tend to polarize relatively rapidly, and to a significant extent. While protons typically exhibit dipolar cross-relaxation with the unpaired electron, leading to negative enhancements, fluorines exhibit mixed scalar and dipolar cross-relaxation with the unpaired electron, leading to positive or negative enhancements respectively.

Following our earlier preliminary studies at 0.35 T on small molecules dissolved in liquid crystals, the applicability of DNP-OE is explored in viscous ionic liquids. DNP-OE experiments typically reflect the cross-relaxation of nuclear spins by unpaired electron spins, leading to a monotonic exponential build-up of nuclear spin polarization under microwave irradiation of the radical ESR transition. It is shown here however that under suitable conditions DNP-OE build-up curves could exhibit extrema. The results are interpreted in terms of a model where the unpaired electron spin interacts relatively weakly with two nuclear spins that cross-relax each other.

The experiment, under these conditions, may be considered as an indirect detection of the Nuclear Overhauser Effect (NOE), and has a number of unique features. Because nuclear spin cross-relaxation is sensitive to molecular motions at the sum and difference of the respective nuclear Larmor frequencies (whereas electron-nuclear cross-relaxation reflects molecular motions essentially at the electron Larmor frequency), the indirect DNP-OE mode of measuring NOE at moderately low fields permits registration of low frequency motions with high dynamic range and sensitivity.

For a nuclear spin pair such as 19F/1H, information about molecular motions at ca. 1 (0.06 nH) and at ca. (1.94 nH) reflects in their cross-relaxation, nH being the H Larmor frequency. This information is now registered starting with an initial state where both nuclear spins are in thermal equilibrium. Motional dynamics in the 0.1 ns – 100 ns range becomes accessible. The experiment is performed in a coil-in-cavity mode to ensure both efficient power transfer of microwaves for ESR irradiation, and efficient NMR excitation and detection at the single NMR RF frequency of relevance. It may be noted that the NMR hardware for this indirect measurement mode of heteronuclear NOE comprises only a single RF channel on the transmitter, probehead and receiver.

DNP Enhanced Solid-State NMR under Fast Magic Angle Spinning

Sachin R. Chaudhari, 1 Dorothea Wisser,1 David Gajan,1 Pierrick Berruyer, 1 Christian Reiter,2 Frank Engelke,2 Christophe Copéret,3 Moreno Lelli,4 Anne Lesage,1 Lyndon Emsley.5

1. Institut de Sciences Analytiques, Centre de RMN à Très Hauts Champs, Université de Lyon (CNRS/ENS Lyon/UCB Lyon 1), France, 2. Bruker Biospin, 76287 Rheinstetten, Germany, 3. ETH Zürich, Department of Chemistry, Vladimir Prelog Weg 1-5, CH–8093 Zürich, Switzerland, 4. University of Florence, Chemistry Department, Magnetic Resonance Centre, 50019 Sesto Fiorentino (FI), Italy, 5. Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. Dynamic nuclear polarization (DNP) has recently developed into a powerful analytical technique to enhance the sensitivity of magic angle spinning (MAS) solid-state NMR spectroscopy of biomolecules and materials.1,2 Most developments and applications of DNP MAS NMR were so far reported at moderate spinning frequencies (up to 14 kHz using 3.2 mm rotors). In this presentation, we will first present our recent results using a 1.3 mm MAS DNP probe operating at 18.8 T (800 MHz) and ~100 K. 3 We will show that how the DNP enhancement varies as a function of MAS signal over a range of sample spinning rates from 10 to 40 kHz with the different DNP mechanism such as Cross and Overhauser effects. The contribution of quenching effects to the overall sensitivity gain at very fast MAS will be discussed, and applications on functionalised mesostructured organic-inorganic materials, alumina surfaces will be presented. In particular, we will show that spinning the sample at 40 kHz leads to a significant increase of coherence lifetimes, substantially increasing the sensitivity of CPMG experiments. 3 These new experimental approaches are expected to significantly broaden the applicability of high-field DNP in the future.

References:

1) Q. Z. Ni, E. Daviso, T. V. Can, E. Markhasin, S. K. Jawla, T. M. Swager, R. J. Temkin, J. Herzfeld, R. G. Griffin, Acc. Chem. Res. 2013, 46, 1933. 2) Rossini, A. Zagdoun, M. Lelli, A. Lesage, C. Copéret, L. Emsley, Acc. Chem. Res., 2013, 46, 1942.

3) Sachin R. Chaudhari, P. Berruyer, D. Gajan, C. Reiter, F. Engelke, D. Silverio, C. Copéret, M. Lelli, A. Lesage, L. Emsley, Phys. Chem. Chem. Phys., 2016, 18, 10616.

Recoupling with phase change Navin Khaneja

IIT Bombay

In this talk, we describe some novel recoupling methods in MAS Solid State NMR. These recoupling methods can be employed for both homonuclear and heteronuclear recoupling experiments and are robust to dispersion in chemical shifts and rf-inhomogeneity. Experimental quantification of these methods is provided with detailed theory.

The Microwave Power and Time Dependence of Static DNP induced NMR Signals

Krishnendu Kundu, Marie R Cohen, Akiva Feintuch, Daniella Goldfarb, Shimon Vega.

Chemical Physics Department, Weizmann Institute of Science, Rehovot, Israel

Dynamic nuclear polarization (DNP) is one of the most efficient methods to increase the sensitivity of NMR and MRI by transferring polarization from electron spins to nuclear spins. For radicals in solid solutions with an EPR line-width smaller or greater than the nuclear Larmor frequency the nuclear signal enhancements during microwave irradiation are interpreted either using the thermal mixing (TM)1 mechanism or a combination of the solid- effect (SE)2 and cross-effect (CE)3 mechanisms. Here, we will focus on the CE process in an effort to follow the time-evolution during MW irradiation and the MW power dependence of the DNP induced nuclear polarization. Although the SE and CE based analysis of the lineshape of the enhancement as a function of the MW frequency ωMW has been possible, it was only realized recently that these lineshapes are directly correlated to the electron polarization distribution in the sample. To quantify this correlation a mathematical model was introduced4 based on an electron spectral diffusion (eSD) mechanism and the indirect-CE (iCE). In the present study we have extended the application of the eSD and iCE models in order to interpret simultaneously experimental ELDOR and DNP results as the function of the MW field intensity ωe1 and irradiation time duration tMW. We will demonstrate the validity of the models in interpreting a collection of non-steady state ELDOR and DNP profiles as a function of tMW with only one set of eSD parameters. The ωe1 dependence of ELDOR and DNP profiles will also be shown for samples with different radical concentrations.

References

(1) Hwang, C. F.; Hill, D.A.; Phys. Rev. Lett. 1967, 19, 1011–1013. (2) Jefferies, C. Phys. Rev. 1957, 106, 164–165. (3) Kessenikh, A.V.; Lushchikov, V.I.; Manenkov, A. A.; Taran, Y.V.; Sov. Phys. Solid State. 1963, 5, 321–329. (4) Hovav, Y.; Kaminker, I; Shimon, D.; Feintuch, A.; Goldfarb, D.; Vega, S. Phys. Chem. Chem. Phys. 2015, 17, 226-224.

A Cost-efficient and Fast Technique of Achieving Sustainable Nuclear Spin Hyperpolarization

Soumya S. Roy and Simon B. Duckett

Centre for Hyperpolarisation in Magnetic Resonance (CHyM) Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK

Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) are two extremely important techniques with applications ranging from molecular structure determination to human imaging. However, in many cases the applicability of NMR and MRI are limited by inherently poor sensitivity and insufficient nuclear spin lifetime. Here we demonstrate a cost-efficient and fast technique that tackles both issues simultaneously. We use Signal Amplification by Reversible Exchange (SABRE) technique to hyperpolarize the 1 target H nuclei by enriched para-hydrogen (p-H2) and store this polarization in long-lived singlet (LLS) form after suitable radio frequency pulses. The method is reversible in nature and proven to work for a large class of biologically relevant potential tracer elements. Compared to the normal scenario, we achieve three-orders of signal enhancement and one- order of lifetime extension, leading to 1H NMR signal detection 15 minutes after state creation. The generation of such hyperpolarized long-lived polarization reflects an important step forward in the pipeline to see such agents used as clinical probes of disease.

References:

1. Roy, S. S.; Norcott, P.; Rayner, P. J.; Green, G. G. R.; *Duckett, S. B., M. H., Angew. Chem. Int. Ed. 2016, (in press) DOI: 10.1002/anie.201609186. 2. Roy, S. S.; Rayner, P. J.; Norcott, P.; Green, G. G. R.; *Duckett, S. B., Phys. Chem. Chem. Phys. 2016, 18, 24905-24911. 3. †Olaru, A. M.; †*Roy, S. S.; Lloyd, L. S.; Coombes, S.; Green, G. G. R.; *Duckett, S. B., Chem. Comm. 2016, 52, 7842-7845. 4. Roy, S. S.; Dumez, J.-N.; Stevanato, G.; Meier, B.; Hill-Cousins, J. T.; Brown, R. C. D.; Pileio, G.; *Levitt, M. H., J. Magn. Reson. 2015, 250, 25-28.

Recent advances in NMR diffusometry Wilfred Kittlera, Stefan Andreas Hertela, Marcel Nogueira d'Eurydicea, Mark Huntera,b, Sergei Obruchkova F. Zonga, L.R. Anceletc,d, I.F. Hermansc,d,e, P. Galvosasa aMacDiarmid Institute for Advanced Materials and Nanotechnology, School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington, New Zealand; bMagritek Limited, Wellington, New Zealand; cMalaghan Institute of Medical Research, Wellington, New Zealand; dMaurice Wilkins Centre, Auckland, New Zealand; eSchool of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. Accounting for the influence of molecular motion in NMR experiments is nearly as old as the discovery of NMR itself [1]. However, with the introduction of pulsed magnetic field gradients [2] NMR became a highly versatile tool for studying diffusion and flow in many areas such as physics, chemistry, material science, medical research and clinical routine. This lecture will revolve around new options offered by NMR diffusometry. We will report on a variant of two-dimensional (2D) Diffusion-Diffusion Correlation Spectroscopy (DDCOSY) [3] which allows for the correlation of molecular displacements in different directions, thus sampling local mobility and confinement. This enables one to measure Fractional Anisotropy (FA) averaged over the whole sample which can be extracted from the DDCOSY results using a particular gradient orientation scheme [4]. We will further discuss the potential of sample averaged FA values for the discrimination of healthy and cancerous biological tissue [4]. We will also discuss recent imaging approaches for porous materials which consist of disconnected pores. While this method is still based on the work horse of NMR diffusometry introduced more than 50 years ago [2] deliberate extension of one gradient pulse [5] allows for Magnetic Resonance Pore Imaging (MRPI) at resolutions well beyond the limits of conventional MRI [6]. As time permits we will also report on the use of second order magnetic fields which allow for the parallel acquisition of q-space [7], thus enabling real time monitoring of averaged propagators [8] and single-shot surface-to-volume ratio measurements in porous media [9]. References [1] Hahn, E. L. Phys. Rev. 80, 580–594, (1950). [2] Stejskal, E. O. and Tanner, J. E. J. Chem. Phys. 42, 288 (1965). [3] P.T. Callaghan, S. Godefroy and B.N. Ryland, Magn. Reson. Imaging, 21, 243–248 (2003). [4] F. Zong et al. Magn. Reson. Chem. (Early View) DOI:10.1002/mrc.4492 (2016). [5] Laun, F. B., Kuder, T. A., Semmler, W., and Stieltjes, B. Phys. Rev. Lett. 107, 048102, (2011). [6] Hertel, S. A. et al. Phys. Rev. E 92 012808 (2015). [7] Kittler, W. C., Galvosas, P., and Hunter, M. W. J. Magn. Reson. 244, 46–52, (2014). [8] Kittler, W., Hunter, M., and Galvosas, P. Phys. Rev. E 92, 023016 (2015). [9] Kittler, W. C., Obruchkov, S., Galvosas, P. and Hunter, M. W. J. Magn. Reson. 247, 42– 49, (2014).

Characterizing, detecting and preserving quantum correlations on an NMR quantum information processor

Kavita Dorai

Email:[email protected] Department of Physical Sciences, Indian Institute of Science Education & Research (IISER) Mohali, India.

Quantum entanglement has long been posited to be responsible for the computational speedup of quantum algorithms and its characterization, detection and protection are important areas of current research. It is important, particularly from an experimental point of view, to be able to detect entanglement by a minimum number of experiments, requiring as little effort as possible. A promising direction of research in the detection of quantum entanglement is the use of local observables to find an optimal decomposition of entanglement witnesses. Random local measurements have also been proposed to construct entanglement witnesses and thereby detect the presence of bipartite entanglement. The first part of this talk will discuss the efficacy of one such scheme on a two-qubit NMR quantum information processor, wherein it will be shown that a set of three random local measurements suffices to detect the entanglement of a general two-qubit state [1]. While entanglement is a signature of quantum correlations, there are other nonclassical correlations which are distinct from their classical counterparts and which display several intriguing features. The next part of the talk will focus on how to experimentally detect the presence of such nonclassical correlations using a recently suggested method based on positive maps. Quantum correlations are extremely susceptible to decoherence and hence several schemes have been designed to protect them from decay, one of the most popular being based on dynamical decoupling. The final part of this talk will focus on how to exploit dynamical decoupling sequences to prolong the persistence of time-invariant quantum discord in a system of coupled NMR qubits, decohering in independent dephasing environments [2]. The application of such schemes to preserve the entanglement of maximally entangled three-qubit states such as the W state and the Greenberger-Horne-Zeilinger state, will also be discussed.

Bibliography 1 Singh, A., Arvind, and Dorai, K. (2016) ArXiv E-print 1610.02472v1. 2 Singh, H., Arvind, and Dorai, K. (2016) ArXiv E-print 1610.02755v1.

Quantum bits and their environment T. S. Mahesh Indian Institute of Science Education and Research, Pune 411008 A quantum bit or a `qubit‘ is a two-level quantum system that holds the smallest unit of quantum information. A set of interacting qubits form a quantum register and unitary operations on the register form quantum gates. Spin-systems in solid or liquid ensembles constitute NMR quantum registers and RF controls implement desired quantum gates. I will talk about several aspects related to qubits and their environment. In particular, I will describe recent developments in characterizing noise, engineering noise, and designing robust controls that suppress noise. I will also describe situations where environmental spins help increase the purity of qubits.

Towards Complete Sequence Specific Assignment of Proteins from a Single Experiment: Applications of 13C-Pyruvate labeling, Non-uniform sampling and Pattern Matching at Greater Than 42 kDa.

Scott Robson

Backbone assignment of large proteins is complicated by spectral overlap and line broadening by slow tumbling and chemical exchange. To solve this problem, we have focused our efforts on improving the resolution of the most sensitive experiment for sequential assignment of deuterated proteins: the TROSY-HNCA. While sensitive, there is little chemical shift dispersion over the 13 sequential-to-non-sequential linkages between spin systems. Further, there is a ~32-42 Hz homonuclear coupling between the 13 13 resolution of the 13 ns. Using perdeuterated [2-13C] pyruvate and [3-13C] pyruvate as sole carbon sources during protein expression and non-uniform data collection, we find we can chemically suppress homonuclear couplings and generate a singlet peak at the true frequency of the uncoupled 13 least twice the height of the surrounding coupled peaks. We have successfully acquired TROSY-HNCA spectra of Maltose Binding Protein (MBP, 42 kDa), with line widths for -fold narrower (4.8 Hz) than that possible with regular glucose labeling. Mock automatic assignments using the HNCA spectrum alone at a resolution of 42 Hz versus 4.8 Hz shows a dramatic increase in unique assignments possible at this higher resolution. Additionally, the ratio of the height of the coupled to uncoupled peaks broadly indicates amino acid type. These ratio patterns match sequentially in sequence specific assignment and are readily discernable, even in Malate Synthase G (82 kDa). Using pattern matching we can automatically assign greater than 85% of the assignable MBP residues from a single HNCA experiment collected over ~3 days. We expect to be able to fully automate this process soon where the only inputs to software would be the amino acid sequence and a high resolution TROSY-HNCA spectrum.

Fast Field Cycling NMR Investigations of Slow Dynamics in Soft Materials

B. V. N. Phani Kumar NMR Laboratory, Inorganic & Physical Chemistry Department Council of Scientific and Industrial Research (CSIR)-Central Leather Research Institute (CLRI), Adyar, Chennai-600020, India. [email protected]

Soft materials, in particular, thermotropic and lyotropic liquid crystals, are the two interesting cases, where rich polymorphism of these systems is mainly induced by temperature and concentration, respectively. A proper understanding of molecular dynamics, in particular, slow dynamics, exhibited by thermotropic liquid crystals is beneficial in designing display devices, sensors etc., while such information on associative copolymers (exhibiting lyotropic liquid crystalline polymorphism) throw light on the functionality of biological membranes. Nuclear Magnetic Relaxation Dispersion i. e., nuclear spin-lattice relaxation rate (R1) data as a function of Larmor frequency, when combined with the judicious choice of theoretical models permits to decouple the various molecular motions contributing to spin-relaxation. In this regard, R1 dispersions were made using a Fast Field Cycling (FFC) NMR spectrometer (at Technical University Ilmenau, Germany) in the Larmor frequency range of 10 kHz – 30 MHz and analyzed using suitable models. The results on thermotropic mesogens indicate the role of local organizations on the nematic phase stability as well as geometrical aspects of cybotactic clusters (local smectic organizations), whereas the importance of short-range order fluctuations noted for a triblock copolymer exhibiting lyotropic liquid crystalline nature. Hence, the present talk is focused on the application of FFC NMR with a view to probing slow molecular processes in such soft systems.

New methods for measuring RCSAs and RDCs for Small Molecule Applications

Nilamoni Nath1, Juan Carlos Fuentes1, Manuel Schmidt1, Armando Navarro-Vázquez2, Roberto R. Gil3 and Christian Griesinger1

1Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; 2Institute of Organic Chemistry and Institute for Biological Interfaces Karlsruhe, Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany; 3Department of Chemistry, Carnegie Mellon University, 4400 Fifth Ave, Pittsburgh, PA 15213 (USA) Wouldn‘t it be great to measure the chemical shifts of carbons and determine from these data the configuration of a natural or synthetic compound of which only several tens of micrograms are available? It has long been clear that this could be achieved if one could accurately measure residual chemical shift anisotropy (RCSA) [1,3], e.g. from the resonance positions under two alignment states. However, until now, it was difficult to differentiate the RCSA, i.e. the shift due to molecular alignment, from the isotropic NMR chemical shift change. The latter originates from a change in environment when switching from alignment state to another. Here we describe a technique to collect pure RCSAs which consists of compressing PMMA gel [3] dissolved in CDCl3 inside a 5 mm NMR tube to obtain different alignments. Although, the sensitive volume is not changed, there is a change in gel-to-solvent ratio causing an isotropic contribution that can be removed post acquisition in a new and robust way. The main achievement obtained is the removal of obstacles for accurate measurement of RCSAs, making them available for routine applications. The simplicity of measuring just a simple 13C 1D NMR experiment should make this method available for every chemist. To demonstrate the robustness of the approach, RCSAs were measured on five different molecules some rigid, some flexible. We were able to determine the correct configuration in each case. To the best of our knowledge, this has never been achieved before. The most notable example is retrorsine in which flexibility aggravates the determination of the relative configuration [4]. Finally, new protocol for measuring RCSAs from polyacetylene liquid crystal and new NMR method for measuring single bond 13C-1H RDCs and two bond 1H-1H RDCs will be presented.

References

1. Kummerlowe, G., Grage, S. L., Thiele, C. M., Kuprov, I., Ulrich, A. S., Luy, B. J. Magn. Reson. 2011, 209, 19. 2. N. Nath, E. J. d'Auvergne, C. Griesinger, Angew. Chem. Int. Ed. 2015, 54, 12706. 3. C. Gayathri, N. V. Tsarevsky, R. R. Gil, Chem-Eur J 2010, 16, 3622. 4. N. Nath, M. Schmidt, R.R. Gil, R.T. Williamson, G.E. Martin, A. Navarro-Vázquez, C. Griesinger, Y. Liu, J. Am. Chem. Soc. 2016, 138, 9548.

The application of NMR spectroscopy in fragment-based drug design

Martin J. Scanlon, Biswaranjan Mohanty, Bradley C. Doak, Stephen J. Headey

Fragment-based drug design (FBDD) has been widely adopted in both industry and academia and has proven to be a robust approach to identify small molecules that bind to a range of protein targets.1 Two compounds derived from programs of FBDD have now been approved for therapeutic use by the US Food and Drug Administration and many others are in advanced clinical trials.

One of the first practical implementations of FBDD was the approach of SAR-by-NMR described by Stephen Fesik‘s group at Abbott,2 which employed protein-detected NMR experiments to identify ligands that bound at adjacent sites on a protein surface. Subsequently, both protein-detected and ligand-detected NMR experiments have been a mainstay of the screening approaches used in FBDD.

The value of NMR as a biophysical tool for screening fragment libraries is clear. However, the application of NMR extends beyond its role in screening. In this presentation I will highlight the use of NMR in the design and curation of fragment libraries, the optimization of conditions for screening as well as the identification of fragments that bind to a protein and a systematic approach to the prioritization of hits for further development.

1. Erlanson, D. A.; Fesik, S. W.; Hubbard, R. E.; Jahnke, W.; Jhoti, H., Twenty years on: the impact of fragments on drug discovery. Nat Rev Drug Discov 2016, 15 (9), 605-19. 2. Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W., Discovering high-affinity ligands for proteins: SAR by NMR. Science 1996, 274 (5292), 1531-4.

Transthyretin-related familiar amyloid polyneuropathy

Yueh-Jung Yen,1 Kon-Ping Lin,2 Yo-Tsen Liu,2 Shing-Jong Huang,3 Yu Chang,1 Tsyr-Yan DharmaYu1*

1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan 2Taipei Veterans General Hospital, Taipei, Taiwan 3Instrumentation Center, National Taiwan University, Taiwan

*[email protected]

Among different mutated precursor proteins involved in familial amyloid polyneuropathy (FAP), transthyretin (TTR) related FAP (TTR-FAP) is the most prevalent and severe genetic subtypes, accounts for ~70% of the FAP cases. For Taiwanese population, a unique Ala97Ser (A97S) TTR mutation was found to account for 90% of Taiwanese patients with TTR-related Familial Amyloid Polyneuropathy (TTR-FAP). To assess the pathogenicity of Ala97Ser TTR, we measured the protein melting temperatures of various mutants. We showed that the melting temperature of A97S TTR is lower than the wild type TTR and the protective mutants including R104H TTR and A97S TTR. We reported a novel way to obtain TTR of the monomeric form at low concentration. We found that TTR monomer can reassemble to tetrameric form when the concentration of TTR is increased. By concentrating protein to a desired concentration, we were able to measure the ratio of TTR tetramer to monomer, which provides another way to assess the protein tetramer stability. Our result suggest Ala97Ser TTR is a pathogenic mutation, leading to a reduced stability of protein tetramer. In addition, we performed measurements at protein level to access the effect of tafamidis, a prescription drug for the treatment of TTR-FAP caused by V30M TTR. Using both ITC and NMR spectroscopy, we showed that tafamidis can stabilize the Ala97Ser TTR tetramer at protein level, which leads to an ongoing clinical trial to exam the drug effect of tafamidis on the disease progress.

NMR views of functional roles of glycoconjugates of biological and pharmaceutical interest

Koichi Kato1, 2, Maho Yagi-Utsumi1,2, Saeko Yanaka1,2, Tatsuya Suzuki1,2, Hirokazu Yagi2, Tadashi Satoh2, and Takumi Yamaguchi1,2,3 1 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 2 Graduate School of Pharmaceutical Sciences, Nagoya City University 3 School of Materials Sciences, Japan Advanced Institute of Science and Technology It has been predicted that more than half of proteins in nature are modified with sugar chains, which not only affect the physical properties of proteins, but also govern their biological functions, and even their fates. Hence, glycosylation is now considered to be one of the most important factors in the design and development of biopharmaceuticals currently typified by antibody medicines [1]. Although 3D structural insights into the protein glycosylation are essential for facilitating the biopharmaceutical design, the structural analysis of glycoproteins has been eschewed until recently because of the complexity, heterogeneity, and flexibility of the glycans [2]. This aversion may have branded structural biologists as glycophobics. In view of the situation, we have been developing a systematic method of structural biology using NMR spectroscopy in conjunction with X-ray crystallography and computational approaches for elucidating structures, dynamics, and interactions of glycoconjugates at atomic level [3-5].

In this presentation, several examples of our structural glycobiology studies will be illustrated dealing with the following topics: ● Glycan-dependent effector functions of antibody ● Glycan-dependent protein-fate determination in cells ● Glycolipid clusters as platform for protein interactions

Our findings will allow new possibilities for atomic descriptions of dynamic structures of functional glycoconjugates of biological and pharmaceutical interests.

References: 1. Y. Kamiya, M. Yagi-Utsumi, H. Yagi, and K. Kato, ―Structural and molecular basis of carbohydrate–protein interaction systems as potential therapeutic targets‖, Curr. Pharm. Des. 17, 1672-1684 (2011). 2. Y. Kamiya, T. Satoh, and K. Kato, ―Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms‖, Curr. Opin. Struct. Biol. 26, 44-53 (2014) 3. T.Yamaguchi, Y.Sakae, Y.Zhang, S.Yamamoto, Y.Okamoto, and K.Kato, ―Exploration of conformational spaces of high-mannose-type oligosaccharides by an NMR-validated simulation‖, Angew. Chem. Int. Ed. 53, 10941-10944 (2014) 4. T.Satoh, T.Yamaguchi, and K.Kato, ―Emerging structural insights into glycoprotein quality control coupled with N-glycan processing in the endoplasmic reticulum‖, Molecules 20, 2475-2491 (2015) 5. T.Satoh, T.Toshimori, G.Yan, T.Yamaguchi, and K,Kato, ―Structural basis for two-step glucose trimming by glucosidase II involved in ER glycoprotein quality control‖, Sci. Rep. 6, Article number: 20575 (2016)

NMR Characterization of Weak Interactions between RhoGDI2 and Fragment Screening Hits Jiuyang Liu, Jia Gao, Fudong Li, Rongsheng Ma, Qingtao Wei, Aidong Wang, Jihui Wu, and Ke Ruan* Hefei National Laboratory for Physical Science at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China Background: The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine- nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis.1

Methods: Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits.

Results: We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening.2 The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis.

Conclusions: Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrative NMR approach.3

General Interests: Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

References

(1) Garcia-Mata, R.; Boulter, E.; Burridge, K. Nat. Rev. Mol. Cell Biol. 2011, 12, 493-504. (2) Wang, N.; Li, F.; Bao, H.; Li, J.; Wu, J.; Ruan, K. ChemBioChem 2016, 17, 1456-1463. (3) Liu, J.; Gao, J.; Li, F.; Ma, R.; Wei, Q.; Wang, A.; Wu, J.; Ruan, K.; Biochim. Biophys. Acta – Gen. Sub. 2016, in press.

Methyl NMR as a tool for determining the

ligand binding modes in weak protein–ligand complexes

Biswaranjan Mohantya, Geqing Wangb, Stefan Nebla, Wesam S. Alwana, Martin L. Williamsa, Bradley C. Doaka, Mehdi Moblic, Martin J. Scanlona aMedicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville Victoria 3052, Australia bLa Trobe Institute for Molecular Bioscience, La Trobe University, Melbourne, VIC, 3085, Australia cCentre for Advanced Imaging, University of Queensland, St Lucia QLD 4072, Australia Fragment-based drug design is now widely used as an approach to identify low molecular weight fragments (~ 200 Da) as starting points for drug discovery against a diverse range of targets.1 Although the structure of the protein target is often known at the beginning of an FBDD campaign, successful elaboration of the initial weak hit compounds generally requires the structures of co-complexes with fragments to be determined. Most commonly this is achieved using X-ray crystallography, either by soaking pre-formed crystals of the protein with a fragment of interest, or by growing co-crystals from a mixture of protein and fragment. In situations where neither soaking nor co-crystallization is feasible, nuclear magnetic resonance (NMR) spectroscopy is an alternative method to determine the ligand binding poses in the complexes. NMR is a powerful approach for characterizing weakly binding complexes,2 but it suffers from much lower throughput compared to X-ray crystallography. We have developed an NMR-based method that enables rapid generation of ligand binding poses for protein–ligand complexes to support programs of structure-based drug design. Our method is based on the use of sparse NOE-based distance restraints derived from protein samples prepared by selective methyl labeling of all methyl-containing residues in a deuterated background. To illustrate the approach, one model will be presented for protein– ligand complex, which was ambiguous by X-ray crystallography.

1. Erlanson, D. A.; Fesik, S. W.; Hubbard, R. E.; Jahnke, W.; Jhoti, H., Twenty years on: the impact of fragments on drug discovery. Nat. Rev. Drug Discov. 2016, 15 (9), 605-19. 2. Mohanty, B.; Williams, M. L.; Doak, B. C.; Vazirani, M.; Ilyichova, O.; Wang, G.; Bermel, W.; Simpson, J. S.; Chalmers, D. K.; King, G. F.; Mobli, M.; Scanlon, M. J., Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data. J. Biomol. NMR 2016, 1-14

ACD Labs

PRAFUL GUPTA, Ph.D. Senior Research Scientist DAIICHI SANKYO INDIA PHARMA PRIVATE LIMITED Gurgaon (India)

Dr. Gupta is currently working as Senior Research Scientist with Daiichi Sankyo India Pharma Private Limited in the Department of Chemistry. He did his Ph.D entitled ―Synthesis of Biologically active compound as 5HT1A receptor antagonists‖ with Dr. Ian A. Cliffe at Ranbaxy Laboratory Limited. Dr. Gupta Started his research career with New Drug Discovery Research center (NDDR), Ranbaxy Laboratory Limited in 2002 in Adrenergic receptor antagonists program. In 2006 he moved to Oncology program where he worked for small molecule kinase inhibitors. In 2010 when Daiichi Sankyo acquired Ranbaxy Labs, He moved to Daiichi Sankyo India (DSIN) research center. For initial few years he worked in anti-inflammation program and in 2013 he led an anti infective project. In 15 years of research experience, apart from publications and patents, Dr. Gupta got several opportunities to show cases his research work at various national and international meetings.

Real-time monitoring of p53’s effects on pyruvate metabolism in live mitochondria using in-organelle NMR metabolomics

Sunghyouk Park Professor and Head Department of Manufacturin gPharmacy College of Pharmacy Seoul National University, Korea Mitochondrial metabolism is at the heart of cellular metabolic activities, and its alteration is increasingly recognized as a critical aspect of cancer metabolism. Still, mitochondrial metabolism has been studied with cellular or mitochondrial extract, hampering the measurement of real time metabolic flux, a true indicator of metabolic activities. Here, we introduce a novel ―in-organelle NMR metabolomics‖ that enables real time metabolomic investigation of live mitochondria. The method makes use of a generally applicable two dimensional heteronuclear NMR techniques and 13C-labeled pyruvate (non-radioactive) for the non-destructive metabolic activity measurement. The method was used to investigate the roles of p53, one of the most well-known tumor suppressors, in mitochondrial pyruvate metabolism, employing p53 KO and WT cells. It was found that p53 can modulate mitochondrial production of lactate, acetate, and acetyl CoA from pyruvate. We also demonstrated p53‘s differential roles in pre-TCA vs. TCA metabolism. Further application of the approach to a p53-activating anticancer agent nutlin revealed its effects that are not expected from its p53 level regulation. The method can be straightforwardly extended to studying organelle-specific metabolism using different tracers and other disease models. It is also expected to contribute to both understanding of the basic disease mechanism and developing therapeutic agents for new mitochondrial metabolic targets.

Exploring Brain Energy Metabolism in Alzheimer's Disease

Anant Bahadur Patel

NMR Microimaging and Spectroscopy, CSIR-Center for Cellular and Molecular Biology, Habshiguda, Uppal Road, Hyderabad 500007, India

Alzheimer‘s disease (AD) is the most common neurodegenerative disorder associated with gradual deterioration of cognitive functions, personality and memory1. Although AD was discovered more than a century ago, there is neither a quantitative diagnosis nor cure for the disease exist. We are investigating neuronal and astroglial metabolic activity in APP-PS1 mouse model1, that exhibits physiology similar to AD subjects, with age to get an insight into AD pathophysiology. We use an approach of 1H-[13C]-NMR spectroscopy in conjunction with an infusion of 13C labeled glucose and acetate to understand metabolic activity of neurons and astrocytes, respectively3. Our analysis indicate that though the neurochemical profile remains unchanged at the age of 6 months in APP-PS1 mice, the rate of glutamatergic and GABAergic neuronal glucose oxidation and neurotransmitter cycle is significantly attenuated at the stage when memory is unperturbed. APP-PS1 mice exhibit decreased level of NAA and glutamate, and increased level of inositol at the stage of severe plaque loading. In addition to reduced neurometabolic activity, APP-PS1 mice exhibit enhanced astroglial metabolic activity at high plaque loading. We have used APP-PS1 mice to understand efficacy of Rasa Sindoor (RS, an Ayurvedic formulation) and dietary restriction (DR) on memory and neurometabolic activity for treatment of AD. Our data suggest that RS and DR improved memory and energy metabolism in APP-PS1 mice. As the neuronal glucose metabolism is stoichiometrically coupled with neurotransmitter cycling in brain4, findings of increase cerebral metabolic in APP-PS1 mice suggest improved neuronal activity with RS and DR interventions in AD. In this presentation, I will be discussing these findings in detail.

References 1. Goedert M and Spillantini MG, Science 2006, 314, 777-781. 2. Jankowsky et al. Hum Mol Genet. 2004, 13, 159-170. 3. de Graaf et al. Magn. Reson. Med. 2003, 49, 409-416. 4. Hyder et al (2005) J. Cereb. Blood Flow Metab. 2006, 26, 865-877.

Acknowledgements: The study was supported by funding from DST (CO/AB/013/2013) and network project (BSC0208).

Design of Small Peptides: Synthesis and Biological Studies for the Development of Anti-inflammatory Agents

Palwinder Singh, Sukhmeet Kaur, Priya Kumari, Jagroop Kaur

Department of Chemistry, Guru Nanak Dev University, Amritsar-143005. India

Filling the gap between small molecule chemicals and the larger proteins, small peptides are identified with anti-cancer [1], anti-inflammatory [2] and anti-fungal [3] activities in addition to their key role as biocatalysts and hormones [4]. Being present in their primary structure, it was envisaged that an appropriate small peptide may occupy the same space in the active site of cyclooxygenase-2 where it‘s natural substrate (arachidonic acid) is normally placed and hence the peptide may act as substrate analogue inhibitor of COX-2. In this direction, a series of peptides were designed on the basis of molecular docking studies and physico-chemical parameters. Their synthesis was followed by the screening for their in-vitro and in-vivo anti-inflammatory activity. Compound 1 was identified as the most potent anti-inflammatory agent. Various physico-chemical techniques including UV-vis, isothermal calorimetry and NMR spectroscopy were used for ascertaining the enzyme – drug interactions. A 3D QSAR pharmacophore model was also developed and steric, hydrophobic factors were found to be the major contributors for the activity of the compounds. The details of all the experiments with conclusive remarks on the use of small peptides as anti-inflammatory agents will be presented.

O H H O N N N H2N H OMe O O

1 IC50 = 0.06 µM crystal coordinates of COX-2 with a peptide

in the active site References 1. Pearce, et al. Adv. Mater, 2012, 24, 3803-3822. 2. Cammusi, et al. J. Exp. Med. 1990, 171, 913-927. 3. Matsunaga, et al. J. Am. Chem. Soc. 1989, 111, 2582-2588. 4. Cloutheir, et al Chem. Soc. Rev. 2012, 41, 1585-1605.

Understanding the toxicity mechanism of Pyrazinamide as revealed by NMR based serum metabolomics and Biochemical analysis

Dinesh Kumar,1,* Atul Rawat,1,2 Swati Chaturvedi,3,4 Ashok K Singh,3 Anupam Guleria,1 Durgesh Dubey,1,2 Amit K Keshari,3 Vinit Raj,3 Amit Rai,3 Umesh Kumar,1 Anand Prakash,2 and Sudipta Saha* 1Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raibareli Road, Lucknow- 226014, India 2Department of Biotechnology and 3Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, India 4Division of Pharmacokinetics & Metabolism (PKMD), CSIR-Central Drug Research Institute, Sitapur Road, Lucknow-226021,India

Pyrazinamide (PYZ) -an essential component of primary drug regimen used for the treatment and management of multidrug resistant or latent tuberculosis- is well known for its hepatoxicity. However, the mechanism of pyrazinamide induced hepatotoxicity is still unknown to researchers. Studies have shown that the drug is metabolized in the liver to pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OHPA) which individually may cause different degrees of hepatotoxicity. To evaluate this hypothesis, PYZ, PA and 5-OHPA were dosed to albino Wistar rats orally (respectively, at 250, 125, and 125 mg/kg for 28 days). Compared to normal rats, PZA and its metabolic products decreased the weights of dosed rats and induced liver injury and a status of oxidative stress as assessed by combined histopathological and biochemical analysis. Compared to normal controls, the biochemical and morphological changes were more aberrant in PA and 5-OHPA dosed rats with respect to those dosed with PYZ. Finally, the serum metabolic profiles of rats dosed with PYZ, PA, and 5-OHPA were measured and compared with those of normal control rats. With respect to normal control rats, the rats dosed with PYZ and 5-OHPA showed most aberrant metabolic perturbations in their sera compared to those dosed with PA. Altogether, the study suggests that PYZ induced hepatotoxicity might be associated to its metabolized products, where the metabolite 5-OHPA contributes more in the overall toxicity of PYZ than PA.

NMR BASED STRUCTURAL BIOLOGY IN IMMUNITY AND NEURODEGENERATION/PROTECTOIN

L. Wong1, L. Russo1, J. Kühn2, S. Pirkuliyeva2, D. Lee1, T.M. Sabo1, S. Ryzanov1,3, L. Antonschmidt1,3, A. Martinez Hernandez4, H.Y. Agbemenyah5, S. Shi6, A. Fischer5, G. Eichele4, S. Becker1, A. Leonov1,3, R. Benz7, M. Zweckstetter1,3, J. Wienands3, A. Giese6, and C. Griesinger1,3

1Dept. for NMR-based Struct. Biology, Max-Planck Institute for Biophysical Chemistry; 2Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen; 3DFG-Center for the Molecular Physiology of the Brain, Göttingen; 4Genes and Behavior Dept., Max-Planck Institute for Biophysical Chemistry, Göttingen; 5 European Neuroscience Institute Göttingen;, 6 Center for Neuropathology and prion research, LMU, Munich, Germany; 7 Jacobs University of Bremen, Germany Then the role of partially disordered proteins in two fields or research is investigated. One is the adaptor protein SLP65 which interacts with CIN85 (1). The two proteins are essential for B cell activation. The protein is found to be mainly unstructured and its various segments entertain different functions or interact with membranes, SH3 domains and forming coiled coils. Based on the structures, a molecular lego will be described that reduces the SLP65/CIN85 interaction to its absolutely necessary essentials. The two proteins can form supramolecular structures which might be related to function.

We are interested in a class of IDPs that are important in neuro- and cellular degeneration, which form oligomers and fibrils. Interference with these aggregates specifically on the oligomer level proves to be a valid concept for treatment of devastating diseases such as Parkinson‘s, Alzheimer‘s, Creutzfeldt Jacob disease and Type II diabetes mellitus.

______

(1) M. Engelke et al. Science signaling: 7 (339) ra79 (2014); J. Kühn et al. Science signaling: 9 (434) ra66 (2016) (2) C.W. Bertoncini et al. PNAS 102, 1430-1435 (2005); P. Karpinar et al. EMBO J 28, 3256-3268 (2009); J. Wagner et al. Acta Neuropath. 125, 795-813 (2013); A.A. Deeg, Biochim. Biophys. Act. 1850 (9), 1884-1890 (2015); S. Shi, J. Neuropath. Exp. Neurol. 74(9) 924-933 (2015)

Structural study of fibroblast growth factor 21 (FGF21)-a potential anti- diabetic drug

Lei Zhu, Hongxin Zhao, Juanjuan Liu and Junfeng Wang

High Magnetic Field Laboratory, CAS, P. R. China

The fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating receptor tyrosine kinases, fibroblast growth factor receptors (FGFRs), that are involved in cell signaling and pathological conditions. Fibroblast growth factor 21 (FGF21) is a newly discovered atypical member of the FGF family that functions as an endocrine hormone. Pharmacologic studies have demonstrated that FGF21 might be an important type II diabetes drug candidate.

Here, we report a structural study of FGF21 and its interaction with FGFR1c receptor by liquid state NMR. Based on the FGF21 structure, we designed FGF21 mutants that successfully locked the FGF21 into a function active conformation with improved protein stability. Further cell and animal experiments demonstrated that this mutation improves the diabetic therapeutic property substantially.

Structural and Functional Studies on Synthetic Peptides

Viji Sarojini

School of Chemical Sciences, University of Auckland, Auckland, New Zealand E-mail: [email protected]

De novo designed synthetic peptides are ideal candidates for understanding protein structure and function. Structural motifs with predictable three-dimensional architecture are ideal tools for the de novo design of synthetic peptides. In many instances, synthetic peptides based on natural sequences or designed de novo also possess therapeutic potential. Particularly important in the context of the alarming increase in multidrug resistance (MDR) are potent 1 antimicrobial peptides and lipopeptides that provide alternatives to conventional antibiotics. Our work focusses on developing peptides for various applications – particularly 2 antimicrobial lipopeptides that eradicate bacterial biofilms and antifreeze peptides (AFPs) 3 that minimize freeze-thaw damage in frozen foods. We have also been working on the challenging synthesis of cyclic tetrapeptides. This talk will summarise results on the structure function analysis of antimicrobial lipopeptides with antibiofilm potential, synthetic antifreeze peptides as well as linear and cyclic tetrapeptides incorporating novel β-turn mimics.

Figure: (left) Membrane lysis and biofilm eradication by synthetic lipopeptides. (right) Ensemble of nine lowest energy conformations of a linear tetrapeptide facilitated by a novel β-turn overlaid with the crystal structure (purple).

References

1. Strieker, M.; Marahiel, M. A., Chembiochem 2009, 10, 607-16. 2. De Zoysa, G. H.; Cameron, A. J.; Hegde, V. V.; Raghothama, S.; Sarojini, V., J Med Chem 2015, 58, 625-39. 3. Kong, C. H.; Hamid, N.; Liu, T.; Sarojini, V., J Agric Food Chem 2016, 64, 4327-35.

Combining EPR and NMR restraints in structure determination of biological systems Gunnar Jeschke

ETH Zürich, Department for Chemistry and Applied Biosciences, CH-8093 Zürich, Switzerland

Both solution-state and solid-state NMR spectroscopy can provide structural information at atomistic resolution for biomolecules or their complexes in states or in environments that are not accessible to x-ray crystallography or even cryo-electron microscopy. However, resolution limitations and assignment problems may put an upper limit on system size, the short-range distance restraints that NMR yields may be insufficient to fully define large structures, and the number of obtainable restraints is reduced drastically for intrinsically disordered proteins and protein domains. None of these problems exists for EPR spectroscopy, which can provide valuable distance distribution information for disordered domains, but the number of obtainable EPR restraints is small, because site-directed spin labelling is required and each sample yields only one or at most a few restraints. Furthermore, translation of label-to-label distance restraints to backbone-to-backbone restraints introduces additional uncertainty. Both the sparsity of EPR restraints and their limited precision preclude the computation of structures at atomistic resolution from only EPR data. Combination of the two spectroscopies is a promising approach to obtaining well-resolved models of larger and partially disordered systems. NMR pseudo-contact shift measurements provide a more accurate spatial distribution of a lanthanide label than computational predictions [1]. EPR distance distributions can reveal the co-existence of several conformations of a large RNA- protein complex [2]. A few long-range distance distribution restraints can stabilize structure computation for discoidal high-density lipoprotein particles [3]. The author thanks F. H.-T. Allain, R. Riek, I. Kuprov, and D. Häussinger for fruitful collaborations.

References

[1] Suturina, E.; Häussinger, D.; Zimmermann, K.; Garbuio, L.; Yulikov, M.; Jeschke, G.; Kuprov, I.; submitted. [2] Duss, O.; Yulikov, M.; Jeschke, G.; Allain, F. H.-T. Nature Comm. 2014, 5, 3669. [3] Bibow, S.; Polyhach, Y.; Eichmann, C.; Chi, C. N.; Kowal, J.; Albiez, S.; McLeod, R. A.; Stahlberg, H.; Jeschke, G.; Güntert, P.; Riek, R. submitted.

Inter-domain communication in protein controlled by intrinsically disordered region (IDR) – a functional role of intramolecular ‘fly-casting’ mechanism in ligand binding

Shin-ichi Tate1,2

1Dept. Mathematical and Life Sciences, School of Science, Hiroshima University 2Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), Hiroshima University, Hiroshima, Japan

Exploring the functional roles of intrinsically disordered regions (IDRs), have become the central focus in protein science. Studies on IDRs in a past decade have demonstrated that IDRs have specific functions that have not been ever found in folded protein units.

Some of the multi-domain proteins have folded units linked by flexible linkers or IDRs. In such proteins, the domains cooperatively work in exerting the function, but it is not well characterized how the IDRs role in such processes.

Prolyl-isomerase, Pin1, has two domains linked by 10-residue IDR. WW-domain specifically binds to phosphor-Ser/Thr-Pro (pS/pT-P) dipeptide motif, while PPIase domain converts Pro in pS/pT-P from cis to trans or vice versa. We have revealed the substrate with pS/pT-P motif migrates between the domains in Pin1 through the extensive NMR titration experiments with numerical simulation to reproduced the NMR titration data. In this migration process, substrate released from one of the domains in Pin1 can be efficiently captured by the other domain, which prevents the substrate from being released back to the solvent, thus the apparent substrate affinity to Pin1 is enhanced by three-order of the magnitudes. The studies with the mutants having the changes in the inter-domain linker have demonstrated the intra-molecular ‘fly-casting’ mechanism mediated by the IDR makes the efficient substrate migration between the domains possible.

The ‘ensemble structure’ analyses of Pin1 done with the combinatorial use of SAXS, PRE and RDC data will be also described to see how the domain dynamics role in the substrate migration within Pin1.

Nanoparticle/biological interface: effect of silica support on membrane surface potential, dynamics of transmembrane peptide,

and effective pKa of ionisable sidechain.

Erkang Ou, Maxim Voinov, Alex Smirnov, and Tatyana Smirnova

North Carolina State University, Raleigh NC, USA

Development of novel lipid-nanostructure hybrids promises to open many new opportunities for analytical and technological applications and paves the way towards living-nonliving interfacing biotechnology platforms. At present, however, little is understood about the influence of a nanostructured support and confinement on electrostatic properties of the membrane-protein interface. In this work we report on spin-labeling EPR studies to 1) evaluate the effect of anionic lipid surface charge density on effective pKa of membrane-burred ionisable sidechains and 2) assess effects of solid inorganic interface, specifically, silica support, on the membrane surface potential and on effective pKa of the membrane- burred ionisable sidechains. The change in the protonation state of the pH- sensitive ionisable nitroxide label was directly observed by CW EPR. We have shown that the effective pKa of the probe increases by 2.1 to 2.3 pH units (depending on the depth of the probe) upon replacing zwitterionic PC with anionic

PG lipids, with almost 80% of that pKa shift observed upon replacing only half of the PC with PG lipids. We have also shown that placing a lipid bilayer with integrated transmembrane α-helical WALP peptide on the surface of silica nanoparticles affects the peptide dynamics and shifts the effective pKa of the probe in a membrane depth-dependent manner. The latter effect was attributed to the negative charge of the silica surface. Supported by NSF 1508607 to TIS.

NMR Investigation of the Molecular Basis of Microtubule Regulation by Neuronal Microtubule-Associated Proteins

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

1 Max Planck Institute for Biophysical Chemistry, 37077 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 is regulated by microtubule-associated proteins (1). The microtubule-associated protein Tau promotes formation and stabilization of axonal microtubules and thus influences intracellular transport, axonal stability and cell morphology (2). The adult human brain contains six isoforms of Tau, which are generated from a single gene by alternative splicing. 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 promotes microtubule assembly by binding to protofilaments at the interface between α-β-tubulin heterodimers using small groups of evolutionary conserved residues (3). 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 proteins.

References

1. Mandelkow, E., and Mandelkow, E.-M. (1995) Microtubules and microtubule-associated proteins, Curr. Opin. Cell Biol. 7, 72-81. 2. Weingarten, M. D., Lockwood, A. H., Hwo, S. Y., and Kirschner, M. W. (1975) Protein Factor Essential for Microtubule Assembly, Proc. Natl. Acad. Sci. USA 72, 1858-1862. 3. Kadavath, H., Hofele, R. V., Biernat, J., Kumar, S., Tepper, K., Urlaub, H., Mandelkow, E., and Zweckstetter, M. (2015) Tau stabilizes microtubules by binding at the interface between tubulin heterodimers, Proc. Natl. Acad. Sci. USA 112, 7501-7506.

NMR study of the left-handed Z-form conformation of nucleic acids induced by Z-DNA binding proteins

Joon-Hwa Lee

Department of Chemistry, Gyeongsang National University, Gyeongnam 52828, Korea.

Z-DNA is a higher energy conformation than right-handed B-DNA and can be stabilized by high salt, negative supercoiling, and complex formation with Z-DNA binding proteins (ZBPs). The ZBPs have been identified in a family of proteins including the human RNA editing enzyme (ADAR1), the mammalian DNA-dependent activator of interferon-regulatory factor (DAI), poxviral E3L protein, and fish ZBP-containing protein kinase (PKZ). The crystal structures of various ZBPs in complexes with 6-base-paired (6-bp) DNA revealed that two Zα proteins bind to each strand of double-stranded (ds) Z-DNA with twofold symmetry with respect to the DNA helical axis. In order to understand how ZBPs specifically recognize Z-DNA sequence in a sea of B-DNA, we performed NMR experiments with complexes of

ZαADAR1 bound to d(CG)3 produced at a variety of protein-to-DNA molar ratios. From this study, we suggested an active B-Z transition mechanism in which the ZαADAR1 first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second ZαADAR1 molecule.

When Z-DNA is formed in a long genomic DNA by ZBP, two B-Z junctions are produced with the extrusion of one base pair from each junction. To answer the question of how ZBPs induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with ZαADAR1. Our study also suggest a three-step mechanism of B−Z junction formation: (i) ZαADAR1 specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT- rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z- DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

The ZαADAR1 domain also binds to Z-RNA and then enhances the editing level of dsRNA by ADAR1. Despite the structural study of ZαADAR1 for Z-RNA, the detailed molecular mechanism by which ZαADAR1 converts a right-handed A-form structure in an RNA duplex to a left-handed Z-form is not well understood. Here, we are undergoing to carry out the NMR study of ZαADAR1 complexed with a 6-bp RNA duplex r(CG)3 at a variety of protein-to-RNA molar ratios and these results were compared to those of DNA substrate, d(CG)3.

Towards a code for single stranded RNA recognition

Neel Sarovar Bhavesh, Akshay Kumar Ganguly, Maruthi Kashyap, Garima Verma and Harshesh Bhatt

Transcription Regulation group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, India. E-mail: [email protected] web: www.neelsb.com

RNA binding proteins or RBPs play an indispensable role in cellular machinery, especially processes such as transcription, post-transcriptional modification of RNA, RNA transport and stabilization, among others. Post-transcriptional modifications of RNA are a major route through which eukaryotes regulate gene expression. These modifications include splicing, mRNA polyadenylation, 5‘ capping and RNA editing. RBPs bind RNA through specific RNA binding domains (RBDs) or modules. Binding affinities and specificities vary throughout this family of proteins. The RNA recognition motif or RRM is the most widely distributed RBD in nature. RRMs are canonically identified through the presence of two RNA binding consensus motifs (RNP). They have a common three-dimensional architecture, which classically consists of a four-stranded β-sheet supported by two α-helices, with the β- sheet serving as the major surface for RNA recognition. In spite of these unifying traits of RRMs, they possess a remarkably diverse RNA recognition capability. Our recent delineation of the RNA-binding mechanism of human TAF15 protein highlights how the concave face of its carboxy terminal RRM recognizes structured loop elements on RNA, in a non-canonical manner. Mutations in this protein have been implicated in familial amyotrophic lateral sclerosis or FALS. The human ETR3 protein on the other hand, possesses three RRMs, which sequence-specifically recognizes CUG and UG-rich RNA. This is carried out through specific interaction of a uracil base by a cleft on the β-sheet surface. The N-terminal RRM of malarial SR1 alternative splicing factor that semi- specifically recognizes pyrimidines exhibits a similar mode of interaction. We have delineated how the latter two instances represent a canonical form of RRM-RNA interaction, with π-π interactions between aromatic amino acids and nucleotide bases being responsible for binding. On the other hand, the TAF15-RRM-RNA interface is largely dominated by hydrogen bonding between charged amino acids and polar groups on the RNA. Our work on RNA recognition by RRMs thus paints an interesting picture of how a single fold is able to recognize different cognate RNAs by virtue of minor but crucial alterations to its binding surface. In addition, delineation of RNA binding specificities of RRMs has provided molecular clues to the progression of debilitating diseases such as myotonic dystrophy, FALS and malaria.

References: 1. Kashyap M, Ganguly AK and Bhavesh NS (2015) Sci. Rep. 5, 17298 (1-14). 2. Kashyap D, Ganguly AK and Bhavesh NS (2015) Biomol. NMR Assign. 9, 103-106. 3. Kashyap M, Sharma A and Bhavesh NS (2013) Acta Crystallogr. F69, 1107-1109. 4. Bhatt H, Kashyap M and Bhavesh NS (2010) Biomol. NMR Assign. 4, 143-145. 88

NMR Characterization of 107 kDa Ternary Complex Involved in Termination of NF-kappaB Signaling

Sulakshana P. Mukherjee1, 3, Pedro O. Quintas1, Reginald McNulty1, Elizabeth A. Komives2 and H. Jane Dyson1 1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037-1000 2Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92037-0378 3Current address: Department of Biotechnology, Indian Institute of Technology Roorkee, Uttarakhand 247667, India Nuclear factor KappaB (NF-κB) is a five member family of inducible transcription factors that is universally present in almost all cell types in higher eukaryotes. In the resting cells, NF-κB remains inactive in the cytoplasm bound to its inhibitor protein IκBα. Upon activation of the pathway through various stimuli, the NF-κB protein enters the nucleus and binds to its cognate κB DNA sites on the promoter/enhancer region of its target genes thereby activating their transcription. The tightly controlled pathway has a robust negative feed-back loop that involves resynthesis of IκBα protein, a target gene of NF-κB itself. The newly synthesized IκBα enters the nucleus, strips NF-κB off its target DNA sites and exits into the cytoplasm as

IκBα:NF-κB complex thus regaining its resting state. The mechanism of this process however remains unclear. In this study we have used NMR spectroscopy to delineate the mechanism of how NF-κB is stripped off its κB DNA by IκBα. Here we show that the process involves the formation of a IκBα:NF-κB:DNA ternary complex. In my talk I will discuss the structural features of about 107 kDa ternary complex as revealed by NMR.

References

[1] Mukherjee SP, Quintas PO, McNulty R, Komives EA, Dyson HJ. Structural characterization of the ternary complex that mediates termination of NF-kB signaling by IkBa. (2016) Proc Natl Acad Sci USA 113:6212-7 [2] Mukherjee SP, Borin B, Quintas PO, Dyson HJ. NMR Characterization of a 72 kDa transcription factor using differential isotopic labeling. (2016) Protein Sci. 25:597-604

Structure, Dynamics, Topology, Membrane-disrupting Mechanism and Function of Antimicrobial as well as Cell Penetrating Peptides

Anirban Bhunia

Bose Institute, P 1/12 CIT Scheme VII (M), Kolkata 700 054, India.

Abstract:

Understanding the mechanisms of biological processes requires precise knowledge of the three- dimensional structures of the executor molecules such as proteins, bioactive peptides and others. Atomic-resolution structures of well-folded proteins or complexes can be obtained from X-ray crystallography. However, a large number of proteins or domains of large proteins (e.g., in signaling cascades) and bioactive peptides (e.g., antimicrobial /cell penetrating peptides) appear to be dynamic, thus limiting the application of X-ray-based methods. A complete understanding of their function can only be accomplished using high- resolution structures. In spite of recent developments in structural biology, membrane peptides/proteins continue to pose tremendous challenges to most biophysical techniques. On the other hand, gaining insights into such molecular systems at the atomic level is possible using nuclear magnetic resonance (NMR) spectroscopy. Our research group has been investigating the high-resolution structures of antimicrobial peptides (AMPs), cell penetrating peptides (CPPs), membrane-peptide interaction and membrane disruption, and the interaction of AMPs with live cell. Our studies also have provided insights into the peptide delivery processes through nano-particle tagged AMPs. I will present the correlation between structure, dynamics and function of the designed antimicrobial peptides for the treatment of human and plant pathogens.

New Methods for NMR of Complex Mixtures

Veera Mohana Rao Kakitaa and Ramakrishna V. Hosura,b*

a University of Mumbai, Department of Atomic Energy, Centre for Excellence in Basic Sciences (UM-DAE-CBS), Vidyanagari Campus, Mumbai-400 098 b Department of Chemical Sciences, Tata Institute of Fundamental Research (TIFR), Colaba, Mumbai-400 005

email: [email protected]

Homonuclear scalar coupling measurements play an important role in structural analysis of complex organic molecules. However, often severely overlapped 1H-1H scalar couplings present in the limited 1H-NMR chemical shift range (~10 ppm) significantly hampers the spectral resolution and that makes both the chemical shift as well as scalar coupling measurements difficult. Under these circumstances, the present work offers various advanced 1D and 2D- homodecoupling NMR methods, which facilitate pure-shift chemical shift information and unambiguous scalar coupling measurements, at ultrahigh resolution.

Structure-activity-relationship of designed peptides using low-resolution spectroscopy and high-resolution NMR technique: Case studies of antifreeze and antiamyloid peptides

Rajiv K. Kar and Anirban Bhunia*

Department of Biophysics, Bose Institute, P-1/12, CIT Scheme VII (M),

Kolkata, 700054, India

* E-mail: [email protected]; [email protected]

Sub-zero temperatures are fatal in most organisms by kinetically slowing down vital biochemical reactions, denaturating biomolecules, or rupturing cell membranes. In this consideration, the natural selection of organisms has offered unique adaptive mechanism by providing antifreeze protein (AFPs) [1]. Our work was focused on structure-activity- relationship, by zooming in on the significant portion of antifreeze proteins that contribute to their functionality. In the first part, we have worked on an Antarctic yeast, Glaciozyma Antarctica, having antifreeze activity. We have designed several peptide fragments based on the sequential strings that show α-helical secondary structure and analysed the antifreeze activity using various biophysical techniques [2]. The solution phase structures of individual AFPs was determined using NMR spectroscopy at an atomic resolution to dictate the relevance of structural helicity with its corresponding antifreeze activity. NMR study also unravels that decrease in temperature results in spanning of resonance (aromatic, Hα, and methyl protons) and are broadened, which suggests that at low temperature, they tend to more α-helical [2,3]. Further, these mesoscopic properties were explored with computational tools [4]. In the continuation work, we have worked on hypothesis that since AFPs are also known for application in cryopreservation of biological samples that might be related to membrane interaction. Thus, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system (zwitterionic DPC and negatively charged SDS) [5]. In another part of our work, the strategy of designing polypeptide antiamyloid inhibitors is adopted where, we have rationalised designing of peptides to target native protein‘s fibrillisation inhibition [6]. Here we have taken lysozyme as model protein and three peptide fragments derived from lysozyme protein sequence R107- R115 to treat lysozyme amyloid fibrillisation [7]. The findings were explored using low- resolution spectroscopy, high-resolution NMR, and STD NMR-restrained docking method

(HADDOCK) to confirm that derived fragments have capability to affect lysozyme fibril formation.

References:

[1] Sicheri F., Yang D.S. (1995) Nature 375; 427–31.

[2] Shah, S.H.H, Kar, R.K.,…Bhunia, A. (2012) Plos One 7(11); e49788.

[3] Kar, R.K., Bhunia, A. (2015) Prog. Biophys. Mol. Biol. 119; 194-204.

[4] Kar, R.K., Bhunia, A. (2015) J. Phys. Chem. B 119; 11485-495.

[5] Kar, R.K., Mroue, K.H.,…Bhunia, A. (2016) J. Phys. Chem. B 120; 902-14.

[6] Banerjee, V., Kar, R.K.,…Bhunia, A. (2013) Plos One 8(8); e72318.

[7] Kar, R.K., Gazova, Z.,…Bhunia, A. (2016) Biomacromolecules 17; 1998-2009.

Solid-state NMR study of live bacteria in the presence of antimicrobial agents

M.-A. Sani, S. Overall and F. Separovic School of Chemistry, Bio21 institute, The University of Melbourne, VIC 3010, Australia [email protected] Antimicrobial peptides (AMPs) have been extensively studied as promising alternatives to traditional antibiotics. Solid-state NMR has been used to characterise their effect on lipid bilayers, their primary target. Such studies are important to provide high-resolution details with a fully controlled and homogenous system, but correlation with in vivo situation remains speculative, especially in sight of the complicated modulations observed with slight change in sample conditions (pH, temperature, lipid composition or peptide concentration). Studying AMPs in live bacteria is, therefore, attractive but challenged by the highly inhomogeneous and complicated architecture of bacteria. We have used 31P solid-state NMR to study the impact of the AMP maculatin 1.1 on live E. coli and S. aureus bacteria. Different levels of dynamic filtering coupled with paramagnetic reagents were used to isolate signals from different molecular species such as nucleic acids, lipids or inorganic phosphate. At certain peptide concentration, but below the minimum inhibition concentration (MIC), a significant impact on the DNA packing of E. coli and S. aureus was observed, which has not been reported to date. Interestingly, the peptide effect on S. aureus was significantly lesser, particularly among lipid signals, despite having a lower MIC than E. coli. Furthermore, peptide treatment induced the production of a new phosphorus species, consistent with a phosphonate, but only in E. coli. Overall, an extensive solid-state31P NMR study of live bacteria will be reported and, although in an early stage of development, has provided new insights into bactericidal mechanisms of AMPs.

Intrinsically disordered regions of proteins mediate their self-association

Tsai-Chen Chen, Hao-Ru Li, Yu-Hao Lin, Wen-Han Chang, De-Chen Chiou, Wan-Chin Jiang, and Jie-rong Huang Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No. 155 Section 2 Li-nong Street, Taipei, Taiwan It is now a well-established concept that proteins do not have to adapt a defined three- dimension structure to function. Those proteins are termed intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs). It is estimated that around half of eukaryotic proteins are IDPs and one-third of eukaryotic proteins contain IDRs. The IDPs or IDRs can fold to specific conformation while binding to their binding partners; they also can adapt different conformations to inter act with different binding partners to act as a hub. Not only in protein-protein interactions, IDPs or IDRs are also involved in protein self- association. One of the most studied types of self-association is amyloid fibrillization, such as amyloid-beta peptide and alpha-synuclein. Recently, increasing evidences showing that proteins with IDRs undergo protein phase separation both in vivo and in vitro. This reversible property is important to the protein function. However, the detailed mechanism governing the self-association is still elusive. Here, we will use the ID C-terminal domains of TDP-43 and the full length Galectin-3 (with a ID N-terminal domain) as examples to demonstrate how the IDRs mediate the reversible self-association.

PROTEIN STRUCTURE DETERMINATION BY MULTI-TIER GLOBAL OPTIMIZATION USING EARLY-STAGE NMR DATA

Jooyoung Lee

Center for In Silico Protein Science & School of Computational Sciences, Korea Institute for Advanced Study, Seoul, 130-722, Republic of Korea Email: [email protected]

In the recent CASP (Critical Assessment of Protein Structure Prediction) 11 experiment, a new challenge, the contact assisted category (called Ts) using simulated sparse NMR contacts was tested. Contacts based on simulations (carried out by the Gaetano Montelione's group) reflect the situation in the initial stage of the NMR experiment. For a fairly large protein (> 160 residues) for NMR, data is typically collected from deuterated samples, which usually results in much spectral overlaps that are difficult to assign properly. The number of simplified spectra straightforward to assign is rather small. Therefore, in the early stage of the protein structure determination by NMR, one is faced with the combinatorial optimization problem to properly assign ambiguous NOE peaks to their corresponding hydrogen atom pairs, which is followed by subsequent structure optimization satisfying all the distance constraint arranged by a given set of the peak assignment. Based on our recent sucesses [1,2] to resolve these combinatorial problems by applying an efficient global optimization method [3,4], we propose a new numerical method to determine protein structures using early-stage NMR data that contains the complication of (1) the presence of false positives (2) the ambiguity in assigning NOE peaks to proper hydrogen atom pairs and (3) exploring the protein conformational space. Benchmarking results from 5 proteins will be discussed.

[1] Keehyoung Joo, InSuk Joung, Jinhyuk Lee, Jinwoo Lee, Weontae Lee, Bernard Brooks, Sung Jong Lee and Jooyoung Lee, ―Protein structure determination by conformational space annealing using NMR geometric restraints‖, Proteins: Structure, Function, and Bioinformatics, Volume 83, Issue 12, 2251–2262 (2015). [2] Keehyoung Joo, InSuk Joung, Qianyi Cheng, Sung Jong Lee and Jooyoung Lee, "Contact-assisted protein structure modeling by global optimization in CASP11", Proteins: Structure, Function, and Bioinformatics, published online 11 JAN (2016). [3] Jooyoung Lee, Harold A. Scheraga, and S. Rackovsky, ―New Optimization Method for Conformational Energy Calculations on Polypeptides: Conformational Space Annealing‖, J.Comput.Chem., Vol. 18, 1222-1232 (1997). [4] Julian Lee, In-Ho Lee, and Jooyoung Lee, ―Unbiased global optimization of Lennard Jones clusters for N <= 201 by conformational space annealing method‖, Phys.Rev.Lett., Vol. 91, 080201 (2003).

NMR studies on the mechanism of epigenetic modification of the biological molecules

Chunyang Cao

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences

345 Lingling Road, Shanghai, 200032, China

Tel: 86-21-54925491(O), Email: [email protected]

The modification on biological molecules, especially epigenetic modification, plays key roles in maintaining CpG DNA methylation and epigenetic code inheritance. In this report, we will discuss three topics listed below. 1) UHRF1 is an important epigenetic regulator for maintenance DNA methylation. We demonstrated two NMR complex structures of PHDUHRF1 binding to unmodified or K9 trimethylated histone tails, which clarify a controversy regarding how the binding of UHRF1 to H3 tails is mediated. Based on our structures, H3R2me0, not H3K9me3, mediates PHD binding. 2) UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Through NMR complex structure of TTD with Spacer peptide, we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. 3) DNA phosphorothioate (PT) modification is a unique epigenetic modification, in which Sulfur is incorporated into DNA backbone by the five-gene dnd cluster (dndA-dndE) products in a sequence- and stereo-specific manner. We demonstrated that DndE was a nicked dsDNA binding protein with new scaffold, while Rp-DNA phosphorothioation does not affect dsDNA B-form conformation, but with weaker antioxidation potential than Sp- phosphorothioation. DNA phosphorothioation is a mark for nuclease to to correctly cleave the PT modified DNA, so that bacteria cannot spread and survive.

References

1) Wang, CK, Cao C, et al, Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger, Cell Res, 2012, 21(9): 1379-1382. 2) Feng J, Cao C, et al, Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition, Nat Comm, 2016, 7: 11197. 3) Hu W, Cao C, et al Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification, Cell Res, 2012, 22(7): 1203-1206. 4) Lan WX, Cao C, et al, Structural investigation into physiological DNA phosphorothioate modification, Sci Reports, 2016, 6:25737.

Structure function studies of SIX3 and its mutants in the disease Holoprosencephaly (HPE) Ravi ampapathi

Only few genes i.e., Drosophila Sine Oculis (so), SIX3, eyes absent (Eya) and dachshund (Dach proteins), have been identified which are essential for compound eye formation and forms a gene network with direct protein interaction and genetic regulation controlling early vertebrate eye development. During eye development, SIX3 has been proven to be a key responsibility in the activation of Pax6, which is a master regulator of eye development. Additionally, SIX3-Homeodomain interacts with specific DNA elements, ATTA core motif in the rhodopsin promoter region and stimulates its transcription, resulting in increased rhodopsin expression. These important findings reveal that SIX3 is a direct stimulator of rhodopsin expression, thus revealing a role of SIX3/rhodopsin pathway in vertebrate eye development. In HPE patients, four different types of mutations in the SIX3-HD were identified. Of these, one is a 9-bp deletion resulting in the deletion of three amino acids in the Homeodomain, and the other 3 are missense mutations. However, lack of structure function studies left a blank in understanding the mechanism of SIX3-HD role in the eye development as well as controlling rhodopsin expression and forebrain development. We will discuss about the solution structures of SIX3-HD and its mutants. We also will share our findings of SIX3-HD, its mutants and their interactions with Rhodopsin DNA.

Exploring the conformational transition of leucine transporter using an all atom structure based Lorentzian potential model

1,2 2,3 1,2,3 Amit Srivastava, Keehyoung Joo, and Jooyoung Lee [1] School of Computational Sciences, Korea Institute for Advanced Study, Seoul, Korea [2] Center for In Silico Protein Science, Korea Institute for Advanced Study, Seoul, Korea [3] Center for Advanced Computation, Korea Institute for Advanced Study, Seoul, Korea

Leucine transporter (LeuT) is a neurotransmitter sodium symporters (NSS) and has been broadly used as a model structure for understanding the dynamic and function of neurotransmitter transporters. LeuT is crystallographically resolved in multiple states: inward-facing open (IFo), outward-facing occluded (OFc) and outward-facing open (OFo). Yet the states visited during the transition from OFo to IFo states and also the transition pathway still remained elusive. In an attempt to visualize the energy landscape of LeuT in between the OFo and IFo states, we performed both the all atom structure based Lorentzian model [1] and a recently introduced adaptive bond bending elastic network model that used the collective modes of motions predicted by bond bending elastic network model. In all atom method, the X-ray structures of OFo and IFo states are used to construct the restraints based on evolutionary information using a machine learning method [2]. The results obtained by using these methods are compared with the conventional MD simulations and experimental data.

[1] Juyong Lee, Keehyoung Joo, Bernard R. Brooks and Jooyoung Lee, The atomistic mechanism of conformational transition of adenylate kinase investigated by Lorentzian structure – based potential, J. Chem. Theory Comput., 11, 3211 (2015). [2] Juyong Lee, Kiho Lee, InSuk Joung, Keehyoung Joo, Bernard R Brooks and Jooyoung Lee, SIGMA-RF: prediction of the variability of spatial restrains in template-based modeling by random forest, BMC Bioinformatics, 16 (2015).

Structural Investigations on Outer Membrane Protein G (OmpG), Channelrhodopsin and Phytochrome Cph1 by Very Fast MAS and Dynamic Nuclear Polarisation (DNP)

Hartmut Oschkinat, FMP Berlin

Structural investigations of membrane proteins in native lipid environment by MAS NMR, including very fast spinning and the application of dynamic nuclear polarisation, are presented. Our studies involve channelrhodopsin and outer membrane protein G form e.coli, OmpG. A solid-state NMR structure of OmpG is presented that deviates from the X-ray structure with respect to loop arrangements. It was obtained by making use of data from fast MAS NMR experiments at 60 kHz spinning, and from amino acid-selectively labelled samples. The value of the various approaches will be discussed.

In future, a major factor facilitating such investigations will be dynamic nuclear polarisation (DNP), which was introduced to increase signal-to-noise by one or two orders of magnitude. During the DNP process, electron polarization is transferred to the surrounding 'core' nuclei, subsequently to the bulk nuclei, and then further on to the molecule of interest. This process depends on several factors; among them are the relaxation behavior of the electrons and protons in the sample. In order to improve the quality of DNP spectra and to obtain maximum signal-to-noise, new radicals were synthesized and employed in measurements of protein samples around 190K. Enhancements in the range of 15-20 were observed in this temperature range while acceptable spectral resolution is observed. In this context, mechanisms of polarization propagation are analyzed and schemes for optimization of samples will be presented. Applications of DNP to a membrane protein system, channelrhodopsin, and to a phytochrome will be presented. It was found that the ground state of the photo cycle of channelrhodopsin is solely consisting of the all-trans form.

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Water Confined in Nano-meter Scale Space of Polymer Electrolyte Membranes Oc Hee Han1,2,3 1Western Seoul Center, Korea Basic Science Institute, Seoul 03759, Republic of Korea 2Graduate School of Analytical Science & Technology, Chungnam National University, Daejeon 34134, Republic of Korea 3Department of Chemistry & Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea

Probing the behavior of chemical species confined in the nano-meter scale spaces can enhance our understanding on the mechanism of ion transport in electrochemical systems[1,2] and the signaling in biological systems.[3,4] Our Overhauser dynamic nuclear polarization (ODNP) nuclear magnetic resonance (NMR) data[5] indicate that water in the middle of the hydrophilic channel moves slower than those close to the channel inner walls. The 1H magic angle spinning (MAS) NMR spectra of the Nafion membranes thermally degraded with the replacement of SO3H groups to H show that the water exposed to more hydrophobic environment moves faster, which is in agreement with the ODNP results described above. In addition, the MAS NMR data manifest that the chemical shift of channel water is an indicator not only of the number of water per SO3H group but also of the degree of hydrophobicity where water is placed. References

[1] Han, O. H. Prog. Nucl. Magn. Reson. Spectrosc. 2013, 72, 1–41. [2] Kreuer, K.-D.; Rabenau, A.; Weppner, W. Angew. Chem. Int. Ed. 1982, 21, 208–209. [3] Belevich, I.; Verkhovsky, M. I.; Wikstrom, M. Nature 2006, 440, 829–832. [4] Chen, Y.; Thorn, M.; Christensen, S.; Versek, C.; Poe, A.; Hayward, R. C.; Tuominen, M. T.; Thayumanavan, S. Nature Chem. 2010, 2, 503–508. [5] Song, J.; Han, O.H.; Han, S. Angew. Chem. Int. Ed. 2015, 54, 3615–3620.

Acknowledgement: This work was supported by the National Science & Technology Council grant (DRC-14-3-KBSI) and the KBSI grants (T34419, T35419, & T35443) to O. H. Han.

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Insights into the mechanical properties of polymers by probing their functional group, and segmental motions using solid-state NMR.

Eldho K Mathai1, Neelima Bullakh2, K. Guruswamy2, Ashish Lele2, P.R. Rajamohanan1 and T. G. Ajithkumar1

1Central NMR Facility, CSIR-National Chemical Laboratory, Pune 411008, India 2Polymer Science and Engineering Division, CSIR-National Chemical Laboratory, Pune 411008, India

Predicting mechanical properties like ductility of polymers is, in general, a very difficult problem. However, it is well known that ductility of a single component amorphous glassy polymer is related to the inter- and intra-molecular cooperative segmental motions that occur in the glassy state. These, in turn, are related to the motions of functional groups in the repeat unit of the polymer. In the case of semi-crystalline polymers, the morphology, in addition is also very crucial to the mechanical property of the polymer.

In this talk the results from studies of functional group, and segmental motions in amorphous and semi crystalline polymers using solid-state nuclear magnetic resonance (SSNMR) will be presented. The separated local field NMR has been used to probe the functional group motion. The Center Band only Detection of Exchange (CODEX) experiments have been used to probe the slow segmental motions. We have carried out studies on polycarbonates and polysulfones which are purely amorphous. Results from the studies carried out on polyoxymethyline, a semi-crystalline polymer will be presented. In this study we have shown that the mechanical property is closely linked to the morphology of this polymer.

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Structure of membrane proteins by solid-state NMR Shenlin Wang College of Chemistry and Molecular Engineering, Peking University, China. Solid-state NMR (SSNMR) is emerging as a powerful tool for investigating membrane proteins. Its main advantage is that it allows for studies of membrane proteins in their native- like, lipid-embedded environments. In my presentation I will review recent progress in SSNMR methodologies towards structure determination of eukaryotic heptahelical membrane proteins, using an example of a rhodopsin from L. maculans (LR).

First, I will discuss the SSNMR sample requirements, focusing on sparsely 13C labeling approaches developed for eukaryotic methyltrophic yeast P. pastrois expression systems. These schemes have advantages of reducing the spectral line width, improving spectral resolution and facilitating in obtaining long-range distance restraints. Secondly, I will discuss the application of paramagnetic relaxation enhancements (PRE) in speeding up solid-state NMR experiments. By reconstitution LR in liposomes containing Gd3+-chelated lipids, the spin-lattice relaxation times of amide protons were shorten by 10 fold, which in turn, improves S/N ratio per time unit by 2 folds. Finally, using the improved strategies, 3D SSNMR spectra were collected to obtain resonances assignments of LR. From the sequential assignments, the secondary structures of LR were analyzed and the SSNMR based H/D exchange experiments were performed to study the topology of LR in lipids environments.

References:

1. Wang, S.; Munro, R.A.; Shi, L.; Kim, S.Y.; Okitsu, T.; Wada, A.; Jung, K.H.; Brown, L.S.; Ladizhansky, V. Nature Methods. 2013, 10, 1007–1012 2. Liu, J.; Liu, C.; Fan, Y.; Munro, R.A.; Ladizhansky, V.; Brown, L.S.; Wang, S.; J. Biomol NMR. 2016, 10, 7-13.

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1H-1H Distance Restraints in fully protonated proteins at 111 kHz and above MAS frequencies.

Vipin Agarwal

TIFR Centre for Interdisciplinary Sciences (TCIS), Hyderabad, India

High-resolution proton spectrum of fully protonated crystalline proteins can be obtained using a combination of high magnetic fields and magic-angle spinning (MAS) frequencies above 100 kHz.1 Recentely, the structure of two proteins were obtained using structral restraints observed with 1H-1H RFDR recoupling. 2,3 In this presentation, we show a new experimental method to obtain long-range proton-proton contacts in fully protonated proteins at fast MAS. The novel method demonstrates that selective 1H-1H contacts on the order of 5-6 Å can be obtained in fully protonated crystalline proteins. The new method is compared with the currently used RFDR sequence in terms of absolute sensitivity, cross-peak intensities and transfer dynamics. A systematic comparison of the experimentally observed and expected 1H- 1H contacts from the X-ray structure will be shown. Some efforts on structure calculation using the 1H-1H restraints obtained from the new experiments will also be discussed.

References:

(1) Marchetti, A.; Jehle, S.; Felletti, M.; Knight, M. J.; Wang, Y.; Xu, Z.-Q.; Park, A. Y.; Otting, G.; Lesage, A.; Emsley, L.; Dixon, N. E.; Pintacuda, G. Angew. Chem. Int. Ed. 2012, 51 (43), 10756.

(2) Andreas, L. B.; Le Marchand, T.; Jaudzems, K.; Pintacuda, G. J. Magn. Reson. 2015, 253 (C), 36.

(3) Nishiyama, Y.; Zhang, R.; Ramamoorthy, A. J. Magn. Reson. 2014, 243 (C), 25.

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SOLID STATE 13C NMR INVESTIGATIONS OF MOLECULAR ORDER AND TOPOLOGY OF THIOPHENE BASED MESOGENS

Nitin P. Lobo1, T. Narasimhaswamy2, and K. V. Ramanathan3

1Inorganic & Physical Chemistry and 2Polymer Science & Technology, CSIR-Central Leather Research Institute, Chennai-600020, India. 3NMR Research Centre, Indian Institute of Science, Bangalore-560012, India.

Thiophene ring has emerged as an essential structural moiety for the functional organic materials. A great deal of interest is generated on thiophene based thermotropic liquid crystals owing to their applications in optoelectronics and photonics. The replacement of the phenyl ring with the thiophene unit in a mesogenic core imparts a dramatic change in phase transition temperatures as well as optical properties. High-resolution solid state 13C NMR has found to be an important tool for thiophene mesogens not only for probing the molecular structure but also in finding the topology. Our studies on topologically different mesogens in which thiophene is structural motif indicated that the 13C-1H dipolar couplings are very sensitive to the location of thiophene in the core. In contrast to the phenyl ring, the insertion of thiophene brings a dramatic change in overall shape of the mesogens. To understand the 13C-1H dipolar couplings of methine carbons of thiophene ring, a new model is proposed from which the location of long axis of the mesogen can be identified. Accordingly, rod-like, U-shape, Fork-like and oligo thiophene based mesogens are examined by the static 1D and 2D 13C NMR in mesophase. The extensive 13C NMR results of thiophene mesogens are explained by considering the irregular pentagonal geometry of thiophene as against to hexagonal geometry of phenyl ring by considering the 13C-1H dipolar couplings and the local order parameter values. Thus, the replacement of phenyl ring with thiophene very much affects the molecular packing and directly influences the phase transition temperatures.

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Structure and Dynamics of Bacterial Peptidyl-tRNA Hydrolase Ashish Arora Senior Scientist Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow-31 Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm, and thereby prevents cell death by alleviating tRNA starvation. Pth are 22 kDa, monomeric, single domain proteins, formed of a central seven stranded mixed β-sheet core, which is enclosed by six α-helices. We have characterized the structure and dynamics of Pth from M. tuberculosis, M. smegmatis, and V. cholerae by using NMR spectroscopy. Our recent work on VcPth and mutants of its key residues involved in catalysis shows that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity as it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24, and are likely to be offset by positioning of a peptidyl-tRNA substrate. The NMR structures of MtPth and MsPth share the canonical Pth fold with the NMR structure of VcPth. The motional characteristics for the lid region and the tip of helix α3, as indicated by MD simulations and NMR data, are similar for these three proteins. However, there are subtle differences in the dynamics of gate loop and base loop, and in the packing of helices α5 and α6, in between these three proteins. The structural and dynamics information will be used to derive a finer understanding of the mechanism of action of this enzyme.

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Two distinct mechanisms of transcriptional regulation by redox sensor YodB

Bong-Jin Lee

College of Pharmacy, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Korea [email protected]

Abstract

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p- benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch. YodB

References 1.Lee SJ, Lee IG, Lee KY, Kim DG, Eun HJ, Yoon HJ, Chae S, Song SH, Kang SO, Seo MD, Kim HS, Park SJ, Lee BJ, Proc Natl Acad Sci USA, ES202~211, 2016

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F1 Bilinear Rotation Decoupled ultra-high resolution diagonal suppressed Total Correlation Spectroscopy

Ajay Vermaa, Bikash Baishyaa

aCenter of Biomedical Research (Formerly Centre of Biomedical Magnetic Resonance), SGPGIMS Campus, Raebareli Road, Lucknow, 226014, India

We report an ultra-high resolution diagonal suppressed F1 Bilinear Rotation Decoupled 1 Total Correlation technique (DS F1 BIRD TOCSY) and its application to complex mixture . Diagonal free homonuclear correlation spectra can be obtained by using heteronuclei at natural abundance or selectively heteronuclei enriched samples2. The method allows identification of cross peaks close to the diagonal which is otherwise obscured by the intense diagonal in regular homonuclear correlation schemes (and leads to loss of correlation information). However, suppression of the diagonal using heteronuclei results in a significant sensitivity penalty in natural abundant samples- two orders of magnitude relative to regular homonuclear correlation when utilizing 13C. Herein, we show that implementation of BIRD in indirect dimension of such diagonal suppressed TOCSY experiment can greatly improve sensitivity and resolution by converting the multiplets into singlets along F1 (except in diastereotopic methylene moieties). Application of the method to a mixture of eight aromatic compounds reveals better identification of cross peaks close to the diagonal relative to other existing TOCSY variants due to the pure shift indirect dimension along with diagonal suppression.. Further, comparison to F1 PSYCHE TOCSY revealed cleaner decoupling effect of BIRD relative to PSYCHE in complex mixture, although F1 PSYCHE TOCSY has higher sensitivity than the former. The covariance processed doubly pure shift ultra-high resolution total correlation maps were also investigated.

References: [1] Verma A, Baishya B*, Submitted to J. Magn. Reson. [2] Baishya B, J. Magn. Reson, 2015, 256, 52-59

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Toxins from TA system of Helicobacter pylori and insight into mRNase activity

Chinar Pathak1, Hookang Im1, Sun-bok Jang1, Ae-Ran Kwon2, and Bong-Jin Lee1

1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 151-742, Korea. 2Department of Herbal Skin Care, Daegu Haany University, Daegu, Korea

The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. The HP0892- HP0893 and HP0894-HP0895 are the only two known TA systems belonging to Helicobacter pylori. In both of these TA systems, the antitoxin binds and inhibits the toxin and regulates the transcription of the TA operon. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remains unclear. Therefore, here an attempt was made to shed some light on the mechanism behind the TA system of HP0892-HP0893 and HP0894-HP0895. Here, we present the crystal structures of apo- and copper-bound HP0894 at 1.28 Å and 1.89 Å, respectively, and the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues involving the mRNase active site was shown. NMR perturbation experiments traced the involved residues in mRNase active site, and mutational analysis coupled with ITC measurement confirmed the involved residues. In the mRNase active site of toxins, the most catalytically important residue His84 of HP0894 or His86 of HP0892 reorients itself acting as a general acid in an acid-base catalysis reaction, while Glu58 acts as a general base. His47 and His60 of HP0894 and/or HP0892 are considered to be involved in enzymatic activity that stabilizes the transition state.

Keywords: Toxin-antitoxin system; Helicobacter pylori; metal.

References: 1. Im, H.*, Jang, SB.*, Pathak, C.*, Yang, YJ., Yoon, HJ., Yu, TK., Suh, JY., and Lee, BJ. (2014) Crystal structure of toxin HP0892 from Helicobacter pylori with two Zn(II) at 1.8 Å resolution, Protein Science, 23(6), 819-832 (*equal contribution). 2. Pathak, C.*, Im, H.*, Yang, YJ., Yoon, HJ., Kim, HM., Kwon, AR., and Lee, BJ. (2013) Crystal structure of apo and copper bound HP0894 toxin from Helicobacter pylori 26695 and insight into mRNase activity, Biochim Biophys Acta, 1834(12), 2579-2590 (*equal contribution).

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NMR Studies of Hierarchical Protein Dynamics

Jayasubba Reddy Yarava a, Baptiste Busia, Albert Hofstettera, Martin Blackledgeb and Lyndon Emsleya*

aInstitut des Sciences et Ingènierie Chimiques, Ecole Polytechnique Fèdèrale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland bUniversité Grenoble Alpes, Institut de Biologie Structurale Jean-Pierre Ebel (IBS), CEA- CNRS-UJF, F-38044 Grenoble, France.

*Corresponding author E-mail address: [email protected] (L.Emsley)

A fundamental challenge in biology is to understand the complex interaction between protein motion and protein function. Recently, Lewandowski and coworkers have shown that temperature dependent magic angle spinning multinuclear solid state NMR relaxation measurements at temperatures ranging from 105 to 280 K can provide a window into the hierarchy of dynamic processes in prioteins.1 Since it is likely that the quantitative description of motions reporting on different components of the system occurring in the protein and the solvent are dependent on a well-controlled experimental conditions, here we evaluate the reproducibility of such measurements, and extend the measurements to additional probes and to other magnetic fields.

We measured at 11.7 T (500 MHz) the same set of sixteen different NMR observables as the previous study twice for each of twenty different temperatures in the range from 105 to 285 K in the fully hydrated crystalline protein GB1 (1H,13C,15N). Our measurements are interpreted using the same analysis as previously and we find excellent reproducibility between the three measurements.

In order to better understand the influence of the magnetic field over the relaxations processes, the same measurements were performed at different magnetic fields, 9.4, 11.7, 14.1 and 18.8 T (400, 500, 600 and 800 MHz, same observables and temperature) with the same experimental protocol. At all fields we are able to identify the same dynamics. Each motion can be characterized by three parameters including the energy of activation. Those information show transitions happening, where different motional process arises together, because their energy of activation is very close.

1.Lewandowski, J. R.; Halse, M. E.; Blackledge, M.; Emsley, L., Direct observation of hierarchical protein dynamics. Science 2015, 348 (6234), 578-581.

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Nonmonotonic sweet taste and molecular conformation of the natural high potency sweetener Rebaudioside A

Bipul Sarma

Department of Chemical Sciences, Tezpur University, Napaam-784028, Assam, India E-mail: [email protected]

The diterpene steviol glycoside, rebaudioside A, is a natural high potency non-caloric sweetener extracted from the leaves of Stevia rebaudiana. This compound shows a parabolic change in sweet taste intensity with temperature which contrasts with the general finding for other synthetic or natural sweeteners whose sweet taste increases with temperature.1 The nonmonotonic taste behavior was determined by sensory analysis using large taste panels. The conformational landscape of rebaudioside A was established at a range of temperatures by means of nuclear magnetic resonance and molecular dynamics simulation. The relationship between various conformations and the observed sweetness of rebaudioside A is described.2

Figure 1 The molecular structure of the Figure 2 Parabolic sweetness variation of natural high potency sweetener rebaudioside-A as a function of temperature was rebaudioside A evaluated by two-alternative forced choice (2- AFC) discrimination tests against sucrose standards with panel comprised of minimum 70 untrained tasters

Reference 1. A. Cruz, B. G. Green, Nature, 2000, 403, 889. 2. P. D. Chopade, B. Sarma, E. E. Santiso, J. Simpson, J. C. Fry, N. Yurttas, K. L. Biermann, J. Chen, B. L. Trout, A. S. Myerson, J. Chem. Phys. 2015, 143, 244301.

111

Cholesterol-mediated allosteric regulation of the structure of membrane-embedded translocator protein from solid-state NMR

Garima Jaipuria1, Andrei Leonov2, Karin Giller2, Rasmus Linser2, Stefan Becker2 & Markus Zweckstetter1,2,3

1 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Von-Siebold-Str. 3a, 37075 Göttingen, Germany. 2 Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany. 3 Department of Neurology, University Medical Center Göttingen, University of Göttingen, Waldweg 33, 37073 Göttingen.

The 18 kDa translocator protein (TSPO) is mainly found on the outer mitochondrial membrane of steroid synthesizing cells 1. TSPO was first described as a peripheral benzodiazepine receptor, a secondary receptor for diazepam. TSPO was subsequently suggested to be important for the transport of cholesterol into mitochondria. Expression of TSPO is strongly up-regulated in areas of brain injury and in neuroinflammatory conditions including Alzheimer‘s and Parkinson‘s disease. TSPO ligands have potential diagnostic and therapeutic applications such as attenuation of cancer cell proliferation and neuroprotective effects. The structures of mammalian and bacterial TSPO protein in different oligomerisation states are now available2, though an insight into how TSPO regulates the transport of cholesterol is missing. Here, we present the latest developments on the study of TSPO in a lipid bilayer to obtain insight into its structural modulation in presence of cholesterol.

1. Rupprecht, R.; Papadopoulos, V.; Rammes, G.; Baghai, T. C.; Fan, J.; Akula, N.; Groyer, G.; Adams, D.; Schumacher, M., Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nature Reviews Drug Discovery 2010, 9 (12), 971-988. 2. (a) Jaremko, L.; Jaremko, M.; Giller, K.; Becker, S.; Zweckstetter, M., Structure of the Mitochondrial Translocator Protein in Complex with a Diagnostic Ligand. Science 2014, 343 (6177), 1363-1366; (b) Li, F.; Liu, J.; Zheng, Y.; Garavito, R. M.; Ferguson-Miller, S., Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism. Science 2015, 347 (6221), 555-558; (c) Guo, Y. Z.; Kalathur, R. C.; Liu, Q.; Kloss, B.; Bruni, R.; Ginter, C.; Kloppmann, E.; Rost, B.; Hendrickson, W. A., Structure and activity of tryptophan-rich TSPO proteins. Science 2015, 347 (6221), 551-555.

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STUDENT ORAL PRESENTATION

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Structural investigations on Helicobacter pylori-Hup and Designing its inhibitors as starting precursors for developing Next Generation Antibiotics Nancy Jaiswal1, Nisha Raikwal1,H.Pandey3, Ashish Arora3, Krishna Mohan Pulori2 and Dinesh Kumar1*

1 Centre of Biomedical Research, SGPGIMS Campus, Lucknow 226014, India 2 Indian Institute of Technology, Haridwar Highway, Roorkee, Uttarakhand- 247667, India 3 CSIR-Central Drug Research Institute, Lucknow 226001, India

*Dr. Dinesh Kumar: [email protected]

Major global healthcare issues in the 21st century are due to infections caused by drug resistant bacteria including Helicobacter pylori (H. pylori). H. pylori is the etiological agent of several human stomach illnesses including gastritis, duodenal/peptic ulcers, and gastric cancer. Half of the world population is chronically infected with H.pylori. Clinically, the H.pylori infections are treated using a combination of different antibiotics. But due to increasing drug resistance it is of utmost importance to find new drugs with novel mode of action for effective eradication of H.pylori infections to cure related gastric pathologies1 In the above context, the Histone like DNA binding protein of H. pylori –referred here as Hup– has been recognized as a novel drug target owing to its capabilities to perform both architectural as well as regulatory cellular functions. Our study (by NMR) has revealed that the Hup (a) exhibits dynamic equilibrium between monomer/dimer in solution and (b) undergoes pH dependent conformational changes. The conformation at low pH (3.5) was found to be very close to the homology modelled structure and thermodynamically most stable. Whereas, at physiological pH, the β- arms forming the DNA binding site were largely disordered. These results led to a hypothesis that at near physiological pH, the beta-arms are highly flexible rendering low affinity for DNA; thus allowing DNA to perform various regulatory and other cellular metabolic activities. Whereas, at acidic pH (where DNA has tendency to precipitate/degrade), the b-arms become structurally stable and inclined towards the DNA binding groove; thus providing mechanical clamping mechanism for its efficient binding to DNA. This low pH conformation of Hup is capable of wrapping DNA around it to form a sturdy nucleoid structure and thus protects it from acidic denaturation. Thus, the protein provides an additional mechanism for H. pylori to rescue and colonize persistently under harsh conditions 114 of human stomach characterized by acidity (transient pH fluctuations). Therefore, suppressing this Hup mediated additional rescue mechanism by the application of inhibitors present a promising mean to limit H. pylori infection in humans and a step towards developing next generation anti-biotics following the rational structure based drug discovery approach.

1 Jaiswal,N.; Raikwal, N.; Pandey,H.; Arora,A.; Ghosh, A.; Bhunia A.; Gulati,K.; Gangele, K.; Poluri, K.; Kumar, D.*, Structural and mechanistic investigations of histone like DNA binding protein (Hup) of Helicobacter pylori, F1000Research 2016, 5:1656 (poster) (doi:10.7490/f1000research.1112555.1)

115

Site Specific Hydration Topology of Native Collagen Protein in Natural Abundance

Akhila Viswan1,2, Neeraj Sinha1

1Centre of Biomedical Research, Lucknow, India 2Faculty of Engineering and Technology, Dr. A. P. J Abdul Kalam Technical University, Lucknow, India

Structural account of the coiled coil triple helical collagen dates back to 60 years but surfeit of information is still pouring in paving grounds for further functional and dynamical insight in native state. Solid state NMR (ssNMR) has emerged out to be one of the powerful techniques providing insight into the insoluble structural protein like collagen1,2 exhibiting significant dynamical fluctuations which are difficult to interpret through conventional physical techniques. ssNMR holds unprecedented advantage over other techniques in measuring multiple anisotropic dependent interaction and deciphering the 13C chemical shift anisotropy (CSA) tensors of complex biological system. 13C CSA tensorial information governs structural parameters in terms of probing molecular motion, orientation and alignment. In our study we demonstrate CSA recoupling employing SUPER3, a robust technique which works under standard MAS condition with CSA scaling factor of 0.155 thereby facilitating a quasi-static powder pattern of native collagen in indirection dimension. This technique offers advantage in terms of MAS resolution whilst retaining anisotropic powder pattern for accurate determination of principal components from inequivalent carbon nuclei well resolved in 13C natural abundance sample. We will be reporting the variation in the tensor components of backbone 13Cα and 13CO regions and sidechain residues of collagen in fresh, dehydrated and D2O exchanged sample of bone recorded in 600 MHz ssNMR spectrometer operating at 150.14 MHz for 13C frequency. Reintroducing CSA under MAS utilizing rotor synchronized 360˚ pulses correlates isotropic chemical shift with the CSA interaction tensor which is obtained as powder pattern in the indirect dimension. The recoupled CSA of glycine, proline, hydroxyproline will yield better interpretation of collagen conformation in terms of dynamics. Structural variation of the shielding tensors exemplified by such rigid yet flexible connective tissue paves way for more direct interpretation of hydrogen bonding network and hydration topology4 of collagen in its native state.

116

References: (1) Rai, R. K.; Sinha, N. J. Phys. Chem. C 2011, 115, 14219−14227. (2) Rai, R. K.; Singh, C.; Sinha, N. J. Phys. Chem. B 2015, 119, 201−211. (3) Liu, S. F.; Mao, J. D.;Schmidt-Rohr, K. J. Magn. Reson. 2002, 155,15-28 (4 ) Wei, Y.; Lee, D.-K.; Ramamoorthy, A. J. Am. Chem. Soc. 2001, 123, 6118- 6126

117

Structural-functional aspects of DRBs in gene regulation of Arabidopsis thaliana.

Upasana Rai, C. Sai Chaitanya, Ramdas Aute and Mandar V. Deshmukh,

Centre for Cellular and Molecular Biology, Hyderabad – 500 007, Telangana, India

.Arabidopsis thaliana has an elaborated and complex RNAi pathway involving four Dicers (DCL1-DCL4) and corresponding five dsRNA-binding domains containing proteins (DRB1-DRB5). DCL1:DRB1 regulates miRNA biogenesis leading to transcript cleavage as primary gene regulation mechanism during developmental stages[1]. Interestingly, under stress, DRB2 outcompetes DRB1 to form DCL1:DRB2 complex which dictates translation inhibition[2,3]. Additionally, DCL3:DRB3 and DCL4:DRB4 regulate siRNA mediated cognate mRNA cleavage[4]. DRBs contain two N- using a tripartite contact. We attempt to understand the structural and mechanistic details that induce the specificity to the seemingly conserved DRBs and corresponding Dicers.

Solution structural studies on DRB2 suggest that dsRBD1 (DRB2D1) adopts a canonical dsRBD fold, whereas, dsRBD2 (DRB2D2) appears to form higher order oligomer. ITC studies for DRB2 suggest that DRB2D2 is the canonical dsRBD fold and is implied as the preferred domain for RNA binding. For DRB4, DRB4D1 binds dsRNA with higher affinity than DRB4D2 due to favora -ms dynamics located at

DRB4D2 during RNA binding, hence, compromising DRB4D2‘s affinity for dsRNA[5].

The study shows that despite higher sequence similarity, subtle structural differences and interdomain dynamics confer DRBs functional variability in which only one dsRBD drives the RNA binding activity while the other may be involved in Dicer or protein:protein interactions.

References:

1. Rodolfo M. Rasia, Julieta Mateos, Nicolas G. Bologna, Paula Burdisso, Lionel Imbert, Javier F. Palatnik and Jerome Boisbouvier. Structure and RNA Interactions of the Plant MicroRNA Processing-Associated Protein HYL1. Biochemistry. 2010, Aug 24; 49, 8237–8239 8237 2. Eamens AL, Kim KW, Curtin SJ, Waterhouse PM. DRB2 is required for microRNA biogenesis in Arabidopsis thaliana. PLoS One. 2012;7(4):e35933. 3. Reis RS, Hart-Smith G, Eamens AL, Wilkins MR, Waterhouse PM. MicroRNA Regulatory Mechanisms Play Different roles in Arabidopsis. J Proteome Res. (2015). 4. Raja P, Jackel JN, Li S, Heard IM, Bisaro DM. Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses. J Virol. 2014 Mar;88(5):2611-22. 5. C. Sai Chaitanya, Ramdas Aute and Mandar V Deshmukh, Communicated, 2016.

118

Interaction of a dimeric Cyclophilin A with Myb1 transcription factor in Trichomonas vaginalis

Tesmine Martin1, 2, 3,Chun-Chi Chou1,4, Shu-Yi Wei1, Jung-Hsiang Tai1, Chun-Hua Hsu1,4, and Chinpan Chen1

From 1Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan 2Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica. Taipei 115, Taiwan 3Department of Chemistry, National Tsinghua University, Hsinchu 300, Taiwan 4 Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan Trichomoniasis, caused by Trichomonas vaginalis is the most common non-viral sexually transmitted disease in humans and is correlated with elevated susceptibility to HIV and HPV transmission. Reliance on a single class of antimicrobial drugs for treatment can worsen the existing problem of drug resistance and hence novel therapeutics are needed1. A recent study revealed that in T. vaginalis, TvCyclophilin A1 (TvCyP1), a homologue of human cyclophilin A, interacts with Myb1 and regulate the release of Myb1 from membrane bound vesicles before being translocated to the nucleus2. Myb1 along with Myb2 and Myb3, control the expression of a hydrogenosomal enzyme, which helps in the parasitic cytoadherence to human vaginal epithelial cells. We have sought to NMR, X-ray crystallography and other biophysical experiments to obtain a detailed understanding of the interaction. Our studies show that TvCyP1 forms a symmetric homodimer with the active sites far away from the dimer interface leading to 1:1 binding of Myb1 to each monomer of the dimeric TvCyP1.We have used NMR titrations and cross saturation transfer experiments to determine the binding site in both proteins. While Myb1 possess a minimum TvCyP1 binding motif, TvCyP1 exhibits specific binding to this region of Myb1. Furthermore, ROESY experiments confirm that TvCyP1 is a functional PPIase and can catalyze the cis/trans isomerization of the GP bond of Myb1 peptide. Determination of the TvCyP1- Myb1 peptide complex throw further light into the design of a specific inhibitor for TvCyP1 which can in fact act as a potential antiparasitic drug.

References

1. Dunne, R. L.; Dunn, L. A.; Upcroft, P.; O'Donoghue, P. J.; Upcroft, J. A., Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis. Cell Res 2003, 13 (4), 239-49. 2. Hsu, H. M.; Chu, C. H.; Wang, Y. T.; Lee, Y.; Wei, S. Y.; Liu, H. W.; Ong, S. J.; Chen, C.; Tai, J. H., Regulation of nuclear translocation of the Myb1 transcription factor by TvCyclophilin 1 in the protozoan parasite Trichomonas vaginalis. J Biol Chem 2014, 289 (27), 19120-36.

119

Recombinant hIAPP (rhIAPP) forms smaller aggregates and is more cytotoxic than synthetic hIAPP – leads obtained from biophysical and cellular studies Richa Dubey1, Pooja Minj2, Nikita Malik1, Shilpy Sharma2, Ashutosh Kumar1

1Department of Biosciences and Bioengineering, IIT Bombay, Powai-400076 2Department of Biotechnology, Savitribai Phule, Ganeshkhind, Pune, Maharashtra-411007

Type 2 Diabetes (T2DM) is one of most prevalent endocrine disorder that is characterized by peripheral insulin resistance, along with deterioration in function of β-cell function and their loss. Human Islet Amyloid Polypeptide (hIAPP1-37); a glucose metabolism associated polypeptide hormone is co-secreted with insulin from secretary granules of pancreatic β-cells under normal conditions [1]. However, in pathological conditions, hIAPP plays a crucial role in amyloid deposition which further leads to β-cell apoptosis [2]. This amyloidogenic protein undergoes conformational transition from its natively unfolded state to the cross β- sheet amyloid fibril [3]. The exact mechanism of hIAPP- induced β-cell apoptosis is not entirely understood till date. To further understand the mechanism of hIAPP- mediated toxicity and to gain structural insight into hIAPP aggregation, it is mandatory to elucidate the structure and function of hIAPP amyloid assemblies.

In this presentation, we report a novel, efficient and cost-effective method for production of uniformly labelled 13C-15N hIAPP fibrils for solid state NMR experiments. The amyloid fibrils thus formed were characterized morphologically by TEM and AFM imaging. Results from biophysical assays confirmed the β- sheet structure of the fibril. The cytotoxic response of hIAPP fibril on INS-1E β-cells was investigated using the MTT cytotoxicity assay and Annexin V staining. rhIAPP fibrils were more potent than synthetic hIAPP in inducing apoptosis in INS-1E cells. An IC50 of 2.4 μM was observed for rhIAPP while it was 5 μM for synthetic peptide. However, cell death was associated with increased oxidative stress, lipid per oxidation and DNA damage in both cases, thereby depicting a similar mechanism of action. In support of these findings, AFM imaging showed that rhIAPP forms smaller aggregates that can be the cause of increased cytotoxicity associated with rhIAPP when compared with synthetic hIAPP. Solid-state NMR magic angle spinning (ssNMR-MAS) studies are currently further being carried out to gain a better understanding into the structural details of these fibrils at the atomic level.

References-

[1] P.Westermark, A.Andersson, G.T.Westermark, Physiol Rev. 91,795-826 (2011). [2] G.J.Cooper, A.C.Willis, A.Clark, R.C.Turner, R.B.Sim, K.B.Reid, Proc. Natl. Acad. Sci. 84,8628-8632 (1987). [3] R.Kayed, J.Bernhagen, N.Greenfield, K.Sweimeh, H.Brunner and W.Voelter, J. Mol. Biol. 287,781-796 (1999).

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Mapping Conformational Dynamics of Transthyretin by Solution NMR Jitendra K. Das, Aritra Bej, Shyam S. Mall, and Sujoy Mukherjee*

Structural Biology and Bioinformatics Division, CSIR - Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal - 700032, India *E-mail: [email protected]

Destabilizing mutations in transthyretin (TTR) induce conformational changes in the native structure leading to tetramer dissociation and formation of partially unfolded intermediates [1]. The significance of these conformational changes caused by amyloidogenic mutations is not well understood as the amyloidogenic propensity cannot be explained by the gross structural difference [2]. In previous work, we have shown a correlation between conformational flexibility and the formation of non-native intermediates with pathogenic propensity of TTR using NMR spectroscopy [3]. Quantifying the backbone dynamics over an extended timescale revealed that the solvent exposed regions of TTR exhibit higher flexibility at picosecond- nanosecond timescale in comparison to the residues at the β-sheets of the central core. MD simulation performed up to 1μs to gain atomistic insights showed good agreement with NMR studies. In contrast, conformational fluctuations at slower (~millisecond) timescale are almost exclusively located in the β-sheets forming the hydrophobic core of the tetramer. The slower motions collectively modulate an exchange process between the native and non-native states, where disease causing mutants were found to have higher population of intermediate states with lower free energy and faster kinetics than the wild-type and the protective mutant.

Acknowledgements: 800 MHz NMR facility at TIFR (Mumbai), CSIR-4Pi Supercomputing facility, & CSIR, ICMR, and DST for funding. References: [1] Hou, X.; Aguilar, M.I.; Small, D. H. FEBS J. 2007, 274, 1637–1650. [2] Hörnberg, A.; Eneqvist, T.; Olofsson, A.; Lundgren, E.; Sauer-Eriksson, A. E. J. Mol.Biol. 2000, 302, 649–669. [3] Das, J. K.; Mall, S. S.; Bej, A.; Mukherjee, S. Angew. Chem. Int. Ed. Engl. 2014, 53 (47), 12781–1278

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Mechanistic insights into the action of nano-conjugates

Indrani Pal, Dipita Bhattacharyya, Rajiv Kumar Kar, Anirban Bhunia and Hanudatta S. Atreya.

It is well established that antimicrobial peptides act as pore-formers to rupture the bacterial cells. This work is focused on studying the mechanism of action of the nano-conjugate with bacterial membrane mimic models. This study for the first time reveals the details of nanoconjugate membrane interaction at an atomic level. The pore formation mechanism and the enhanced efficiency of the nanoconjugate were explored using fluorescence spectroscopy, CD spectroscopy, and NMR spectroscopy. Structural changes of the peptide and the nanoparticle bound peptide have been captured which infers the propensity of the peptide to form a helical structure upon interacting with the membrane. The calculated structure of the peptide and nanoparticle bound peptide remains almost identical in presence of the membrane mimic environment. In the case of the nanoconjugate, the increase in local positive charge concentration makes the system to penetrate the bacterial membrane faster which further allows the nanoparticle to access the intercellular organelles easily. This dual mode of mechanism thus makes this nano-conjugate a promising antibacterial agent towards multi drug resistant bacteria.

References:

(1) Brahmkhatri, V. P., Chandra, K., Dubey, A., and Atreya, H. S. (2015) An ultrastable conjugate of silver nanoparticles and protein formed through weak interactions. Nanoscale 7, 12921–31. (2) Pal, I., Brahmkhatri, V. P., Bera, S., Bhattacharyya, D., Quirishi, Y., Bhunia, A., and Atreya, H. S. (2016) Enhanced stability and activity of an antimicrobial peptide in conjugation with silver nanoparticle. J. Colloid Interface Sci. 483, 385–393. (3) Brogden, K. a. (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250.

122

Complete eradication of axial peaks and Falls couplings with Ultrahigh resolution in an NMR correlation experiment

Sandeep Kumar Mishra and N. Suryaprakash

NMR Research Centre, Indian Institute of Science, Bangalore-560012

Couplings between two nuclei is crucial for accurate structural assignment and conformational information. More often the large number of 1H-1H couplings and small dispersion of hydrogen chemical shift, results the severe overlap of peaks hampering the n 1 accurate measurement of JHH from the 1D H NMR spectrum. The most of the J-correlation experiments reported for coupling extraction suffer from the intense axial peaks and undesirable extra couplings creating ambiguity in the peak assignment and accurate couplings measurement. Due to small chemical shift dispersion and complicated coupling pattern 1 n restrict the resolution of H NMR experiments that also affect the preciseness of JHH measurement. In this work we have developed two new different 2D NMR experiments which are able to eradicate all the axial peaks and unwanted couplings, while retaining only the couplings of selected proton to its partners with achieving the high resolution by homonuclear broadband decoupling during acquisition time. The application of this novel method is confirmed by extracting the couplings from the overcrowded spectra of complex molecules and the molecular mixtures.

References 1. Giraud, N.; Béguin, L.; Courtieu, J.; Merlet, D. Angew. Chem. Int. Ed. 2010, 49, 3481; Angew. Chem. 2010, 122, 3559. 2. Giraud, N.; Pitoux, D.; Ouvrard, J. M.; Merlet, D. Chem. Eur. J. 2013, 19, 12221. 3. Sinnaeve, D.; Foroozandeh, M.; Mathias, N.; Morris, G. A. Angew. Chem. Int. Ed. 2016, 55, 1090.

123

Pyrimidine favored recognition by RRM1 of PfSR1

Akshay Kumar Ganguly, Garima Verma, Neel Sarovar Bhavesh*

Transcription Regulation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110 067 India

The complexity of proteome of Plasmodium falciparum, which is quite diverse despite a genome size of merely 23 mega base pairs, encoding 5300 genes is a big challenge to drug and vaccine development. This proteomic diversity is majorly due to the process of alternative splicing (AS). AS leads to synthesis of different mature mRNA isoforms and consequently, different translation products, from the same pre-mRNA transcript.

The P. falciparum Ser/Arg-rich protein 1(PfSR1) is the first protein to be functionally characterized as an alternative splicing factor in any Apicomplexans. Here, we have performed study to understand molecular basis of RNA recognition by PfSR1 alternative splicing factor. We have determined its three-dimensional solution-structure if free and RNA bound state and used thermodynamic parameters to understand the specificity and affinity of RNA recognition by PfSR1. This is the first structure of a binary protein-RNA complex from any Apicomplexans. Mutagenesis and calorimetry studies suggest RNA recognition motifs-1 (RRM1) of PfSR1 is biased toward pyrimidine during RNA recognition.

124

An Unusual Calcium Binding Protein From Entamoeba histolytica That Exhibits GTPase Activity

Deepshikha Verma,1 Aruna Murmu2, Alok Bhattacharya2 and Kandala V. R. Chary1

1Tata Institute of Fundamental Research, Mumbai, India and TIFR Center for Interdisciplinary Sciences, Hyderabad, India 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

GTPases are a large family of hydrolysing enzymes which act as molecular switches and regulate a wide variety of signal transduction pathways. They participate in the regulation of cell polarity, gene transcription, G1 cell cycle progression, microtubule dynamics, vesicular transport pathways and a variety of other enzymatic pathways. GTPases are involved in cell- cycle regulation. They regulate cytokinesis by regulating assembly of the actin and myosin contractile ring, which constricts to form the two daughter cells. Here, we characterize a Ca2+-binding protein (EhCaBP6) from E. histolytica, as a novel GTPase. EhCaBP6 hydrolyses GTP without any sequence similarity or domain organization that resembles known GTPases. EhCaBP6 is mainly localized in the nucleus and present at the microtubule end and intercellular bridge during cytokinesis. It forms a two-domain structure similar to CaM and requires full length protein for its GTPase activity. EhCaBP6 shows overall conformational change upon binding to GTP. The fact that the cell cycle of E. histolytica showed many unusual features, and also the kinases and GTPases vary as compared to other eukaryotes, the Ca2+ dependent GTPase activity of EhCaBP6 may have a bearing in the unusual biology of this organism.

125

Characterizing Quantum Correlations, Beyond Entanglement, on an NMR Quantum Information Processor

Amandeep Singh, Arvind, Kavita Dorai*

Department of Physical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, P.O. Manauli, Punjab-140306, India *[email protected]

Key words: Non-Classical Correlations, Positive Maps.

Quantum correlations are at the heart of almost all quantum computational tasks utilized to achieve computational speedup in contrast to classical computations. Entanglement is one of the most explored type of quantum correlation but is not the only one. There have been cases where certain types of separable states showed quantum correlations, captured by quantum discord [1]. Creation of quantum states possessing such quantum correlations on physically realizable systems and thereby detecting them are of utmost importance in experimental quantum-information-processing research. In the present study we experimentally create a class of Bell diagonal states [2], which are otherwise separable with a non-zero quantum discord, and detect the presence of quantum correlations with a recently suggested method of positive map [3].

References:

[1] H. Ollivier and W.H. Zurek, Phys. Rev. Lett. 88, 017901 (2001) and L. Henderson and V. Vedral, J. Phys. A 34, 6899 (2001). [2] M. D. Lang and C. M. Caves, Phys. Rev. Lett. 105, 150501 (2010). [3] R. Rahimi and A. SaiToh, Phys. Rev. A 82, 022314 (2010).

126

Altered metabolism in Parkinson’s disease based on serum metabolomics

Sadhana Kumari, S. Senthil Kumaran

Department of NMR, All India Institute of Medical Sciences, New Delhi

Introduction: Parkinson‘s disease (PD) is a neurodegenerative disorder caused by dopaminergic loss of neurons. We used 1H NMR technique to investigate the metabolic profile in sera samples, with the hypothesis that the some metabolites would be different in PD as compared to controls. Methods: Blood samples from PD patients (n= 20; mean age: 57 ± 7.0 years) and healthy controls (HC) (n=10; mean age: 49 ± 2.22 years) after 12 hours fasting were obtained. Proton NMR spectra were acquired using 700 MHz NMR spectrometer (M/s. Agilent Technologies, USA) with 1D CPMG using a 90˚ pulse, echo time=15ms; 64 scans; relaxation delay=70s. Data were estimated using MestReNova software (version10.0, Mestrelab Research, Spain) and Multivariate partial least square discriminate analysis (PLS-DA) was carried out using MetaboAnalyst, a web-based metabolomics data processing tool.

Figure 1. (A) PLS-DA plot of PD (red colour) and HC (green) in 3D representation and (B) representative 1D NMR spectra (red: PD, Black: HC). Results and Discussion: Fifteen metabolites were assigned and integral values evaluated. Significant increase in Lactate, Glutamine, Methyl Guanidine and decrease in Tryptophan were observed in PD as compared to HC (non-parametric t- test - Wilcoxon rank-sum test). PLS-DA analysis depicts clear separation between PD and HC (Figure 1). Lower levels of Tryptophan suggest elevated oxidative stress1, higher levels of Lactate suggest an affected energy and glucose metabolism, elevated Methyl Guanidine concentration may be attributed to oxidative stress and protein metabolism impairment2 and increased levels of glutamate may be ascribed to impaired mitochondrial function, due to increased vulnerability of affected neurons3 in PD as compared to healthy controls. References 1. Hatano, Taku, et al. Journal of Neurology, Neurosurgery & Psychiatry 2015 2. Raquel D, et al. Neurodegenerative Diseases 2011; 8: 109-116 3. Beal, MF, Lang AE, and Ludolph AC. Neurodegenerative Diseases: Neurobiology, Pathogenesis and Therapeutics. Cambridge University Press, 2005; ISBN: 9780511113741

127

Noise Suppression in Spin Noise NMR using Wavelet Transform Analysis

D.Adilakshmi1, 2, Kousik Chandra 2 and K. V. Ramanathan,2

Department of Physics1, NMR Research Centre2, Indian Institute of Science, Bangalore-12

([email protected])2

The fluctuations in spin systems are to be viewed as an intrinsic quantum mechanical property of systems immersed in random magnetic environment's and are observed as spin noise in the absence of any radio frequency excitation[1,2]. Spin Noise NMR bears great application potential at low spin number regime as it exceeds thermal polarization. Here we have introduced wavelet transform to overcome the inherent low sensitivity of Spin Noise NMR for high spin numbers by distinguishing real peaks from the noise contaminated NMR data. Symlet wavelet transform as a discrete and orthogonal wavelet transform (DWT) are used for noise suppression along with the soft threshold function, used for obtaining smooth denoised spectra [3]. We are also exploring application of denoising NMR spectra through wavelet transforms to metabolomics. The details will be presented.

References

[1] F. Bloch. Phys. Rev., 1946,70,460–474.

[2] Tycho Sleator, Erwin L. Hahn, Claude Hilbert, and John Clarke. Phys.Rev. Lett., 1985, 55,1742–1745.

[3] Xinmin Ge, Yiren Fan, Jiangtao Li, Yang Wang, and Shaogui Deng.Noise reduction of nuclear magnetic resonance (nmr) transversal data using improved wavelet transform and exponentially weighted moving average(ewma). Journal of Magnetic Resonance,2015,251,71–83.

128

Amyloid betaA peptide fragments analysis by NMR

Dillip Kumar Senapati a,b, Priya Narayan c, R. Dongre a, T. Nireekshana a, S. Raghothama a a NMR Research Centre, Indian Institute of Science, Bangalore – 560012, India. b Department of Physics, Indian Institute of Science, Bangalore – 560012, India. c Department of Bio-Technology, Sir M.Visvesvaraya Institute of Technology, Bangalore – 562 157, India.

Email id - [email protected], [email protected]

Polymerization of the β-amyloid (ADAEFRHDSGYEVHHQKLVFFAEDVGS- NKGAIIGLMVGGVVIA) peptide into fibrils is hypothesized to be a causative event in the development of Alzheimer‘s Diseases. Structural information of β-amyloid fibrils formation is fundamental for elucidating mechanism for protein folding and assembly of A. We plan to undertake such analysis by obtaining fragment structures starting from N-terminal and systematically increasing the fragment size and look for any deviation in propagation of structure as the length increases. Such analysis is also useful for development of diagnostics and therapeutic approaches as we wish to study interaction of these A peptide fragments with different metal ligands [1].

We have collected 1D and 2D NMR data on A1-6(DAEFRH), A1-8, A1-10, A1-12, and A1-16. We are in the process of collecting data on few more fragments. We also plan to carry out metal titration NMR data on all these fragments. Resonance assignments are done using combination of TOCSY and ROESY spectrum. First indication from Chemical shift Index plot comparison for all fragments shows no drastic discrepancies in deviation of structures.

Solid state NMR [2] analysis on these fragments is yet another approach we wish to pursue as it is well suited for understanding amyloid fibril formation. Our initial attempts at recording 13C-CPMAS resulted in broad unresolved spectrum probably due to amorphous nature of these peptides. Hence we plan to re-crystallize them and carry forward SSNMR work with advanced techniques such as SQ-DQ (Single Quantum-Double Quantum) correlation methods developed in our laboratory along with other standard hetero-nuclear 2D correlation experiments.

References :

1. Does Aluminium bind to Histidine? An NMR investigation of Amyloid β 12 and Amyloid β 16 Fragments: Priya. N, Krishnarjuna.B, Vinaya.V, Jagadeesh Kumar.D, Sudhir Babu,Ramanathan. K.V, Easwaran.K.R.K, Nagendra.H.G, and Raghothama. S. Chem Biol Drug Des. 2013; 82: 48–59.

2. ―Solid State NMR Studies of Amyloid Fibril Structure‖ Robert Tycko, Annu Rev Phys Chem. 2011 May ; 62: 279–299.

129

Description of Multi Quantum (MQ) excitation in Half Integer Quadrupolar nuclei using Effective Floquet Hamiltonians

Vinay Ganapathy and Ramesh Ramachandran Department of Chemical Sciences Indian Institute of Science Education and Research Mohali

The utility of solid-state NMR (ssNMR) as a tool for characterizing molecular structure depends on the measurement of molecular constraints from experiments. From an experimental perspective, spectral resolution seems to be the main determining factor in the availability of molecular constraints. Since spectral resolution in the solid state is inherently controlled by the strength of the anisotropic interactions, experimental methods centered on resolution enhancement remain an active pursuit. From a theoretical perspective, extraction of molecular parameters from experimental data requires a comprehensive understanding of the spin dynamics in NMR experiments. Here in this talk, we confine our discussion to NMR experiments involving quadrupolar nuclei. In contrast to their spin ½ counterparts, analytical description of the excitation process in quadrupolar nuclei are often fraught with difficulty owing to the presence of the quadrupolar interactions. Employing spin I=3/2 nucleus as an example, the nuances of multiple-quantum (MQ) excitation/reconversion1,4 in solids will be discussed based on the theory of effective RF Hamiltonians2,3. References:

1. Frydman, L.; Harwood. J. Am. Chem. Soc. 1995, 117, 5367. 2. R. Venkata SubbaRao ; Deepansh Srivastava; Ramesh Ramachandran. Phys. Chem. Chem. Phys. 2013, 15, 2081. 3. Deepansh Srivastava; R. Venkata SubbaRao; Ramesh Ramachandran. Phys. Chem. Chem. Phys. 2013, 15, 6699. 4. G. Vinay, R. Ramachandran, Analytic Theory of Multiple-Quantum NMR of Quadrupolar Nuclei. In Graham A. Webb, editor: Annual Reports on NMR Spectroscopy, Vol 89, ARNMR, UK: Academic Press, 2016, pp. 123-184.

130

Design and Solid-state NMR/DFT Characterization of Self-assembled 13C, 15N-labelled Small Cyclic-Peptides as Mimics of Amyloid type Aggregation /Inhibition.

V. S. Phani Babu, K. V. Mohana Rao, J. K. Lakshmi, and B. Jagadeesh* NMR & Structural Chemistry Division, CSIR-Indian Institute of Chemical Technology Hyderabad-500007, India. [email protected], [email protected]

Self-assembly of designed short peptides into various molecular nanostructures is becoming a growing interest in nanobiotechnology and therapeutic models. In order to gain insight into the bio-molecular self-assembly processes involved Amyloid peptides such as 306VQIVYK311, and to propose plausible inhibitors, newer peptide scaffolds as models are constantly reported. Solid-state NMR plays important role in gaining structural insights of these self-assembled structures. Herein, we present the design, synthesis, and solid-state NMR/DFT characterization of a new class of uniformly 13C, 15N-labelled unnatural peptides that self-assemble as needle-like structures and their synthetically modified analogs as inhibitors.

References: 1) Lu, J. X.; Qiang, W.; Yau, W. M.; Schwieters, C. D.; Meredith, S. C.; Tycko, R. Cell 2013, doi:10.1016/j.cell.2013.08.035. 2) Sievers, S. A.; Karanicolas, J.; Chang, H. W.; Zhao, A.; Jiang, L.; Zirafi, O.; Stevens, J. T.; Münch, J.; Baker, D.; Eisenberg, D. Nature 2011, 475, 96-100. 3) Jagannadh, B.; Reddy, M. S.; Rao, Ch. L.; Prabhakar, A.; Jagadeesh, B.; Chandrasekhar, S. Chem. Comm. 2006, 4847-4849. 4) Thureau, P.; Sauerwein, A. C.; Concistré, M.; Levitt, M. H. Phys. Chem. Chem. Phys. 2011, 13, 93-96.

131

POSTERS

132

P1:

1H NMR-based Serum Metabolomics reveals Erythromycin -induced hepatotoxicity in Albino Wistar rats

Atul Rawat1,2, Durgesh Dubey1,2, Anupam Guleria2, Umesh Kumar2, Amit Kesari3, Swati Chaturvedi3, Anand Prakash1, Sudipta Saha3, Dinesh Kumar2*

Departments of 1Biotechnology and 3Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, 2Centre of Biomedical Research, Sanjay Gandhi Post Graduate Institute of Medical Sciences Campus, Lucknow, Uttar Pradesh, India,

Erythromycin (ERY) belongs to the class of macrolide antibiotics which are widely used to treat and prevent different types of bacterial infections. One of the major side effects of ERY treatment is liver injury. In this study, 1H NMR based serum profiling and integration of histopathological and SEM examination were employed to systemically assess the toxicity of ERY. Metabolic profiles of sera collected from ERY and control group were obtained using high-resolution 1D 1H CPMG and diffusion edited NMR spectra. The metabolic profiles were compared using multivariate statistical analysis to identify the metabolic disturbances associated with ERY treatment and thus the perturbed biochemical pathways in this condition. The key metabolites responsible for class separation were mainly characterized (a) by the increased levels of dimethylamine, malonate, choline, and phospholipids; and (b) by the decreased levels of isoleucine, leucine, valine alanine, glutamate, citrate, glycerol, lactate, threonine, HDL, LDL, VLDL, N-acetyl glycoproteins (NAG), unsaturated lipids. The observed metabolic perturbations in the sera of ERY group were involved in multiple metabolic pathways, associated with dyslipidemia, Krebs cycle, and amino acid metabolism, alluding to reduced energy biogenesis from all sources. These results exhibit the toxicity effects of the ERY treatment in rats. However, further studies in animals and in humans are needed in order to have sufficient safety evidence for its use in humans or susceptible individuals on long term medication.

133

P2:

Advanced 1H-1H Homodecoupling Methods for Spectral Simplification of Complex Molecules

J K Lakshmi1, K. Veera Mohana Rao1, V. S. Phani Babu1, Sangram S. Kale2, Gangadhar J. Sanjayan2, S. Chandrasekhar3, P.V. Srinivas4 and B. Jagadeesh1* (e-mail: [email protected]) 1 Centre for NMR and Structural Chemistry, CSIR-IICT, Hyderabad 500 007, India 2 Division of Organic Chemistry, National Chemical Laboratory, 3 Natural products Division, CSIR-IICT, Hyderabad 4 Cipla API R&D , Pathalaganaga ,Mumbai Unambiguous assignment of the individual 1H chemical shifts and establishment of through-bond connectivity of the corresponding atomic sites are very crucial in solution state NMR for accurate structure analysis of drug molecules. However, spectral crowding due to the scalar coupling multiplicity spread over the inherently limited chemical shift range of 1H nuclei, the conventional NMR methods fail to give precise structural information. The problem is more acute for complex molecules. Here we discuss advanced ―pure shift‖ NMR methods developed in our group and demonstrate them for synthetic/natural organic molecules and unnatural peptidomimics.

References: (1) K. Veera Mohana Rao, R. Kavitha and B. Jagadeesh Anal. Chem., 2015, 87, 7258–7266; (2) K. Veera Mohana Rao, V S Phani Babu and B. Jagadeesh B. Magn. Reson.Chem., 2016, 54, 308-314 (3) K. Veera Mohana Rao and B. Jagadeesh B. Magn. Reson. Chem, 2014, 52, 389-394.

134

P3:

The Correlation between the single point mutation and Alpha-Synuclein aggregation

Priyatosh Ranjan and Ashutosh Kumar

Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai-400076

Parkinson‘s disease (PD) is the second most common neurodegenerative disorder after Alzheimer‘s disease. It is characterized by the loss of dopaminergic neurons and the presence of intracellular proteinaceous inclusions known as Lewy bodies [1, 2]. Lewy bodies consist mainly of proteinaceous aggregates of natively unstructured 140 amino acids residue presynaptic amyloidogenic protein α Synuclein (α-Syn) [3].Despite being little or no ordered structure under physiological conditions, α Syn undergoes conformational transition from random coil to the highly organized fibrillar structure i.e. cross β sheet structure. α-Syn adopts an ensemble of conformations that are stabilized by long range contacts involving the N (1-60) and C termini residues (109-140). These interactions shields the highly amyloidogenic NAC region from the solvent and prevents aggregation.Esteban-Martin et al [4] using paramagnetic relaxation enhancement (PRE) identified fibrillar like contacts in soluble monomeric α-Syn between residues forming β1-β2 and and β4-β5 strands of the amyloid fibril. They also found nonfibrillar contacts between residues present in β1-β4, β1- β3, β2-β4, and β2-β5 strands of the amyloid fibril. A redistribution of this network of fibrillar and nonfibrillar contacts is required for the aggregation to take place. Conditions such as change in pH, temperature and single point mutation perturb these long range interactions leading to the formation of aggregation prone partially folded intermediate. Uversky et al [5] for the first time reported the presence of aggregation-competent partially folded intermediate under decreased pH or increased temperature which is strongly correlated with the enhanced formation of α-Syn fibrils. Our objective in the current research work is to understand why and how a single point mutation in case of the familial mutants of α-Syn accelerates or retards the rate of fibril formation. Even though these mutations are supposed to perturb the native, autoinhibitory, long range tertiary contacts, the correlation between the perturbation and fibrillization does not go well. While A53T, E46K, and H50Q aggregate faster, the other familial mutants such as A30P, G51D, and A53E aggregates slower compared to the wild type. Our aim in the current research is to understand how these single point mutations modulate the structural and dynamic properties of the monomeric free protein. . In my current research study, I will be focussing mainly on the two novel familial mutants i.e., H50Q and G51D and will try to understand how these single point mutations affect their aggregation in an opposite manner.

References

135

[1] M. Goedert, Alpha-synuclein and neurodegenerative diseases, Nature Reviews Neuroscience, 2 (2001) 492-501. [2] M.G. Spillantini, M.L. Schmidt, V.M.-Y. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert, α- Synuclein in Lewy bodies, Nature, 388 (1997) 839-840. [3] P.H. Weinreb, W. Zhen, A.W. Poon, K.A. Conway, P.T. Lansbury, NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded, Biochemistry, 35 (1996) 13709-13715. [4] S. Esteban-Martín, J. Silvestre-Ryan, C.W. Bertoncini, X. Salvatella, Identification of fibril-like tertiary contacts in soluble monomeric α-synuclein, Biophys. J., 105 (2013) 1192- 1198. [5] V.N. Uversky, J. Li, A.L. Fink, Evidence for a partially folded intermediate in α- synuclein fibril formation, J. Biol. Chem., 276 (2001) 10737-10744.

136

P4:

UNDERSTANDING THE MECHANISM OF FAT10-MEDIATED 26S PROTEASOMAL DEGRADATION PATHWAY

Hitendra Negi1, Ranabir Das1

1National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bellary Road, Bengaluru-56, INDIA

Ubiquitin Proteasome system (UPS) regulates protein levels by degrading proteins responsible for cell cycle regulation, transcription factors, metabolic enzymes etc., outside the lysosome. The protein substrates mediated for degradation gets modified with a transferable degradation signal, such as Ubiquitin (Ub) which forms K-48 poly-ubiquitin chain that can now enter 26S Proteasome complex. Beside Ubiquitin, several other Ubiquitin like modifiers (ULM) have been identified and all of them modifies the substrate in a similar fashion. One such ULM is Human Leukocyte Antigen (HLA)-F Adjacent Transcript 10 (FAT10) that solely act as degradation signal. Unlike Ubiquitin, FAT10 does not form any chain and a single moiety is potent enough to take the substrate to 26S proteasome complex. Another unique feature of FAT10 is its self-degradation along with the target substrate hence it does not get recycled like ubiquitin.

Despite being identified more than a decade ago, the structure of FAT10 and its mechanism for degradation is poorly understood. In this study, we have addressed this question by understanding the biophysical properties of FAT10 using NMR and other biophysical methods.

Keyword: Ubiquitin, ULM, FAT10, NMR

137

P5:

Saliva metabolic profiling of oral cancer using NMR spectroscopy Bolaji Fatai Oyeyemi1, Amit Paramraj2, Abinav Kumar2, Chintamani2*and Neel Sarovar Bhavesh1*

1International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, Aruna Asaf Ali Marg, New Delhi 110 067. 2Vardhman Mahavir Medical College (VMMC)-Safdarjung Hospital, New Delhi 110 029.

Oral cancer (OC) is one of the most debilitating forms of cancer with high mortality rate. It represents a well-defined subgroup of head and neck cancer frequently associated with tobacco usage and excessive alcohol intake. Understanding the metabolic profile of oral cancer is germane for proper monitoring and molecular diagnosis which in-turn might lead to early detection that is critical for survival. We have used 1H NMR spectroscopy based metabolic profiling and multivariate analysis of saliva from oral cancer (n= 22), tobacco chewer (n= 21) and healthy control (n= 20) with the aim of demystifying differential global snapshot of all small-molecule metabolites. We obtained 1H NMR spectra and processed with unsupervised principal component analysis (PCA), the supervised partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) to elucidate the 1H NMR for identifying distinguishing metabolites of oral cancer. Good separation was obtained and distinguishing metabolites (VIP scores > 1) were observed among the different groups (cases, tobacco chewer and healthy control) and several putative metabolites were annotated. Trimethylamine-N-oxide, Succinic acid, Glycine, Creatine, Thymine, 2-Furoylglycine and Hydroxy-2-methylbutyric acid were upregulated in OC cases while Dimethylglycine, Trimethylamine, Creatinine, Propanedinitrile and Allantoic acid were downregulated. More so, the following metabolites, N-Nitrosodimethylamine, Sarcosine and 7-Methyladenine, 1-Octacosanol were found in higher quantities among tobacco chewers. Finally, Pyridoxal 5'-phosphate, P- Dichlorobenzene, Erythrose were upregulated in the healthy control group. All differential metabolites were statistically significant at p < 0.05. The results indicated that 1H NMR saliva-based metabolic profiling approach is a feasible and efficient method for differentiating the OC patient from healthy controls. It thus provides a potential novel strategy and a more convenient noninvasive procedure for accurate diagnosis and monitoring of OC.

138

P6:

Insights into gallbladder cancer: what NMR can provide?

Raj Kumar Sharma1, 2, Neeraj Sinha1, Anu Behari2, V.K Kapoor2

Author’s affiliations:

1. Centre of Biomedical Research, SGPGIMS Campus, Lucknow- 226014 2. Integral University, Kursi road, Lucknow-226026 3. Department of Surgical Gastroenterology, SGPGIMS, Lucknow-226014

Gallbladder cancer (GBC) is the fifth most lethal cancer of the biliary tract and commonest biliary cancer worldwide. Due to late exposure and asymptomatic behaviour, it has been challenging to diagnose. Surgical removal of gallbladder (GB) remains the only option. Gallstone relationship with GBC has been stablished now and considered as a major risk factor for GBC1-2. Nowdays NMR based metabolomics are being applied for study of various biofluids (serum, bile, Balf, urine, saliva, pus etc.), tissue and many kinds of biological samples3. These samples could be associated with different kinds of disease i.e. inflammatory disease, lung disease and cancer. NMR based metabolomics can give the exact mechanism of what is happening in metabolic levels. This could be important in screening of specific metabolites and their characterization, pattern recognition with metabolic alteration. In spite of other cancers there are very limited reports present in literature emphasizing on GBC.

Due to late exposure and asymptomatic behaviour, it is important to identify biomarkers for early diagnosis of GBC. Recently several NMR based methodology have been applied to study the GBC in our institute4-7. We are continuously involved in NMR based metabolomics to increase our understanding on gallbladder cancer and its correlation with gallstone. Here we present different NMR based approaches applied to analysis of GBC. We have looked biomarkers in serum, bile, urine samples. We have also analysed gallstone samples and results will be presented.

References:

(1) Misra, S.; Chaturvedi, A.; Misra, N. C.; Sharma, I. D., Lancet Oncol 2003, 4, 167-76. (2) Wistuba, I. I.; Gazdar, A. F., Nat Rev Cancer 2004, 4, 695-706. (3) Nicholson, J. K.; Lindon, J. C., Nature 2008, 455, 1054-1056. (4) Jayalakshmi, K.; Sonkar, K.; Behari, A.; Kapoor, V. K.; Sinha, N., Solid State Nuclear Magnetic Resonance 2009, 36, 60-5. (5) Jayalakshmi, K.; Sonkar, K.; Behari, A.; Kapoor, V. K.; Sinha, N., NMR in Biomed 2011, 24, 335-342. (6) Sonkar, K.; Behari, A.; Kapoor, V. K.; Sinha, N., Metabolomics 2013, 9, 515-528. (7) Srivastava, M.; Sharma, A.; Kapoor, V. K.; Nagana Gowda, G. A., Hepatology Research 2008, 38, 997-1005.

139

P7:

Structural and Functional studies of Non structural proteins of Influenza A virus: Implication to rational design of potent anti-influenza drugs

Nancy Jaiswal1, Medha Karnik2 and Dinesh Kumar1*

1 Centre of Biomedical Research, SGPGIMS Campus, Lucknow 226014 2 Yuvaraja’s College, Jhansi Lakshmi Bai Road, Mysuru, Karnataka 570005

Influenza A virus is associated with significant morbidity and mortality and is a continuing worldwide public health problem with 3-5 million severe cases reported annually, including 250000-500000 deaths worldwide. There are two basic mechanisms by which influenza may occur and cause pandemics: (a) by direct transmission (of mutated virus) from animal (bird) to humans and (b) through re-assortment of an avian influenza virus with a human influenza virus. But it has not yet acquired the ability for efficient human-to-human transmission. Influenza A virus evolve constantly, and new mutant strains replace the old, in the process known as genetic drift. However, due to the high mutation rate and emerging resistance against neuraminidase inhibitors and amantadine / rimantadine, it is of utmost importance to investigate viral proteins as potential drug targets. In the above context, Non Structural protein 2- namely NS2- has been recognised as potential target for designing antiviral therapies. The NS2protein is ubiquitous in all influenza –A virus subtypes and are directly involved in modulating the important aspects of the virus replication cycle. NS2 mediates directional nucleo-cytoplasmic trafficking of the newly synthesized viral ribonucleoproteins (vRNPs) by acting as an adaptor between viral ribonuleoprotein complexes and the nuclear export machinery of the cell therefore known as Nuclear Export Protein (NEP), thus facilitates the viral replication cycle. The crystal structure of the M1 protein binding domain (C-terminal) has been solved using X-Ray crystallography. The full length structure of NS2 protein is still unknown. Overall, Non-structural protein 2 have been recognized as potential targets for designing antiviral therapies. Working in this direction NMR based structural and functional studies of NS2 will be done for structure based drug discovery.

140

P8:

Probing Water-Amyloid Interactions by Solid State NMR

Renuka Ranjan1, 2, Neeraj Sinha1 1Centre of Biomedical Research, SGPGIMS Campus, Raebareily Road, Lucknow -226014, India. 2School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi -221005, India. Amyloid ß peptides are the component of extracellular plaque characteristic of Alzheimer‘s disease as well as Cerebral Amyloid Angiopathy. The plaque forming peptides are the length variants composed of 40 and 42 amino acid residues, the Aß(1-42) being more cytotoxic and having more aggregation propensity than Aß(1-40)[1]. The amyloid fibrils of Aß(1-42) have a cross-ß sheet arrangement composed of two parallel ß-strands connected by a ß- hairpin turn which constitutes a hydrophobic core[2,3] that has been proven water accessible by probing dynamics of L17,L34,V36,M35 residues in Aß(1-40) and D23N mutant of Aß(1-42) amyloid fibrils using solid-state NMR [4]. Solid state NMR is a promising technique for studying structure, dynamics and water- protein interactions of amyloids.

In the proposed study, we shall look at the dynamics of individual amino acids common to each region of the amyloid assembly of Aß(1-42) as well as the disordered region and therefore compare the changes in mobility which may indicate relative water accessibility. A comparison between amyloid fibrils formed by a mutant of domain B1 in Immunoglobulin Binding protein G (GB1) is likely to provide information on the dynamics and interactions with water of same amino acids constituting the core of amyloid fibrils formed by mutant GB1 as well as Aß(1-42). GB1 amyloid fibrils have served as a model to study aggregation by domain swapping mechanism, also applicable to aggregation of Amyloid-ß peptides [5]. Initial results of expression, purification, aggregation of mutant GB1 and Aß(1-42) and characterization of amyloid fibrils formed by these peptides using solid state NMR spectroscopy shall be presented.

References:

[1] Suzuki, Nobuhiro, et al. "An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants." Science 264.5163 (1994): 1336-1340. [2] Kirschner, Daniel A., Carmela Abraham, and Dennis J. Selkoe. "X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation." Proceedings of the National Academy of Sciences 83.2 (1986): 503-507. 141

[3] Zhang, Shengsheng, Nicole Casey, and Jonathan P. Lee. "Residual structure in the Alzheimer's disease peptide: probing the origin of a central hydrophobic cluster." Folding and Design 3.5 (1998): 413-422. [4]Vugmeyster, Liliya, et al. "Flexibility and Solvation of Amyloid-β Hydrophobic Core." Journal of Biological Chemistry 291.35 (2016): 18484-18495. [5] Li, Jun, et al. "Amyloid-like fibrils from a domain-swapping protein feature a parallel, in- register conformation without native-like interactions." Journal of Biological Chemistry 286.33 (2011): 28988-28995.

142

P9:

Role of salt and temperature in the oligomerization of the mouse prion protein

Suhas Bhate1,Ishita Sengupta1,Jayant Udgaonkar1, Ranabir Das1

1National centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bellary Road, Bengaluru-65, INDIA

Prions are proteinacious infectious particles which in the oligomerized form cause neurodegenerative diseases known as transmissible spongiform encepohalopathies (TSE) which include CJD, FFI, GSS and Kuru. The mouse prion protein has an alpha-helix rich structure which converts to a beta-sheet rich structure upon oligomerization. It oligomerizes in the presence of 150mM NaCl at pH4 and 37C. To this end, we have used solution NMR to investigate the effect of salt and temperature on the monomeric form of the mouse prion protein on a residue specific basis.

Keyword: Prion protein, NMR spectroscopy

143

P10:

Dynamics of SUMO upon interaction with SIM (SUMO Interaction Motif)

Kiran Sankar Chatterjee1, Ranabir Das1 1National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bellary Road, Bengaluru-65, INDIA Dynamics of biomolecules can be crucial for its function. Post-translational modifications like SUMO (Small Ubiquitin like Modifiers) modification can alter the interaction and function of its target proteins. One mode of interaction between a SUMO- modified protein and its co-factor is through the non-covalent interactions between SUMO and SBMs/SIMs (SUMO Binding/Interacting Motifs). SBM/SIMS is characterized as hxhh or hhxh (h=val/Ile/leu and x=any amino acid) sequence flanked by acidic amino acids, and in some cases serine residues. In an interesting fashion, SBM/SIM interaction has been shown to induce rigidity in SUMO. In this work we investigate the altered dynamics of SUMO proteins upon binding with SIM peptides by solution NMR spectroscopy.

Keywords: SUMO, SIM, NMR

144

P11:

On the exactness of Effective Hamiltonians derived from the method of Contact transformation

Rajat Garg and Ramesh Ramachandran

Department of Chemical Sciences Indian Institute of Science Education and Research (IISER) Mohali Sector 81, Mohali- 140306, Punjab, INDIA

Development of analytic methods has remained an active pursuit in spectroscopy for a variety of reasons ranging from the design/optimization of experiments to quantitative interpretation of spectroscopic data. From a theoretical standpoint, most of the proposed analytic methods in spectroscopy have centered on the derivation of ―effective Hamiltonians‖ within the framework of perturbation theory [1]. In particular, derivation of effective Hamiltonians based on the contact transformation [2], [3] procedure has become a standard practice in molecular spectroscopy. While the utility of the method in NMR spectroscopy has been demonstrated in simpler cases under idealized conditions [4], [5], a more generalized procedure facilitating analytic descriptions beyond the perturbation limit remains desirable [6]. For illustrative purposes, the analytic methods currently employed in the derivation of effective Hamiltonians in heteronuclear decoupling will be discussed.

References

1. D. Papousek and M. R. Aliev, Molecular Vibrational-Rotational Spectra-Elsevier, Amsterdam, 1982. 2. J. H. V. Vleck, Phys. Rev. 33, 467 (1929). 3. R. Ramesh and M. S. Krishnan, J. Chem. Phys. 114, 5967(2001). 4. J. R. Sachleben et.al, J. Chem. Phys. 104, 2518(1996). 5. J. R. Sachleben et.al, Solid State Nucl. Mag. Resonance 29, 30(2006). 6. C.N. Banwell and H. Primas. Mol. Phy.,6, 225(1962).

145

P12:

Switch in the Handedness Influenced by Solvent in Mixed β-Peptides Sirisha K1, Prashant T2, G. V. M. Sharma2, Kallaganti V. S. Ramakrishna1 , Hans-Jörg Hofmann3, and Akella V. S. sarma1

1. Nuclear Magnetic Resonance and Structural Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India 2.Organic and Biomolecular Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India 3.Institute of Biochemistry, Faculty of Biosciences, University of Leipzig, Germany.

Abstract: The present study describes the effect of solvent on helix type and handedness in mixed β-peptides derived from C-linked carbo-β-amino acids [β-Caa(l)] with a D-lyxo furanoside side chain and β-hGly in 1:1 alternation . In chloroform solvent, the peptides with an (S)-β-Caa(l) monomer at the N-terminus formed a right-handed 10/12-mixed helix, whereas peptides with β-hGly constituent at the N-terminus displayed a left handed 10/12 helix. An unprecedented solvent-directed ‗switch‘ from a 10/12- into a 12/10-helical pattern connected with a change of handedness was observed in these later β-peptide sequences with a β-hGly constituent at the N-terminus in the solvent methanol. Based on NMR and CD studies, a left-handed 10/12-helix in the solvent chloroform, changes into a right-handed 12/10-helix in more polar protic solvent methanol (Fig. 1). In acetonitrile, a polar aprotic solvent, the NMR data indicated the co-existence of both the helix types. Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the left-handed 10/12- and the right-handed 12/10-helix, which may explain the possibility to influence the structure equilibrium by external factors like solvents. A solvent effect like this, which changes both the hydrogen bonding pattern and the handedness of helices, is rather rare for foldamer 10/12-helices.

References: 1.Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; idmer, H. Helv. Chim. Acta , 79, 913-941, 1996. 2.Gellman, S. H. Acc. Chem. Res. 31, 173-180, 1998. 3. Sharma, G. V. M.; Subash, V.; Narsimulu, K.; Ravi Sankar, A.; Kunwar, A. C. Angew. Chem. Int. Ed. 45, 8207-8210, 2006. 146

P13:

RDC as a tool for finding transient dimer formation

Shine A. and Vinesh Vijayan*

Large number of cellular proteins are found as oligomers and proteins in oligomeric state are critical for performing their functions2. So the examination of protein oligomerization such as its structure, binding interface and orientation of subunits etc., is important. The noncovalent association of protein ubiquitin in solution was studied using Nuclear Magnetic Resonance Spectroscopy. The concentration dependant measurement of Residual Dipolar Couplings(RDCs) of ubiquitin and subsequent analysis revealed that, the noncovalent dimer of ubiquitin resembles K11 linked diubiquitin in structure.

References

1. Liu, Z., Zhang, W. P., Xing, Q., Ren, X., Liu, M., & Tang, C. (2012). Noncovalent dimerization of ubiquitin. Angewandte Chemie International Edition, 51(2), 469-472. 2. Mei, G., Di Venere, A., Rosato, N., & Finazzi‐Agrò, A. (2005). The importance of being dimeric. FEBS journal, 272(1), 16-27. 3. Lee, H. W., Wylie, G., Bansal, S., Wang, X., Barb, A. W., Macnaughtan, M. A., ... & Prestegard, J. H. (2010). Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping. Protein Science, 19(9), 1673-1685.

147

P14:

Mapping metabolic perturbations in Mycobacterium smegmatis in response to different stress conditions using NMR spectroscopy

Saleem Yousf1, Arshad Rizvi3, Sharmistha Banerjee3,* and Jeetender Chugh1,2,*

1Department of Chemistry, 2Department of Biology, Indian Institute of Science Education and Research (IISER), Dr Homi Bhabha Road, Pashan, Pune 411008, India 3Department of Biochemistry, School of Life Sciences, South Campus, University of Hyderabad, Gachibowli, Hyderabad, Telangana State 500046, India

*[email protected] & [email protected]

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), although is a preventable and curable disease; it is responsible for ~2 million total deaths worldwide. Mtb is known to inhabit the alveolar macrophages immediately after infection. Though in most of the cases the activated alveolar macrophages are able to clear the pathogen, in several cases, the pathogen in return develops intelligent strategies to avoid eradication and persists within. While residing in the macrophages, Mtb faces several hostile conditions represented by an environment that is nutritionally deprived, highly hypoxic, acidic and under oxidative stress. In this study, we used the 1H NMR based metabolomics to reveal the metabolic profile of M. smegmatis (used as model organism for Mtb) under normal growth and environmental conditions simulating infection, such as nutrient deprivation, acidic and oxidative stress. This study identified and provided information on the critical metabolites that are associated with the adaptation in Mycobacteria under different conditions. This information would be extrapolated to construct metabolic pathways that are crucial for the survival of the pathogen in stress conditions. Leads from this study would eventually help in identifying attractive drug targets for the development of new treatments against tuberculosis.

References:

1. Halouska S, Fenton RJ, Zinniel DK, Marshall DD, Barletta RG, Powers R. J Proteome Res. (2014), 13(2):1065-76

148

P15:

Dynamics of double stranded RNA binding domain1 (dsRBD1) of TRBP

Harshad Paithankar1, Pankaj Jadhav3, Amit Naglekar3, Shilpy Sharma3and Jeetender Chugh1,2*

1Department of Chemistry, 2Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India 3Department of Biotechnology, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India *[email protected] TAR (Trans-activation response) RNA binding protein (TRBP) is a double stranded RNA binding protein (dsRBP), which plays key roles in vital biological processes in cells ranging from development, proliferation to death. It is one of the main components of the ribonucleoprotein complexes involved in the biogenesis of the small RNAs like miRNA and endo-siRNAs. In the miRNA biogenesis pathway, TRBP is known to assist the Ribonuclease III enzyme –Dicer – to cleave ~33bp precursor RNA (pre-miRNA) to shorter 22bp RNA duplex. One of the strands of this duplex, called guide strand is then loaded on to the Argonaute protein as mature miRNA while the other stand is degraded. TRBP is known to interact with a wide range of pre-miRNAs each having a different conformation due to presence of various helical perturbations.2 For such interactions, either the target RNA or TRBP needs to undergo conformational change to adapt for efficient binding. A recent report suggested that RNA does not show any bending upon binding with TRBP1 as is observed for many DNA binding proteins.3 Therefore, it may be possible that TRBP adapts different conformations to interact with various dsRNAs. With this background, we present the dynamics of the dsRBD1 of TRBP at different time scales studied using NMR relaxation experiments including spin relaxation, relaxation dispersion and real time NMR.

References:

1 R. Acevedo, D. Evans, K. A. Penrod, and S. A. Showalter, Biophys J, 2016, 110, 2610-7. 2 R. Acevedo, N. Orench-Rivera, K. A. Quarles, and S. A. Showalter, PLoS One, 2015, 10, e0116749. 3 R. S. Spolar, and M. T. Record, Jr., Science, 1994, 263, 777-84.

149

P16: Study of βγ-Crystallins by NMR Spectroscopy Sujeesh K. S., Kausik Chandra, Yogendra Sharma & Hanudatta S. Atreya

The mammalian eye lens is a rich source of crystallin proteins which are two major types; α- crystallins and βγ-Crystallins.1 βγ-Crystallins of eye lens have many structural homologs in various organisms including eyeless organisms. βγ-crystallins of eyeless organisms have the ability to bind calcium whereas vertebrates lack this ability. By studying the variation in calcium binding properties of eye lens βγ-Crystallins and its structural homologs in various organisms, it is possible to understand the evolution of these eye lens proteins. We are trying to solve high-resolution structure and study the calcium associated dynamics of βγ-Crystallin of Ciona intestinalis, a structural homolog of human βγ-Crystallin, using Nuclear Magnetic Resonance (NMR) Spectroscopy and other biophysical techniques. In order to study the structure and dynamics of this protein, the complete chemical shift assignments have been obtained by NMR spectroscopy using a 15N-13C labeled protein.2 Primarily we have used standard sequential backbone resonance assignment strategy which involves the measurement of HSQC, HNCO, HNCACB, and CBCA(CO)NH NMR experiments. But there were a few ambiguities in assigning some residues. Henceforth we have implemented new methods such as selective unlabeling3 and D, N-Filtered HSQC experiment to overcome the problem.

References:

1. Bloemendal, H., de Jong, W., Jaenicke, R., Lubsen, N. H., Slingsby, C., & Tardieu, A. (2004). Ageing and vision: structure, stability and function of lens crystallins. Progress in biophysics and molecular biology, 86(3), 407-485. 2. Cavanagh, J., Fairbrother, W. J., Palmer III, A. G., & Skelton, N. J. (1995). Protein NMR spectroscopy: principles and practice. Elsevier. 3. Krishnarjuna, B., Jaipuria, G., Thakur, A., D‘Silva, P., & Atreya, H. S. (2011). Amino acid selective unlabeling for sequence specific resonance assignments in proteins. Journal of biomolecular NMR, 49(1), 39-51

150

P17:

Synthesis, 1D and 2D NMR spectral studies of 2- Thiophenecarba nicotinic hydrazone and 2- Thiophenecarba benzhydrazone Jyothy G Vijayan and N M Nanje Gowda Department of Chemistry, Christ University, Bengaluru-29 [email protected]

Synthesis and NMR spectral studies of bidentate N and S heterocycles of 2- Thiophenecarba nicotinic hydrazone and 2- Thiophenecarba benzhydrazone have been carried out .The compounds 2- Thiophenecarba nicotinic hydrazone and 2- Thiophenecarba benzhydrazone was synthesized by reacting stoichiometric quantities of nicotinic hydrazide and benzhydrazide with 2-thiophene carboxaldehyde in methanol in the presence of glacial acetic acid at refluxing temperature .On cooling the reaction mixture, the product was obtained as colorless solid. 1H, 13C, 1H-1H COSY and 1H-13C HSQC experiments have been conducted to characterize the compounds. Keyword: 2- Thiophenecarba nicotinic hydrazone , 2- Thiophenecarba benzhydrazone and 2D NMR References

1) H.Mimoun, L.Saussine and Edgier, J.Am.Chem.Soc. 105(1983) 3101-3110. 2) A. Datta, K. Das, Y.M. Jhou, J.H Huang and H.M. Lee, Acta Cryst, 66(2010) 1271. 3) L.L Koh, O.L Kon, K.W. Loh and Y.C. Long, Biochem, 72 (1998 )155.

151

P18:

13C NMR Chemical shifts and DFT studies on 5-methoxy/ethoxy-2- mercaptobenzimidazoles

Manju Pandey and N M Nanje Gowda

Department of chemistry, Christ University, Bangalore-560029

5-alkoxy-2-mercaptobenzimidazoles are pharmacologically important derivatives of benzimidazole. 5-methoxy derivative serves as a precursor required for the synthesis of antiulcer drug omeprazole whereas 5-ethoxy analog is the main component of antiarrhythmic drug bradizol. Intra-molecular interactions and conformations in such molecules play a key role in understanding the efficacy of their biological activity. Conformational aspects of organic compounds are being investigated by NMR and DFT methods in solution and gas phase respectively (1, 2). 5-methoxy/ethoxy carbons may attain planar or nonplanar conformation with respect to the aryl ring of 2-mercaptobenzimidazole (Fig. 1). 13C chemical shifts of ring carbons are influenced by the substituent orientations (Anti, Syn or Skew) as indicated by substituent chemical shifts (SCS). The SCS values for the ortho carbons induced by the methoxy/ethoxy group are negative and in the range -12 to - 15 ppm implying the shielding effect. The complete geometry optimization of the molecules was performed using DFT (B3LYP)/6-311++G (d, p) method. Gauge-Independent Atomic Orbital (GIAO) 13C chemical shift calculations were done by the same method. Conformational analysis was done by potential energy scans (PES). The energies, bond lengths, Wiberg bond orders, dipole moments, atomic charges and rotational energy barrier were also calculated.

Anti Syn Skew R= CH3, C2H5 Fig. 1

1. Bogdan Musielak, Tad A. Holak and Barbara Rys, J. Org. Chem., 2015, 80 (18), 9231. 2. Ryszard B. Nazarski, Piotr Wałejko and Stanisław Witkowski, Org. Biomol. Chem., 2016, 14, 3142.

152

P19:

Investigating structural flexibity of TRBP2 RBD2 to understand dsRBD-dsRNA interaction

Himani Rawat1, Harshad Paithankar1, Pankaj Jadhav3, Amit Naglekar3, Shilpy Sharma3 and Jeetender Chugh1, 2*

1Department of Chemistry, 2Department of biology, Indian Institute of Science Education and Research (IISER) Pune, Dr. HomiBhabha Road, Maharashtra 411008, India 3Department of Biotechnology, SavitribaiPhule Pune University, Ganeshkhind, Pune, Maharashtra 411007, India *[email protected]

Many diseases including cancer and Alzheimer‘s have been shown to be associated with altered miRNAs expressions, suggesting their big role in these diseases. To perceive the significance of various roles played by up- and down-regulation of miRNAs in cells, it is important to understand the biogenesis pathway of miRNAs that includes nuclear and cytoplasmic processing of larger RNA transcripts regulated by a variety of proteins. Trans- activation response RNA binding protein (TRBP) is known to play significant roles in cytoplasmic processing of miRNAs1. TRBP is a 366 residues long protein with three double- stranded RNA binding domains (dsRBDs) known to have typical dsRBD fold (α1-β1-β2-β3- α2) connected by the linker regions. dsRBD-1&2 interact directly with pre-miRNAs whereas dsRBD3 interacts with RNase-III enzyme Dicer to aid in catalytic cleavage of pre-miRNAs to miRNA/miRNA* duplex. Following this, TRBP forms a part of RNA induced silencing complex (RISC) to help in guide-strand selection and hence play a direct role in RNA interference. Though dsRBD2 shares a similar structure with dsRBD1 but α1 helix of dsRBD1 is known to be slightly displaced compared to dsRBD2. Reported biophysical studies hint towards a greater rigidity of dsRBD2 domain compared to dsRBD1, which might be because of stronger inter-residue interactions within the domain2. Here, we attempted to investigate differential binding affinity of the two domains of TRBP towards dsRNA binding using NMR dynamics as a probe. These results would also shed light on the mechanism of TRBP binding to multiple RNA targets in the miRNA biogenesis pathway.

References

1. Wilson, Ross C.;Tambe, A; Kidwell, MA; Noland, CL; Schneider, CP; Doudna, JA. Molecular cell 57.3 (2015): 397-407. 2. Yamashita, S; Nagata, T; Kawazoe, M; Takemoto, C; Kigawa, T; Guntert, P; Kobayashi, N; Terada, T; Shirouzu, M; Wakiyama, M; Muto, Y; Yokoyama, S. Protein Science 20.1 (2011): 118-130.

153

P20:

Structural Studies and Expressed Protein Ligation of OsVDAC Isoform 4 using NMR

Parvathy Jayan, Vinesh Vijayan

School of Chemistry, IISER- Thiruvananthapuram, Thiruvananthapuram-695016

The voltage-dependent anion-selective channel(VDAC) is the most abundant protein in the outer mitochondrial membrane.It is the major conduit for metabolite transport across the membrane. The largely studied isoforms of VDAC are human VDAC (hVDAC), mouse VDAC (mVDAC), and rice VDAC (OsVDAC). Three dimensionalstructures of hVDAC (hVDAC1) and mVDAC (mVDAC1) have been resolved by NMR spectroscopy and X-ray crystallography respectively. It consists of 19 β-strands forming a β-barrel, and a α-helix N- terminal domain that lies within the pore of β-barrel. Since the mechanism of gating and function of N-terminal helix in the gating mechanism is not clearly known, it is important to study the dynamics of helix and also study the interaction of helix with rest of the β-barrel. We attempt to study this mechanism using solution NMR spectroscopy using native chemical ligation method for better control over the isotope labeling of the helix. Our initial results on the expression and chemical ligation of OsVDAC4 will be presented in the poster.

References 1. Mathew et al., Journal of Membrane Biology, 2011, 244,67–80. 2. Thomas Raschle et.al.,J. Am. Chem. Soc. 2009, 131, 17777–17779. 3. Tsyr-Yan Yu et.al., Biochim Biophys Acta. 2012, 1818(6): 1562–1569.

154

P21:

NMR-based metabolomics study on Drosophila model of Huntington’s disease

Virender Singh1, Raj Kumar Sharma‡2, Thamarailingam Athilingam ‡1, Pradip Sinha1, Neeraj Sinha2, Ashwani Kumar Thakur1*

1Biological Science & Bioengineering, IIT Kanpur, Kanpur-208016, India 2Centre of Biomedical Research, SGPGIMS Campus, Raibareli Road, Lucknow-226014, India

Huntington‘s disease (HD) is a neurodegenerative disease with characteristic accumulation of polyglutamine (polyQ) rich aggregates in neurons.1 The onset and disease severity is directly proportional to length of polyQ stretch. Accumulation of polyQ rich aggregates leads to neurodegeneration. To develop better understanding of disease, researchers have developed different Huntington‘s disease models like yeast, Drosophila, C. elegans and mouse. In this study, we have used Drosophila as HD model.2 We have established fly metabolomics as novel tool to understand the disease pathology at metabolite level which is ultimately translated to phenotype. We have used Q20 flies expressing polyQ with 20 glutamines as control, Q93 flies expressing polyQ with 93 glutamines as intermediate and Q127 flies expressing polyQ with 127 glutamines as disease category. Polyglutamine was expressed in all cells of fly eyes under GMR-GAL4 driver. With the help of multivariate analysis performed on proton NMR spectra, we could see separation of data into three categories using both PCA (unsupervised) and PLS-DA (supervised) approaches.3 We could identify 25 metabolites, of which 6 metabolites showed maximum contribution to group separation. Trend followed by changes in metabolites hold true for both male and male-female combined data sets and maximum variance is explained by first component itself. Flies expressing Q93 showed intermediate level of metabolites compared to control and Q127 flies. Multiple pathways were found to be implicated in intermediate and disease groups combined (p<0.05, FDR<0.05). We have established NMR based metabolomics as useful tool for better understanding the Huntington‘s disease pathology in Drosophila model.3

References 1. Ross, C. A. and Tabrizi, S. J. Huntington's Disease: from Molecular Pathogenesis to Clinical Treatment. Lancet Neurol. 2011, 10, 83-98.

155

2. Marsh, J. L.; Pallos, J. and Thompson, L. M. Fly Models of Huntington's Disease. Hum. Mol. Genet., 2003, 12, R187-R193. 3. Beckonert O.; Keun, H. C.; Ebbels, T. M.; Bundy, J.; Holmes, E.; Lindon, J. C. and Nicholson, J. K. Metabolic Profiling, Metabolomic and Metabonomic Procedures for NMR Spectroscopy of Urine, Plasma, Serum and Tissue Extracts. Nat. Protoc., 2007, 2, 2692-2703.

156

P22:

A new approach for the study of Desmotropy in Olmesartan medoxomil by Solid State NMR

Yashwantsinh Jadeja, Bhagyawanti Chomal, Madhavi Patel, Hetal Jebaliya, Ranjan Khunt* and Anamik Shah*

Centre of Excellence, National Facility for Drug Discovery Complex, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India.

*Correspondence to: Dr. Ranjan C Khunt, Centre of Excellence, NFDD Centre, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India. E-mail: [email protected]

―Desmotropy‖, the theoretical phenomena of tautomerism is new trends to study in tetrazole containing pharmaceutical bioactive potent molecules. Olmesartan medoxomil is member of angiotensin receptor blockers, containing tetrazole ring. Present work is to introduce new plead for desmotropy study with the help of 1H-15N HETCORE and 15N CPMAS solid state nuclear magnetic resonance, which is novel path for the characterization and identification.

References:-

1. W. Holzer, R.M. Claramunt, C. López, I. Alkorta, J. Elguero, A study in desmotropy, Solid state nuclear magnetic resonance, 34 (2008) 68-76. 2. M. Skotnicki, D.C. Apperley, J.A. Aguilar, B. Milanowski, M. Pyda, P. Hodgkinson, Characterization of two distinct amorphous forms of valsartan by solid-state NMR, Molecular pharmaceutics, 13 (2015) 211-222. 3. Rao, D. Apperley, C. Rodger, NMR study of desmotropy in Irbesartan, a tetrazole- containing pharmaceutical compound, Journal of the Chemical Society, Perkin Transactions 2, DOI (1998) 475-482. 4. M. Skotnicki, D.C. Apperley, J.A. Aguilar, B. Milanowski, M. Pyda, P. Hodgkinson, Characterization of two distinct amorphous forms of valsartan by solid-state NMR, Molecular pharmaceutics, 13 (2015) 211-222. 157

P23:

Method development and validation: quantitation of telmisartan bulk drug and its tablet formulation by 1H NMR spectroscopy

Yashwantsinh Jadeja, Bhagyawanti Chomal, Madhavi Patel, Hetal Jebaliya, Ranjan Khunt and Anamik Shah*

Centre of Excellency, National Facility for Drug Discovery (NFDD) Complex, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India.

The quantitative NMR spectroscopy is nowadays a new tool for determination of pharmaceutical potent biologically active molecules in bulk drug and its tablet formulation than the other analytical techniques. Herein, qNMR method was developed for an anti- hypertensive drug, telmisartan in bulk drug and its tablet formulation. The précised method was developing by using malononitrile as an internal standard.

The methylene signal of telmisartan appeared at δ= 5.46 ppm (singlet) relative to the signal of malononitrile at δ= 3.59 ppm (singlet) in CDCl3, as a NMR solvent. The development and validation of the method were carried out as per ICH guidelines. The method was found to be linear (r2 = 0.9999) for 0.5 to 3.5 mg/ml in the drug concentration range. The relative standard deviation for Accuracy and Precession was not more than 2.0 %. The sensitivity of the method was carried out by LOD and LOQ, at 0.05 and 0.2 mg/ml respectively concentration. The robustness of the method was studied by changing parameters as well as different solvent manufacturer company. The result shows that method was accurately developed for quantification of Telmisartan in pharmaceutical dosage form. The developed method by 1H NMR spectroscopy is comparatively easy and more precise with respect to the other analytical tools.

158

P24:

Do intermediate states of a protein always lead to its misfolding??

Juhi A. Rasquinha, Soumyadeep Chatterjee and Sujoy Mukherjee

Structural Biology and Bioinformatics Division, CSIR - Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, West Bengal, India, 700032

Amyloidoses is a pathological condition in which the protein misfolds and forms insoluble aggregates which get deposited in the extracellular space of major organs like the heart and kidneys (1). LEN is a human Ig VL protein of ƘIV sub-type that has been isolated from the urine of a patient suffering from multiple myeloma but without any sign of amyloidoses. However, it forms amyloid fibrils under destabilising conditions such as acidic pH along with vigorous agitation (2). Studies had shown earlier that the intermediate states of LEN often correspond to its misfolding (3). However, it has now been observed that it is not necessary for all intermediate states to be in a misfolded state.

To probe the conformational dynamics of LEN, the wild type gene was cloned, expressed in E. coli C41 cells and grown in (2H/15N) enriched minimal media. The protocol for expression was fine tuned to ensure uniform labelling of the LEN backbone. LEN was extracted from the periplasmic fraction and purified in a two-step ion-exchange chromatography. 15N-CPMG relaxation dispersion experiments were performed to study the chemical exchange rates involving intermediate state conformers at three different temperatures. Using CPMG relaxation dispersion trajectories, the rate of exchange between the native and intermediate conformers (kex) and population of intermediate conformers (PI) were obtained for the three temperatures, from which the magnitude of 15N chemical shift difference between the exchanging conformers |Δw|, the forward rate constant (kf) and the backward rate constant (kb) were calculated. Finally, the free energy difference between the native and intermediate states (ΔGN→I) at the three temperatures were calculated and fitted to obtain the change in heat capacity between the native and intermediate states (ΔCp). The sign of (ΔCp) was used to infer the nature of the intermediate state of LEN.

References:

1. Blancas, Annu Rev Biochem; 2013; 82, 745-774.

2. Mukherjee, Biomol NMR Assign; 2009; 3, 255-259.

3. Mukherjee, Biochemistry; 2011; 50, 5845-5857.

159

P25:

NMR Studies of α/-Peptides from -Dideoxy C-linked-Carbo--Amino Acids (β-Caa(dd)) and L-Ala : Generation of 11/9-Helix, Helix-Turn and Helix-Turn-Helix Structures

Kanakaraju Marumudi, Pothula Purushotham Reddy, Adepu Ramesh, Gangavaram V. M. Sharma and Ajit C. Kunwar

CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail: [email protected]

Keywords: foldamers ∙ α/-peptides ∙ β-Caa(dd )∙ 11/9-helix ∙ helix-turn ∙ helix-turn-helix

Hetero peptides derived from mixed α/-peptides mimic the α-helices and inhibit protein-protein interactions, providing the impetus to investigate such ‗foldamers‘. α/β Hybrid peptides derived from alternating C-linked-carbo-β-amino acids (β-Caa), with sugar side chains and Ala residues display a variety of helical structures. The role of side chains in stabilizing the folds in such regular 1:1 peptides derived from β-Caa containing deoxy [1] arabinose (3-deoxy-L-ara) side-chain and the D-ala was recently investigated. It was observed that with no substitutent at C3 position in the sugar ring, very stable helices were formed. Since electrostatic and steric interactions play an important role in the stabilization of the folds, it was felt worthwhile to investigate foldamers containing β-Caa with1,2-dideoxy furanoside side chain (β-Caa(dd)). α/β-Peptides (1-3) containing L-Ala and β-Caa(dd) were investigated to explore the role of side chains, without an acetonide group, in their folding propensities. The penta-peptide 1, with a C-terminus ‗α-β-α‘ motif, folds as a regular 11/9- helix where as the hexa-peptide 2, with ‗β-α-β‘ at the C-terminus, as expected,[2] spawned a 11/9-helix at the N-terminus, which nucleated a ‗turn‘ at the C-terminus, thus generating a ‗helix-turn‘ (HT) motif. The turn in the HT motif was stabilized by a 13/7-membered H- bonded pseudo-ring. The octa-peptide 3, has two sequential β residues, with fragments ‗-- -‘ and ‗---‘. Such a design generated a ‗helix-turn-helix‘ (HTH) super-secondary structure,[2] the turn is stabilized by an11/15-membered bifurcated H-bond and a 9-membered H-bond. The peptide provided the shortest sequence for a HTH motif.

1. Boc-L-Ala-β-Caa(dd)-L-Ala-β-Caa(dd)-L-Ala-OMe

2. Boc-L-Ala-β-Caa(dd)-L-Ala-β-Caa(dd)-L-Ala-β-Caa(dd)-OMe

3. Boc-L-Ala-β-Caa(dd)-L-Ala-β-Caa(dd)-β-Caa(dd)-L-Ala-β-Caa(dd)-L-Ala-OMe References: 1. G. V. M. Sharma, G. Anjaiah, M. Kanakaraju, B. Sudhakar, D. Chatterjee, A. C. Kunwar, Org. Biomol. Chem. 2016, 14, 503–515. 2. G. V. M. Sharma, P. Nagendar, K. V. S. Ramakrishna, N., M. Choudhary, A. C. Kunwar, Chem. Asian J. 2008, 3, 969–983.

160

P26:

Application of 13C-NMR spectroscopy in the study of plant volatile organic compounds

R. Ananthakrishnan, A.P. Anu Aravind and K.B. Rameshkumar

Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram-695562, Kerala, India.

Plant derived volatile organic compounds have been used for their perfume, flavour, preservative and insecticidal properties. These compounds are economically important and have wide industrial applications. Gas chromatographic methods such as GC-FID and GC- MS are the most widely accepted methods for the identification of plant derived volatile organic compounds. However, GC methods are quite difficult to distinguish between the isomeric forms of compounds due to their close similarity in mass fragmentation pattern and close retention index (RI) values. Recently, 13C-NMR spectroscopy has been evolved as an efficient characterisation tool for unambiguous identification of plant derived volatile organic compounds, especially from crude essential oils. The present paper describes the application of 13C-NMR spectroscopy in identification of closely related and isomeric volatile compounds from aromatic plants. The major tetrahydrofuran derivatives furopelargone A and B were distinguished from Cinnamomum macrocarpum leaves essential oil using 13C-NMR spectroscopy. α-Copaene and α-ylangene are stereo-isomeric compounds common in essential oils. However, the unambiguous characterisation of these compounds by GC-MS analysis is quite difficult due to the close RI and similar mass spectra. The sterio-isomeric compounds were distinguished from crude essential oil of Garcinia talbotii leaves by 13C- NMR spectroscopic studies. The stereochemistry of volatile compounds from plants is important, especially in chemical ecological aspects, where the activity of isomeric forms vary considerably. 13C-NMR spectroscopy is an inevitable and reliable tool in analysing such closely related isomeric volatile compounds from crude essential oils.

References

Kubeczka, K. H.; Formacek, V. Essential Oil Analysis by Capillary Gas Chromatography and Cabon-13 NMR Spectroscopy, John Wiley and Sons: Chichester, 2002.

Atta-ur-Rahman.; Ahmad, V.U. 13C- NMR of Natural Products, Vol.1.; Springer Science: New York, 1992.

161

P27:

Structure and Dynamics insight of Human Skp1 protein and its role in recognition of F-box binding partner in SCF complex

Nitin Nathubhai Kachariya, Sarath Chandra Dantu, Rupali Shinde, Ashutosh Kumar

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai 400076, Mumbai, India. Email: [email protected]

Protein synthesis is an energy consuming process as substantial amount of energy is invested in the formation of peptide bonds. All this energy is dissipated when the protein is degraded. The process of protein degradation is very specific in animal cells and it takes place via the Ubiquitin Proteasomal System (UPS) or lysosomal degradation. UPS is involved in biological processes such as signal transduction, DNA repair, and control of cell division, induction of immune response, regulation of transcription, stress response, apoptosis, inflammation, and embryonic development (M. Pagamo group, 2014). In proteasomal system, E1-activating enzyme, E2-conjugating enzyme, and E3 ligase enzyme work in cascade to target the substrate for ubiquitination. SCF (Skp1-Cullin-Fbox) complex is one of the E3 enzymes that ubiquitinate the cell cycle regulatory proteins. Skp1 protein is the adapter protein which N-terminal interact with Cullin1 and C-terminal interact with F-box protein. More than 65 F-box proteins have been discovered in human genome and there is only one Skp1 protein. X-ray crystal structure of Skp1was solved in complex with five different FBPs (PDB: 1FQV, 2ASS, 2E21, 2OVR, 3WSO). But, the precise conformational properties of standalone Skp1 and the mechanism employed by Skp1 to recognize various FBP‘s is not yet clear. Hence, structure and dynamics of Skp1 at atomic level resolution is required for complete understanding the Ubiquitination.

Uniformly 15N and 13C isotopic label human Skp1 protein was purified by affinity chromatography. Recorded double and triple resonance NMR experiments specifically, HSQC, HNCO, HN(ca)CO, HNCACB, HN(co)CACB, HNN, HNCN, for sequence specific backbone assignment; TOCSY-HSQC, HCC(co)NH, 13C HSQC, CC(co)NH experiments for side-chain assignment; 15N NOESY-HSQC, 13C NOESY-HSQC, deuterated aromatic NOESY-HSQC experiments for structure calculation and T1 and T2 relaxation experiments for dynamics calculation. Sequence specific backbone and side chain assignment was completed and residue specific secondary structure as well as dynamic nature of Skp1 protein from chemical shift was obtained. Additionally we also performed three molecular dynamic simulations (each 200ns long) of Skp1 protein using Gromacs 4.6 package. It will further increase our understanding of the standalone Skp1 structure and will pave way for the future characterization of protein structure-dynamics and will assist in ascertaining mode of interaction of Skp1 with various F-box proteins.

Publication:

1. Kachariya NN, Dantu SC, Kumar A. Backbone and side chain assignments of hu-man cell-cycle regulatory protein S-phase kinase associated protein-1. Biomol NMR Assign. 2016. Doi: 10.1007/s12104-016-9699-2

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2. Dantu SC, Kachariya NN, Kumar A. Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex (2016). Proteins, 2016; 84 (1): 159- 171. Doi: 10.1002/prot.24963

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P28:

NMR resonance assignment of 73 kDa NF-kB protein complex

Tahseen Raza1, Prikshat Dadhwal1 Manish K Choker1, Kandala V. R. Chary2, Hanudatta S. Atreya3 and Sulakshana P. Mukherjee1

1Biomolecular Interaction and Structural Dynamics Lab, Department of Biotechnology, IIT Roorkee, Roorkee, 247667, India. 2Department of Chemical Sciences, Tata Institute of Fundamental Research, 1, Homi Bhabha Road, Colaba, Mumbai 400005, India. 3NMR Research Centre, Indian Institute of Science Banglore, 560012, India.

NF-kB is a protein complex, which regulates transcription of many mammalian genes and is ubiquitously present in almost all the mammalian cell types. NF-kB is a five membered family of proteins, which form dimers in various combinations. These dimers are present in the cytoplasm in the resting state of the cells. NF-kB signaling pathway is vital for immune response and it involves a variety of biomolecular interactions. This family of protein is recently found to cross-talk with other signaling pathways such as the IRF pathway. It is observed that NF-kB proteins also regulate the transcription of a subset of IRF3-target genes. However, the mechanism of this gene regulation by NF-kB is not clear, which we intend to study using NMR spectroscopy.

The first step to study the above mentioned mechanism of gene regulation by NMR spectroscopy is to obtain maximum assignments of the NMR resonances of NF-kB dimer. Here we present the assignments of p50 subunit of NF-kB dimer (73 kDa). To assign this huge protein complex we have used the strategy of transfer of assignments from smaller domains to the full-length complex along with various amino-acid unlabeling techniques. The resonance-assignments of this protein complex provide valuable insight into the structure of NF-kB complex in DNA-free conformation.

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P29: qNMR method development and validation for Betamethasone 17-valerate by 1H NMR spectroscopy

Bhagyawanti Chomal, Princy Wadhwaniya, Yashwantsinh Jadeja, Ranjan Khunt and Anamik Shah*

Centre of Excellency, National Facility for Drug Discovery (NFDD) Complex, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India. Email:- [email protected], [email protected]

The quantitative NMR method development and validation for Betamethasone 17- valerate carried out by using malononitrile as a reference standard. The selection of reference material should not interfere with the analyte. Methylene signal of Betamethasone (BET) 17- valerate appeared at δ= 3.9 ppm (singlet) relative to the signal of malononitrile at δ= 4.45 2 ppm (singlet) in DMSO-d6, as a NMR solvent. The method was found to be linear (r = 0.9999) for BET concentration range from 0.5 to 3.5 mg/ml. The relative standard deviation for accuracy and precession was found to be not more than 2.0 %. The sensitivity of the method was carried out by LOD and LOQ, at 0.05 and 0.2 mg/ml respectively concentration. The robustness of the method was studied by changing in different parameters as well as different solvent manufacturer company. The result shows that method was accurately developed for quantification of Betamethasone 17-valerate in pharmaceutical dosage form. The present accuracy and precision data set of developed method by 1H NMR spectroscopy is conventionally more precise with respect to the other analytical method.

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P30:

Addition of KNKSR Moiety Enhances the Antimicrobial Activity of Designed Peptides: A Combined Spectroscopy and Microscopy Study

Sk. Abdul Mohid1, Anirban Bhunia1*

1Department of Biophysics, Bose Institute, P-1/12, CIT Road, Scheme VII (M), Kolkata 700054, India.

Key words: AMP, TOCSY, NOESY, LPS

In this report, an extension of a previously published work has been carried out to identify more potent next generation peptides. We aimed to modify a potent 16 residue long AMP VG16KRKPA to a shorter equally potent 13 residue analogue VG13P. Subsequent N- or C- terminal addition of a ―KNKSR motif‖ isolated from Bovine Lactoferrin peptide (WR17) B, known to play a key role in peptide anchoring to the microbial cell surface, was performed to give rise to KG18 and VR18, respectively. MIC# and MHC# assays showed that the VG13P, VR18 and KG18 are non-hemolytic peptides with broad range of activity against Gram- negative bacteria such as E. coli, P. aeruginosa and plant pathogens such as X. oryzae and X. campestris and human fungal pathogens C. albicans and C. neoformans var. grubii. Further studies revealed that the peptides have the potential to disintegrate the lipopolysaccharide (LPS) micelles and permeabilize the outer and inner membrane of multi drug resistant P. aeruginosa. The solution NMR structure of KG18 and VR18 in LPS show a folded amphipathic conformation with a separation of polar and hydrophobic faces. A turn-type structure was observed at the C-terminal of KG18 while VR18 was mainly stabilized by aromatic – aromatic packing and hydrophobic interactions between Trp, Phe and Leu in both cases with extended N- and C- terminal, respectively, populated with charged residues. The motif based design of KG18 and VR18 from VG13P provides valuable insights into the development of more potent antibacterial peptides to fight against emerging MDR pathogens.

References:

A) Datta A, Ghosh A, Airoldi C, Sperandeo P, Mroue KH, Jiménez-Barbero J, Kundu P, Ramamoorthy A, Bhunia A, Sci Rep. 2015 Jul 6; 5:11951. B) Ghosh A, Datta A, Jana J, Kar RK, Chatterjee C, Chatterjee S, Bhunia A, Mol Biosyst. 2014 Jun; 10(6):1596-612.

* To whom correspondence should be addressed:

Dr. Anirban Bhunia

Email: [email protected] / [email protected]

Tel: +91-33-2569 3336.

#MIC: Minimum Inhibitory Concentration; MHC: Minimum Hemolytic Concentration.

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Molecular Insight into the Lipid Directed Interaction of A Designed Antimicrobial Peptide Dipita Bhattacharyya++, Dong-Kuk Lee+, Anirban Bhunia++

++Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, +Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul, Korea

Key words: Antimicrobial Peptide (AMP), Membrane lipids, Membrane specificity.

Our studies with a 16 residue long, designed antimicrobial peptide (AMP), VG16KRKP have shown it to have activities against a host of Gram negative bacterial strains, mainly via membrane perturbation. However, haemolytic assays performed with the same did not show any haemolytic activity against human blood cells. Hence the properties of lipid compositions of the membranes that help mediate interactions with the AMP must be well understood to unravel the underlying mechanism of the peptide‘s selectivity towards microbial membrane. Thus in order to understand the impact of the lipid head groups of the predominantly occurring phospholipids in bacterial and/or mammalian membrane on their interaction with the peptide, we prepared lipid vesicles with varying concentrations of the anionic POPG and the zwitterionic POPC phospholipids. Firstly, fluorescence based dye leakage assay was performed for studying the affinity of the peptide towards the heterogenous mixture of phospholipids. The results provide a clear correlation between the total content of anionic lipids in combination with the zwitterionic phospholipids, thus highlighting the direct role played by the charged head groups of the lipids in interaction with the cationic AMP. Next, 31P NMR experiments confirmed the disorientation of the lipid vesicles with increasing concentrations of VG16KRKP. This result was further supported by DSC experiments that helped in understanding the thermodynamics of this interaction, beyond the general physical alignment of the membrane lipids or the phospholipid head groups. Taken together, this study helps elucidate the mechanisms responsible for the peptide‘s specificity towards microbial membranes at the lipid-peptide interface.

Abbreviations: NMR: Nuclear Magnetic Resonance; POPG: 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol; POPC: Palmitoyl-Oleoyl Phosphatidyl Cholin; DSC: Differential scanning calorimetry

References: 1. Ramamoorthy, A.; Thennarasu, S.; Lee, D.K.; Tan, A.; and Maloy, L. Solid-State NMR Investigation of the Membrane-Disrupting Mechanism of Antimicrobial Peptides MSI-78 and MSI-594 Derived from Magainin 2 and Melittin. Biophysical J. 2006, 91, 206–216. 2. Cheng, J.T.J.; Hale, J.D.; Elliott, M.; Hancock, R.E.W.; Straus, S.K. The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants. Biochimica et Biophysica Acta. 2011, 1808, 622–633.

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3. Datta, A.; Ghosh, A.; Airoldi, C.; Sperandeo, P.; Mroue, K.M.; Jiménez-Barbero, J.; Kundu, P.; Ramamoorthy, A.; Bhunia, A. Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control. Scientific Reports, 2015, 5:11951, 10.1038/srep11951

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Targeting Gram-Negative Bacterial Pathogens Using Nanoparticle-Tagged Antimicrobial Peptide: Structural and Functional Insights. Humaira Ilyas, Rimi Chowdhury, Santa Sabuj Das, Anirban Bhunia*

Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054

KEYWORDS: Antimicrobial peptides, gold nanoparticle, anti-salmonella peptide, Nuclear Magnetic Resonance. ABSTRACT: In an earlier report, a designed 16-residue AMP, VG16KRKP (VARGWKRKCPLFGKGG), showed potent activity against plant pathogenic Gram-negative bacteria and opportunistic fungal pathogens1. However, it failed to inhibit growth of the pathogenic bacterial strain Salmonella typhi. In the present study, we report an approach of tagging gold nanoparticles to VG16KRKP for increasing its potency against Salmonella typhi. Our study reveals that nanoparticle-tagged VG16KRKP exhibits potent anti-Salmonella activity at low concentrations in both in-vitro and in-vivo models. We have also extended our study to provide structural insights at the atomic resolution in the context of live cells and S. typhi LPS to enable a correlation between its structure and function. Structural elucidations in S.typhi LPS reveal a compact loop-type structure stabilised by hydrophobic and aromatic interactions between Val1, Ala2, Trp5, Cys9, Pro10, Leu11 and Phe12 forming a closed web like network that probably helped in docking the peptide at the LPS hydrophobic groove formed of acyl chains. The positively charged residues R3, R7, K8 and K14 formed a cationic site being oriented at a distance close to 12 Å from each other hinting at plausible electrostatic interactions with the phosphate head groups of LPS a key step in AMP- membrane interaction initiation. Altogether, our work provides a novel gold-NP tagged peptide having the potential to serve as anti-Salmonella agent. Additionally, crucial structural information may aid in development of novel antimicrobial agents for use as drugs. REFERENCE: 1. Datta A, Ghosh A, Airoldi C, Sperandeo P, Mroue KH, Jiménez-Barbero J, Kundu P, Ramamoorthy A, Bhunia A. Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control. Sci Rep. 2015 Jul 6;5:11951. doi: 10.1038/srep11951.

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ACCELERATED RELAXOMETRY AT EARTH’S FIELD RASHMI RAO, NITHIN VAJUVALLI, SAIRAM GEETHANATH* Medical Imaging Research Centre, Dayananda Sagar Institutions, Bangalore Pulse sequence design in polarizing field systems involve inclusion of a polarizer pulse of 3 to 4s long which results in long acquisition times for T1 and T2 measurements. Current work demonstrates Fast, Robust and SimulTaneous T1 and T2 (FRoST) on an Earth Field MR system (Terranova, Magritek Inc.). It measures T1 using ‗T1Bp‘ and ‗T1Be‘ sequences (called ‗macros‘) with acquisition times of 98 and 130 seconds respectively for 1 average for a sample of water. T2 measurements require 100 and 6 seconds respectively. FRoST employs variable polarizing pulse durations using 2 schemes for fast and robust measurements. For fast measurements, polarizing time (τp) was varied linearly from 1000ms to 4000ms with a step of 1000ms. Another fast scheme of FRoST involves τp chosen as 1000, 2000, 4000 and 5000ms to acquire exponentially increasing and saturation regions of the T1 relaxation curve. Results from a 500ml of water sample are shown in Figure 1. For –τp/T1 each value of τp, echos from a CPMG macro was curve fitted using S=S0 (1- e ) to obtain T1. T2 value is obtained directly as a result of CPMG macro. For robust measurements, τp was varied from 500ms to 5000ms with a step of 500ms. T1 and T2 of 1.2mM CuSO4 phantom was measured using FRoST sequence. τp for fast measurements was varied as 100ms to 400ms with a step of 100ms and 100,200,400 and 500ms. Robust measurements were carried out by varying τp from 100ms to 500ms with a step of 50ms and the results are shown in Figure 2. FRoST employs series of CPMG acquisitions which inherently enables averaging of T2 4 times in the case of fast acquisitions and 10 times in the case of robust acquisitions. FRoST provides fast and robust relaxometry measurements which are close to those obtained by standard macros available in Terranova in a much lesser time. Since variation of τp in a polarizing field system is analogous to variation of TR in conventional systems, this method can be scaled to high field systems as well. Future work involves in application of FRoST for ex-vivo characterization of tissues like tumours.

Figure 1: T1 and T2 results of FRoST on water Figure 2: T1 and T2 results of FRoST on 1.2mM CuSO4 phantom

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P34:

Structural change in retinal- and sodium ion-binding site of Na+ pump rhodopsin at neutral and acidic pH studied by solid-state NMR

Arisu SHIGETA1, Shota ITO2, Okitsu TAKASHI3, Akimori WADA3, Keiichi INOUE2,4, Hideki KANDORI2, Izuru KAWAMURA1 1Yokohama Natl. Univ., 2Nagoya Inst. Tech., 3Kobe Pharm. Univ., 4JST・PRESTO Krokinobacter rhodopsin 2 (KR2) is a sodium ion pumping rhodopsin extracted from K. eikastus [1]. Comparing with other H+ or Cl- pumps, the mechanism of cation transportation should be unique as an electrostatic barrier around retinal Schiff base (SB) should be removed during transport process. Crystallography showed two conformations of counterion, Asp116, at neutral and acidic pH [2]. KR2 and other sodium ion pumping rhodopsin (NaR) has conserved residues of Asn112, Asp116 and Gln123 (NDQ motif) which is DTD motif instead in the case of Bacteriorhodopsin (BR) with Asp85, Thr89 and Asp96 [3]. Thus, these NDQ motif of NaR are thought to be important for sodium ion pumping, hence, we focused on the structural information of retinal binding pocket. Here, we present solid-state NMR results of [14, 20-13C]Retinal, [Phenyl-4-13C]Tyr, [6-13C]/[7-15N]Lys- labeled WT-KR2 sample at pH 8.0, 6.0, 5.0, 4.0 and 3.0 which were applied to Dipolar Assisted Rotational Resonance experiment to observe magnetization transfer between 13C-13C spin pairs close each other [4]. From DARR and CP-MAS spectra, we assigned the chemical shifts of Retinal C20, Retinal C14, Tyr218Cζ, Lys255Cε, Lys255Nζ and Tyr25Cζ at each pH. 15N NMR signal of protonated Schiff base showed torsion around N-Cε at neutral condition compared to Bacteriorhodopsin (BR) because of the unique location of counterion, which is one helical pitch away towards the cytoplasmic side. Chemical shift of Lys255Nζ indicated the pKa of Asp116 to be between 5.0 and 6.0. Tyr218 forms weak hydrogen bond with Asp251 which allows to work as a temporal binding site of Na+ during photocycle. The chemical shift of Tyr218 did not change, thus suggesting the pKa of Asp251 is lower than 4.0. In addition, Na+ binding site at extracellular side releases Na+ at acidic pH observed by the change of hydrogen bonding strength of Tyr25. These features may give us new insights about unique mechanism of sodium pump.

[1] Inoue, K. et al. Nat. Commun. 2013, 4, 1678. [2] Kato, H. E. et al. Nature 2015, 521 (7550), 48–53. [3] Inoue, K. et al. Angew. Chemie Int. Ed. 2015, 54 (39), 11536–11539. [4] Shigeta, A. et al. (2016) submitted.

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P35

Study of NF-κB dimer formation by NMR spectroscopy

Manish K. Chhoker1, Prikshat Dadhwal1 Tahseen Raza1, Kandala V. R. Chary2 and Sulakshana P. Mukherjee1

1Biomolecular Interaction and Structural Dynamics Lab, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India

2Department of Chemical Sciences, Tata Institute of Fundamental Research, 1, Homi Bhabha Road, Colaba, Mumbai 400005, India

NF-κB is a family of mammalian transcription factors, which regulate the expression of a large number of genes involved in cell growth, immune response, inflammatory viral response, and apoptotic cell-death. NF-κB activates transcription through binding to huge number of DNA κB sites which are distributed in the mammalian genome. This family comprises of five members, namely, RelA (also known as p65), p50, p52, c-Rel, and RelB, which form dimers in various combinations. The heterodimer p65/p50 being the most abundant form. Each NF-κB monomer contains a conserved DNA binding Rel homology region (RHR) consisting of a N-terminal domain and a C-terminal dimerization domain. The different composition of the dimers add to the diverse set of genes that are recognised and thus activated by this family. However, the exact mechanism of various combinatorial dimer formation remains unclear. In this study, I have endevoured to determine the role of preferential dimer composition on the NF-κB target gene regulation using NMR spectroscopy. The aim of this project is to study the dynamics that the individual NF-κB members undergo in solution and further to determine the mechanism of formation of various combinatorial dimers.

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P36:

Non-covalent interaction of SUMO with amyloidegenic protein α-synuclein inhibits its aggregation

Rajlaxmi Panigrahi, Dev Thacker, Jai Shankar Singh, Priyatosh Ranjan, Ashutosh Kumar

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India

α-Synuclein is a natively unfolded neuronal protein that is expressed abundantly in the presynaptic neurons. It has been associated with synaptic vesicle trafficking, but its physiological function remains largely unknown. However, it plays a central role in the pathology of Parkinson‘s disease, as well as Lewy Body Disease, and multiple system atrophy. α-synuclein is the major component of the intracellular Lewy bodies that are the histopathological hallmark of Parkinson‘s disease (Lasheul et al., 2013). Posttranslational modifications including ubiquitination (Bennett et al., 1999; Lee et al., 2008)., phosphorylation (Okochi et al., 2000; Smith et al., 2005, and nitrosylation (Giasson et al., 2000; Hodara et al., 2004) of α-synuclein have been reported to play a role in α-synuclein aggregation. A recent study showed that SUMOylation, a post-translational modification involving covalent attachment of SUMO to the target protein, abolishes α-synuclein fibril formation. In our study, we show that SUMO-1 interacts non-covalently with α-synuclein and it alters the aggregation kinetics and fibril morphology of α-synuclein as confirmed by nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC) and atomic force microscopy (AFM). We also determine the mechanism by which the inhibition of α-synuclein fibril formation occurs upon SUMO-1 and α-synuclein non-covalent interaction.

References

1. Lashuel, H.; Overk, C.; Oueslati, A.; Masliah, E. Nature Reviews Neuroscience 2013, 14, 38-48. 2. Stefanis, L. Cold Spring Harbor Perspectives in Medicine 2012, 4:a009399 3. Bennett, M.C., Bishop, J.F., Leng, Y., Chock, P.B., Chase, T.N., Mouradian, M.M., J. Biol. Chem 1999, 274, 33855–33858. 4. Lee, J.T.,Wheeler, T.C., Li, L., Chin, L.S. Mol. Genet. 2008, 17, 906–917. 5. Okochi, M.,Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P.J., Haass, C. J. Biol. Chem. 2000, 275, 390–397. 6. Giasson, B.I., Duda, J.E., Murray, I.V., Chen, Q., Souza, J.M., Hurtig, H.I., Ischiropoulos, H., Trojanowski, J.Q., Lee, V.M. Science 2000, 290, 985–989.

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P37:

Quantum State Tomography on NMR Quantum Computer

Aditya Mishra* and Anil Kumar**

*BS-MS Student, Department of Physics ,IISER, Mohali * *Department of Physics, Indian Institute of Science, Bangalore

The ability of experimental techniques for preparing initial states and implementing the universal set of quantum logic gates are two major important features for its use in quantum information processing by NMR. The characterization of output quantum state at the end of algorithm or a protocol is equally important2. To completely characterize the state of the system one has to obtain all elements of the density operator. The vanishing nature of Tr(ρI+)or Tr(ρI-) terms unless ρ represents single quantum terms, make NMR sensitive only to the single quantum coherences. Therefore one has to design set of pulse sequences that map the higher order quantum terms to readout position in order to obtain full density matrix3.The number of pulse sequences increases as dimension of the system increases and one also has to reconstruct the density matrix those many times. Therefore, the Quantum State Tomography is a challenging task for higher dimensions of the system.

In this study, we have designed new set of pulse sequence to tomograph the density matrix for a 4-qubit system. We have also verified the tomograph by using given set of pulse sequence for 2-qubit case1 by applying unitaries to the initial density matrix.

References :

(1)Avik Mitra ,Arindam Ghosh,Ranabir Das, Apoorva Patel and Anil Kumar Experimental implementation of local adiabatic evolution algorithms by NMR quantum information processor, Journal of Magnetic Resonance 177(2005)285-298. (2)Ivan S.Oliveira,Tito J. Bonagamba,Roberto S.Sarthour,Jair C.C. Freitas and Eduardo R. DeAzevedo, NMR Quantum Information Processing, ELSEVIER, ISBN:978-0-444-527820. (3)Isaac Chuang and Michael Nielsen, Quantum Computation and Quantum Information, Cambridge university press.

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Implication of the intrinsic dynamics of CENP-A (Cse4) N-terminus tail in the recognition and assembly of kinetochore proteins

Nikita Malik1, Sarath Chandra Dantu1, Mamata Komberbail2, G Krishnamoorthy2, Ashutosh Kumar1 1Dept. of Bioscience & Bioengineering, IIT Bombay, Mumbai, 2Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai. Protein-protein interactions are of utmost significance for all cellular processes like, cell signaling, cell cycle regulation, and other biosynthetic pathways. The signaling molecule often induces conformational changes in the protein, which is a cue for further relay of information. The centromeric region of chromosome is also a hub for different protein-protein and protein-DNA interactions that in- turn regulate the cell division. A Histone H3 variant protein CENP-A (Cse4) is an important component of the specialized nucleosome at the centromere that is responsible for successful chromosome segregation. The structure of C-terminus of Cse4 in complex with Histone H4 and chaperone protein, Scm3 and has been elucidated by NMR [1], however the structure of the N- terminus is not known. The N-terminus is disordered and is the longest tail amongst all known histones. Due to the lack of structural studies there is no information available about the interaction of Cse4 N-terminus with other centromeric proteins, neither is the function of this tail completely established.

In our study, we are focusing on probing the structure and recognition dynamics of Cse4 and its interaction with other centromeric components. NMR studies have been carried out and the denatured protein (229 aa) has been assigned [2]. This information is used to map the change in the residue specific dynamics of both the N- and C- terminus domains. Time resolved fluorescence and MD simulations have been used to study the dynamics of both domains in the presence and absence of other centromeric proteins. Our results suggest that the binding of one component to Cse4 leads to conformational changes in the unique N-terminus tail, shedding light on the steps that lead to formation of specialized nucleosome. This offers a structural model to explain the functions of the unique N-terminus of Cse4.

References-

1. Zhou Z, et al. (2011). Nature 472:234–237. 2. Malik and Kumar (2016). J Biomol NMR 66(1):21-35.

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P39:

Synthesis and characterization of N-(5-methylisoxazol-3-yl)-2- (phenylsulfonyl)acetamide derivatives

Ramu Kakkerlaa, Srinivas Marri b,c and M. P. S. Murali Krishnac

aDepartment of Chemistry, Satavahana University, Karimnagar-505001,Telangana, India. bDepartment of Chemistry, Siddhartha Degree & P.G. College, Narsampet- 506132,Telangana,India. cDepartment of Chemistry, Andhra Polytechnic College, Kakinada-533003, Andhra Pradesh,

The chemistry of isoxazole derivatives continues to draw the attention of synthetic and medicinal chemists due to their varied biological activities. A large number of isoxazole derivatives exhibited anticancer, anticonvulsant, analgesic, antibacterial and antifungal activity. On the other hand, sulfonamide derivatives have been reported to possess significant antibacterial activities through competitive inhibition of dihydropteroate synthetase enzyme (DHPS) which is involved in folate synthesis. Moreover, some sulfonamides work as ant- inflammatory drugs like celecoxib which works as a COX-2 inhibitor and acetazolamide which works by diuretic mechanism .On light of these findings, we planned to prepare the target compounds as hybrid molecules. These molecules contain the isoxazole ring system and sulfonamide moiety.

The key intermediate 2-chloro-N-(5-methylisoxazol-3-yl)acetamide required for the preparation of the title compounds, was obtained by the interaction of 3-amini-5- methylisoxazole with chloroacetyl chloride in the presence of triethylamine in dry benzene. The 2-chloro-N-(5-methylisoxazol-3-yl)acetamide coupled with different substituted thiophenols(2a-d) in the presence of K2CO3 in dry acetone to give N-(5-methylisoxazol-3-yl)- 2-(phenylthio)acetamide, these coupled product undergoes the oxidation in the presence of hydrogen peroxide in glacial acetic acid to give title compounds. The structure of all the synthesized compounds were established on the basis of their spectroscopic data (IR, 1H NMR, 13C NMR and mass spectra).

O O O Cl SH O R HN HN K2CO3 S HN H2O2 S O N Dry Benzene N N O O H3C H3C O R R H3C

R = H, Cl, F, CH3

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Exploring the diffusion dynamics of tri-block copolymer –nanoparticle system using PFG-NMR spectroscopy Jyotsana Ojha1, Dr Raju Nanda2, Dr Kavita Dorai3

Department of Physical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81 SAS Nagar, Manauli PO 140306 Punjab India. [email protected],[email protected],[email protected]

Diffusion Order NMR-Spectroscopy (DOSY) has been used in this work to understand the dynamics of aqueous triblock co polymeric (F127:PEO100-PPO65-PEO100) mixture of gold nano particles (AuNPs). The interactions of F127 with the AuNPs are fundamentally important to study as these systems are used in a wide range of application starting from medicines to materials sciences. In this context PFG NMR is one of the simplest methods to study the intermolecular interactions probing through their self-diffusion measurements. Both the temperature and concentration dependent self-diffusion studies of F127 have been performed in its F127-H2O and F127-H2O-AuNPs mixtures. The aqueous solutions of F127 shows interesting signal attenuation with the increase of the applied gradient strength indicating the anomalous diffusion of the polymer. The signal attenuation is not following the Stejskal-Tanner (ST) equation in this case; however stretched-ST equation has been applied to estimate the self- diffusion coefficient of the polymer. Interestingly, this behaviour has been disappears with the change in both the concentration and temperature of the medium. On the other hand, the self- diffusion of polymer is following the ST equation in its F127-H2O-AuNPs mixtures; however the estimated self-diffusion coefficients shows significant variation with the change in the diffusion time, suggesting an anomalous diffusion of the polymer in the system. 2D NOESY and TOCSY (1H-1H) study indicates the strength of the binding between the AuNPs with the F127. It is expected that this NMR study advance the understanding of polymer-H2O-AuNPs systems for their potential applications in various areas of medicines and material sciences.

References:

[1] Ulrich, K; Galvosas, P; Karger, J; Grinberg, F. Effects of Self-Assembly on Diffusion Mechanisms of Triblock Copolymers in Aqueous Solution. Phy. Rev. Lett., 2009, 102, 037801. [2] Walderhaug, H; Nystrom, B. Anomalous Diffusion in an Aqueous System of a Poly(ethylene oxide)-Poly(propyleneoxide)-Poly(ethylene oxide) Triblock Copolymer during Gelation Studied by Pulsed Field Gradient NMR. J. Phys. Chem. B, 1997, 101, 1524. [3] Xu, J; Han, Y; Jie, Cui; Jiang, W. Size Selective Incorporation of Gold Nanoparticles in Diblock Copolymer Vesicle Wall. Langmuir. 2013, 29, 10383. [4] Shukla, M; Dorai, K. Resolving Overlaps in Diffusion Encoded Spectra Using Band- Selective Pulses in a 3D BEST-DOSY Experiment. J. Magn. Reson. 2011, 50, 69. [5] Shukla, M; Dorai, K. Disentangling Diffusion Information of Individual Components in a Mixture with a 3D COMPACT-IDOSY NMR Experiment. Magn. Reson. Chem. 2012, 50, 341.

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Inhibition of Insulin Amyloid Fibrillation by a Novel Amphipathic Heptapeptide: Mechanistic Details Studied by Spectroscopy in Combination with Microscopy

Bhisma N Ratha, Anirban Bhunia Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata-54, India; Tel: +91-33-25693336; E-mail: [email protected] and [email protected]

The aggregation of insulin into amyloid fibers has been a limiting factor in the development of fast acting insulin analogues, creating a demand for excipients that limit aggregation. Despite the potential demand, inhibitors targeted specifically at insulin have been few in number. Here we report a designed, non-toxic and serum stable heptapeptide, KR7 (KPWWPRR) that differs significantly from the primarily hydrophobic sequences that have been previously used to interfere with the insulin amyloid fibrillation. ThT fluorescence assays, CD spectroscopy, and one-dimensional proton NMR experiments suggest KR7 primarily targets the fiber elongation step with little effect on the early oligomerization steps in the lag time period. From confocal fluorescence and AFM experiments, the net result appears to be the arrest of aggregation in an early, non-fibrillar aggregation stage. This mechanism is noticeably different from previous peptide-based inhibitors, which have primarily shifted the lag-time with little effects on later stages of aggregation. As insulin is an important model system for understanding protein aggregation, the new peptide may be an important model compound for understanding peptide-based inhibition of amyloid formation.

Reference

Ratha, B. N.; Ghosh, A.; Brender, J. R.; Gayen, N.; Ilyas, H.; Neeraja, C.; Das, K. P.; Mandal, A. K.; Bhunia, A. Journal of Biological Chemistry 2016.

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Detecting a Late Folding Intermediate of Ubiquitin

Parag Surana and Ranabir Das

National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore 560065, India

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by Nuclear Magnetic Resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with co-factors as well as for assembly of poly-Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein‘s core, the long loop converges on the core in the final step of the folding process.

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Hand-shake” model is used for recognition of F-box proteins by Skp1 in SCF ubiquitin E3 ligase

Sarath Chandra Dantu, Nitin Nathubhai Kachariya, Amrita Bhattacharya, Rupali Shinde, Ashutosh Kumar

Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, 400076

SCF E3 ligase is a multi-protein enzyme complex endowed with the final step of ubiquitination process; i.e. transfer of multiple ubiquitin‘s from the E2-Ubiquitin complex onto the target substrate bound to the F-box protein (FBP) on the other end of the SCF, separated by a 6 nm distance. S-phase kinase-associated protein 1 (Skp1) is the link, i.e. the adapter protein which presents the FBP to the rest of the SCF complex formed by Cullin-1, Ring box protein-1, and the E2-Ubiquitin. More than 70 different FBP‘s are encoded by human genome and Skp1 has to recognize such a diverse class of proteins and then further contribute to the ubiquitination.1 Using molecular dynamics (MD) simulations we have initially proposed that the C-terminal helices H7 and H8 of Skp1 lose their helicity in absence of FBP Skp2 and when in complex with Skp2 it modulates its conformational properties.2 The solution state NMR experiments confirm the above prediction.3

Here we present the solution structure of Skp1. We will also present the residue specific dynamics extracted from model free analysis of NMR relaxation experiments. Further, using the Force distribution analysis on molecular dynamics data along with NMR data, we propose a model of how Skp1 uses the conformational flexibility of its C-terminal helices to recognize various FBP‘s.

References

1. Cardozo, T.; Pagano, M., The SCF ubiquitin ligase: insights into a molecular machine. Nat Rev Mol Cell Biol 2004, 5 (9), 739-751. 2. Dantu, S. C.; Kachariya, N. N.; Kumar, A., Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex. Proteins: Structure, Function, and Bioinformatics 2016, 84 (1), 159-171. 3. Kachariya, N. N.; Dantu, S. C.; Kumar, A., Backbone and side chain assignments of human cell cycle regulatory protein S-phase kinase-associated protein 1. Biomolecular NMR Assignments 2016, 10 (2), 351-355.

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Influence of Alkyl Chain Length on Binding and Dynamics of Ionic liquids with Collagen and Human Serum Albumin – A detailed NMR Investigation

R. Ravikanth Reddy,a,† B. V. N. Phani Kumar,a,† Ganesh Shanmugam,b,† Balaraman Madhanc,† aNMR Lab, Inorganic & Physical Chemistry Department, bOrganic & Bioorganic Chemistry Laboratory, cCHORD, a,b,cCSIR-Central Leather Research Institute, Adyar, Chennai-600 020, India. †Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhavan, 2 Rafi Marg, New Delhi 110 001, India.

The molecular insights on the interaction of greener solvents such as ionic liquids (ILs) with collagen are beneficial as ILs could be alternative solvents for leather making in future. Similarly, IL interaction with globular proteins could be important as ILs can stabilize native protein which may increase the efficiency of enzyme, catalytic activity, etc. Hence, the binding and dynamics of the green ILs such as imidazole alkyl sulfate based ILs viz, 1- butyl 3-methylimidazolium alkyl [where alkyl = Hydrogen, Methyl, Octyl and Dodecyl] sulfate with the collagen and a model protein such as Human Serum Albumin (HSA) in aqueous solution, have been made with the aid of solution NMR. Various NMR Parameters i.e., 1H chemical shifts, non-selective spin-lattice relaxation times (T1NS), spin-spin relaxation times (T2) and selective spin-lattice relaxation times (T1SEL) have been employed to probe the molecular level interactions of ILs with HSA and Collagen. In addition, STD NMR has been used to monitor the spatial proximity of IL‘s with HSA and Collagen. The results indicate the selective binding of anionic part of IL with HSA and Collagen. Selective T1 data analysis revealed the global binding affinity between IL and proteins enhanced with an increase in alkyl chain length of the anionic part of IL.

References:

1. Reddy, R. R.; Phani Kumar, B. V. N.; Shanmugam, G.; Madhan, B.; Mandal, A. B., Molecular Level Insights on Collagen-Polyphenols Interaction Using Spin-Relaxation and Saturation Transfer Difference NMR. J Phys Chem B 2015, 119, 14076-85. 2. Meyer, B.; Peters, T. NMR Spectroscopy Techniques for Screening and Identifying Ligand Binding to Protein Receptors. Angew. Chem. Int. Ed. 2003, 42, 864-890. 181

Mehta, A.; Rao, J. R.; Fathima, N. N., Electrostatic Forces Mediated by Choline Dihydrogen Phosphate Stabilize Collagen. J Phys Chem B 2015, 119, 1281

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NMR and EPR studies on the stabilization of SDS in presence of the pluronic L61 triblock copolymer G. K. S. Prameela,a B. V. N. Phani Kumar,a V. K. Aswal,b J. Subrahmanian,a A B Mandala and S. P Moulikc

aNMR Lab, Inorganic and Physical chemistry department, Council of Scientific and Industrial Research (CSIR)-Central Leather Research Institute (CLRI), Chennai 600020, Adyar, India.. bSolid State Physics Division, Bhabha Atomic Research Center, Mumbai 400085, India cCentre for Surface Science, Department of Chemistry, Jadavpur University, Kolkata 700032, India.

The stabilization of Sodium dodecylsulfate (SDS) microstructure in presence of the pluronic L611in aqueous medium was examined using NMR and EPR techniques. EPR measurements were done by using a spin probe 5-Doxyl Steric Acid (5-DSA) incorporated in the micelle system. Both NMR and EPR results indicated the presence of L61 facilitated mixed micelles, whose onset occurred at CAC ([SDS] ~4 mM). This mixed micelle formation in both SDS and L61 is well evidenced by variable diffusion binding isotherms in NMR and rotational correlation times measured for the nitroxide spin label 5-DSA in EPR. Furthermore, 2H NMR spin-relaxation studies evidenced non-spherical nature of the microstructure of SDS-L61 mixed aggregates which is well supported by Small Angle Neutron Scattering (SANS) measurements. These results when compared with our earlier results on SDS/L31,2 supported that higher hydrophobicity of L61 (PPO/PEO ~10) played a significant role in inducing highly non-spherical microstructures when mixed with SDS micelles. Representative NMR self-diffusion (Figure A) and EPR rotational correlation time (Figure B) results are presented in the figures below.

References 1. Kostarelos, K.; Tadros, Th. F.; Luckham, P. F. Physical Conjugation of (Tri-) Block Copolymers to Liposomes toward the Construction of Sterically Stabilized Vesicle Systems. Langmuir 1999, 15, 369-376. 2. Prameela, G. K. S.; Phani Kumar, B. V. N.; Pan, A.; Aswal, V. K.; Subramanian, J.; Mandal, A. B.; and Moulik, S. P. Physicochemical perspectives (aggregation, structure and dynamics) of interaction between pluronic (L31) and surfactant (SDS). Phys. Chem. Chem. Phys., 2015, 17, 30560-30569.

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Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide Fragments from Human Lysozyme: A Combined Spectroscopy, Microscopy, and Docking Study Kar, R.K. and Bhunia, A* Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India Tel: +91-33-25693336. E-mail: [email protected] and [email protected]

Degenerative diseases, such as Alzheimer‘s and prion diseases, as well as type II diabetes, have a pathogenesis associated with protein misfolding, which routes with amyloid formation.1 Recent strategies for designing small-molecule and polypeptide anti-amyloid inhibitors are mainly based on mature fibril structures containing cross β-sheet structures.2 Several applications of polypeptide agents with anti-amyloid properties have been reported in the literature, whereby these agents are designed based on their properties like β-sheet binders/breaker and hydrophobicity.3 In the present study, we have tackled the hypothesis that the rational design of anti-amyloid agents that can target native proteins might offer advantageous prospect to design effective therapeutics. Lysozyme amyloid fibrillization was treated with three different peptide fragments derived from lysozyme protein sequence R107- R115 (Lz-peptide) or to a modification by the point mutation to change). The interference of the studied peptides with formation of lysozyme amyloid aggregates has been examined using experimental and computational methods. Using low-resolution spectroscopic, high- resolution NMR, and STD NMR-restrained docking methods such as HADDOCK4, we have found that these peptide fragments have the capability to affect lysozyme fibril formation. The present study implicates the prospect that these peptides can also be tested against other amyloid-prone proteins to develop novel therapeutic agents.5

References: (1) Knowles, T. P.; Vendruscolo, M.; Dobson, C. M. Nat. Rev. Mol. Cell Biol. 2014, 15 (6), 384−96. (2) Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8 (2), 101−112. (3) Takahashi, T.; Mihara, H. Acc. Chem. Res. 2008, 41 (10), 1309−18. (4) Dominguez, C.; Boelens, R.; Bonvin, A. M. J. Am. Chem. Soc. 2003, 125 (7), 1731−7. (5) Kar, R.K.; Gazova, Z.; Bednarikova, Z,; Mroue, K.H.; Ghosh, A.; Zhang, R.; Ulicna, K.; Siebert, H.C.; Nifantiev, N.E.; Bhunia, A. Biomacromolecules 2016, 17; 1998-2009

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Residue Specific dynamics of Skp1 in SCF ubiquitin E3 ligase

Amrita Bhattacharya, Nitin Kachariya, Sarath Chandra Dantu, Ashutosh Kumar

Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400076

The S-phase kinase-associated protein 1 (Skp1) is an adapter protein in the multi-protein SCF (Skp1-Cullin-1-F-box) ubiquitin E3 ligase complex. Skp1 recognizes and binds to the F-box protein (FBP), which in turn binds and presents the substrate meant for degradation to the rest of the E3 ligase. Skp1 binds to the conserved F-box domain of the FBP via its C-terminal helices H6, H7 and H8. FBPs are the only variable component of the SCF complex and thereby determine the substrate specificity. Many of these FBPs along with Skp1 are implicated in cell cycle dysregulation, DNA damage1,cancer2,3 along with several other diseases. Therefore, the Skp1-Fbox domain interface is a potential new drug target.4 Skp1 has been co-crystallized with five different FBP's, however the interaction and recognition dynamics of this interface has not been investigated yet. To design drugs for this interface, the first step is to understand the dynamics of Skp1 alone. In this work we present the residue level dynamics of various residues of Skp1 in ps-ms regime studied using various solution state NMR experiments.

1. Santra M., Wajapeyee, N., and Green, M. F-box protein FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage, Nature(2009), 459, 722-725 2. Randle S. and Laman, H. F-box protein interactions with the hallmark pathways in cancer, Seminars in Cancer Biology (2016), 36, 3-17 3. Yong-Qiang Liu1, Xiao-Lu Wang1, Xin Cheng1, Yong-Zhi Lu2, Gui-Zhen Wang et al. Skp1 in lung cancer: Clinical significance and therapeutic efficacy of its small molecule inhibitors, Oncotarget (2015), 6(33), 34953–34967 4. Cardozo and Abagyan, R. Druggability of SCF Ubiquitin Ligase–Protein interfaces, Methods in Enzymology (2005), 399,634-53

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Understanding the structural and dynamics basis of interaction between Plasmodium falciparum and Human sumoylation machinery

Jai Shankar Singh1, Vaibhav Kumar Shukla2, Mansi Gujarati3, Ram Kumar Mishra3, Ashutosh Kumar1

1Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai, 2Centre for Excellence in Basic Sciences, Mumbai. 3Department of Biological Sciences, Indian Institute of Science Education & Research, Bhopal.

Malaria is one of the deadliest infectious diseases which takes millions of lives annually and is caused by parasitic protozoans of the genus Plasmodium. Deadliest amongst these forms is the ―cerebral malaria‖ which is afflicted upon by Plasmodium falciparum (1). Plasmodium adopts various survival strategies including post-translational modifications (PTMs) to stabilize and potentiate its crucial proteins for successful infection cycle in the mosquito and human host. Recently, sumoylation has been characterized as one of the important PTMs and many of its putative substrates have been identified in Plasmodium (2). Sumoylation is the covalent attachment of SUMO protein to the substrate protein, which is mediated by three distinct enzymes, E1-activating (hetero-dimeric Aos1/Uba2), E2-conjugating (Ubc9) and E3 ligase. These act in a sequential manner to attach SUMO to the substrate protein in an ATP dependent manner (2). Since the structure of Pf-SUMO is still unknown, we elucidated its structure by solution state NMR and studied the residue specific interactions of Pf-SUMO with Pf- Ubc9 and the possibilities of cross-interaction of host and parasite sumoylation machineries. Further, biophysical studies, like ITC (Isothermal titration calorimetry) have been done to verify the interactions between Plasmodium and the human proteins. We have identified the important residues of Pf-SUMO and proteins involved in sumoylation that governs the specificity of interaction in Plasmodium as well as human host.

References:

1. Murray CJL et al. (2012) Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet 379:413-431 doi: 10.1016/S0140-6736(12)60034-8 2. Herhaus L, Dikic I (2015) Expanding the ubiquitin code through post‐translational modification. EMBO Rep 16:1071-1083 doi: 10.15252/embr.201540891 3. Hay RT (2005) SUMO: a history of modification. Mol Cell 18:1-12 doi:10.1016/j.molcel.2005.03.012

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Structural comparison of the DNA-binding domain of several telomere- binding proteins to illustrate the evolutional mechanism

Zeyu JIN, Ji-Hye Yun, Sunjin Moon, Sunggeon Ko, Weontae Lee

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins (TBP), they play an essential role in genome stability. With the evolution of the living beings, there are large amounts of evidences telling us the correlation between different species. Within our studies, we determined the solution structure of DNA binding domain of TBP in different species, such as, NgTRF1 in tobacco[1], RTBP1 in rice[2], CEH-37 in nematode Caenorhabditis elegans[3], and AtTRB2 in Arabidopsis thaliana[4][5]. The structural information shows the mechanism of how those TBP interact with specific telomere sequence in different species. The overall structure and functions of them show the correlation by evolutional relationship between the species. The NgTRF1 and RtBP1 belongs to the plantae subkingdom show the similar structure which have four α-helixes and specifically bind to the plant telomere sequences, (TTTAGGG)n as opposed to human telomeric proteins TRF1 and TRF2, as the another side of evolution tree, have three α-helixes and target on the (TTAGGG)n specifically. CEH-37, belonging to Nematode Caenorhabditis elegans, located in unique branch on evolution tree, specifically binds double stranded C. elegans telomeric DNA

(TTAGGC)n, which is different from that of other eukaryotic species. Interestingly, AtTRB2, the domain of TBP from Arabidopsis thaliana, has three helixes, the same as human TBP, and could recognize both human and plant telomere sequences. Through all of those studies, we could draw the story of how evolutional changes happened in the TBP by species for structural stability and specific DNA recognition.

Reference:

[1] Ko, S., Jun, S. H., Bae, H., Byun, J. S., Han, W., Park, H., ... & Kim, W. T. Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins. Nucleic acids research, 2008, 36(8), 2739-2755.

[2] Ko, S., Yu, E. Y., Shin, J., Yoo, H. H., Tanaka, T., Kim, W. T., ... & Chung, I. K. Solution Structure of the DNA Binding Domain of Rice Telomere Binding Protein RTBP1. Biochemistry, 2009, 48(5), 827-838.

[3]. Moon, S., Lee, Y. W., Kim, W. T., & Lee, W. Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans. Biochemical and biophysical research communications, 2014, 443(2), 370-375.

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[4] Yun, J. H., Lee, W. K., Kim, H., Kim, E., Cheong, C., Cho, M. H., & Lee, W. Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana. Biochemical and biophysical research communications, 2014, 452(3), 436-442.

[5] Lee, I., Kim, H., Ko, Y. J., & Lee, W. NMR Characterization of the DNA‐binding Domain of Arabidopsis thaliana Telomere Repeat Factor.Bulletin of the Korean Chemical Society, 2016, 37(4), 485-489.

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Developing Solid-State NMR Techniques for Structural Characterization

Kshama Sharma a, Kaustubh R. Mote a, Vipin Agarwal a, Matthias Ernst b, P. K. Madhu a,c

aTIFR Centre for Interdisciplinary Sciences, bETH Zurich, cTata Institute of Fundamental Research

Solid-state NMR is a very flexible and powerful technique for elucidation of geometry and dynamics information on a variety of samples. However, there is still need to overcome sensitivity and resolution aspects along with the necessity to carry out multidimensional experiments in a short span of time. In order to overcome these challenges, we have made use of three approaches. First approach includes the improvement of heteronuclear spin decoupling efficiency at high magic-angle spinning (MAS) frequencies as high MAS has led to improved resolution and sensitivity due to better averaging of dipolar interactions. The appropriate use of low-power (~10 kHz) decoupling sequences shows comparable performance as high-power (~195 kHz) decoupling sequences which is deleterious for most of the biological samples.1 Secondly, the structural investigation techniques relying on the recoupling of various interactions such as dipolar coupling. The use of symmetry-based dipolar-recoupling sequences has shown great success in determining structures up to a picometer resolution for 13C-13C distances. We have shown that the asynchronous implementation of (a symmetry-based dipolar recoupling sequence) outperforms the rotor-synchronized and supercycled version of especially for spin pairs with small dipolar couplings and large chemical-shift anisotropies. Thirdly, to speed up the data acquisition process, the pulse sequences that implement sequential acquisition strategies on one and two radio radiofrequency channels with a combination of proton and carbon detection to record multiple experiments under magic-angle spinning have been coded. 2

References

1. K. Sharma, P. K. Madhu, V. Agarwal, J. Magn. Reson., 270,136-141. 2. K. Sharma, P. K. Madhu, K. R. Mote, J. Biomol NMR., 65, 127-141.

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Multiband 19F Overhauser DNP Study of a Highly Viscous System: 1, 4- difluorobenzene and Poloxamer (P188)

Arnab Dey, Abhishek Banerjee and N. Chandrakumar*

MRI-MRS Centre, Department of Chemistry, IIT Madras, Chennai-600036, Tamil Nadu E-mail: [email protected]

Nuclear magnetic resonance (NMR) offers insights into molecular structure and dynamics, although it suffers from low sensitivity. The inherently low sensitivity of NMR may however be overcome by hyperpolarization. Overhauser dynamic nuclear polarization (ODNP)1 is one possible hyperpolarization technique. ODNP exploits cross relaxation of nuclear spins by electron spins in solution state. As ODNP arises from the interaction of the electron spin with nuclear spins in the substrate molecule, it can give direct information about molecular dynamics and intermolecular interactions, besides the gain in sensitivity. In the last few years ODNP is getting considerable renewed attention from research communities in the context of studies on biomolecules, biofluids, polymers etc2,3.

ODNP enhancements are highly influenced by translational and rotational molecular motions in liquids, and hence also change with viscosity. ODNP experiments in a highly viscous medium at moderately low fields could be very interesting as they allow us to probe slow molecular motions4, which are difficult to probe directly with high field NMR.

Here we report a multiband 19F ODNP study of a system composed of 1, 4- difluorobenzene (1,4-DFB) and poloxamer P188. We have investigated the changes in DNP parameters (including enhancement, leakage factor, coupling parameter) with different compositions of solute P188 and solvent (1,4-DFB). Our studies are in the context of the wide applications of poloxamers, which it may be recalled, are triblock copolymers with both hydrophilic and hydrophobic groups, which are widely used for drug delivery. Further, poloxamers in association with different fluorinated molecules are popular in biomedical studies such as in vivo imaging and in vivo cell tracking with magnetic resonance, etc.

Our preliminary findings suggest that at X-band 19F enhancements are in line with anticipated viscosity changes, resulting in reduced dipolar contribution to the cross relaxation at higher viscosity, and thus leading to stronger positive enhancements. At Q-band, however, ‗leakage‘ relaxation, which would be more than an order of magnitude stronger, appears to lead to reduced scalar enhancements at higher viscosity.

References

1. Overhauser, A. W. Polarization of Nuclei in Metals. Phys. Rev. 1953, 92, 411-415. 2. Neudert, O.; Reh, M.; Spiess, H. W.; Münnemann, K. X-Band DNP Hyperpolarization of Viscous Liquids and Polymer Melts. Macromol. Rapid. Commun. 2015, 36, 885- 889.

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3. Grucker, D.; Guiberteau, T.; Eclancher, B.; Chambron, J.; Chiarelli, R.; Rassat, A.; Subra, G.; Gallez, B. Dynamic Nuclear Polarization with Nitroxides Dissolved in Biological Fluids. J. Magn. Reson. 1995, 106, 101-109. 4. Banerjee, A.; Dey, A.; Chandrakumar, N. Slow Molecular Motions in Ionic Liquids Probed by Relaxation of Nuclear Spins during Overhauser Dynamic Nuclear Polarization. Angew. Chem. Int. Ed. 2016, 55, 14756-14761.

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1H NMR based serum metabolomics analysis of hepatitis C virus-infected patients of Liver Cirrhosis

Amar Deep1,2, Atul Rawat3, Durgesh Dubey3, Suchit Swaroop1, Ajay Kumar2#,

1Experimental and Public Health Laboratory, Department of Zoology, University of Lucknow 2Department of Gastroenterology, King George’s Medical University, Lucknow 3Centre of Biomedical Research, Lucknow

Background: Liver-related conditions due to chronic hepatitis C virus infection are the third leading cause of nonsurgical hospitalizations after cardiovascular diseases and infections. Chronic HCV carriers are at risk of progressive liver fibrosis, carcinoma, and up to 30% can develop cirrhosis after decades of infection with significant risk of morbidity and mortality. Metabolomics is increasingly being used for studies on infectious diseases. Aim & Objective: To see the metabolic changes in serum using 1H NMR in hepatitis C infected patients. Methods: We performed metabolome profiling of serum samples to identify potential biomarkers for diagnosing HCV. Results: The study group comprised of HCV patients (n=20) and age and sex matched healthy control (n=20). Serum samples were collected from the study group and processed according to the standard protocol. Two types of high-resolution 1D 1H NMR spectra were acquired, CPMG and diffusion edited to explore the metabolic perturbation. A 1H NMR-based metabolic profiling approach coupled with multivariate statistical analysis was employed to establish early markers of HCV infection. Our results revealed the multiple metabolic pathways were affected in these patients, mainly involving lipid metabolism leading to an increase in cholesterol, altered phospholipid metabolism, and N-acetyl glycoproteins. Conclusion: The present pilot study will be the guiding attributes for a larger sample size and to discriminate between various stages of fibrosis, necrosis, inflammation and steatosis.

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Differential Metabolic Signatures for Lupus and Lupus Nephritis identified using NMR based serum metabolomics Durgesh Dubey1, Anupam Guleria1, Avadhesh Pratap2, Smriti Chaurasia2, Atul Rawat1, Ramnath Misra2 and Dinesh Kumar1 1Centre of Biomedical Research and 2Department of Clinical Immunology, SGPGIMS, Raibareli Road, Lucknow-226014, India *Author for Correspondence: [email protected] Systemic lupus erythematous (SLE) is a chronic inflammatory autoimmune disease which affects kidney. Lupus nephritis (LN) is a serious manifestation of SLE. Conventional biomarkers in current use have not been very successful in early accurate recognition of renal involvement within SLE. Thus, novel biomarkers are necessary to enhance the diagnostic accuracy and sensitivity of lupus renal disease, prognostic stratification, monitoring of treatment response and detection of early renal flare.

Metabolic profiles of serum obtained from 22 SLE patients and 40 LN patients were analyzed using high-resolution 1D 1H CPMG NMR spectra combined with multivariate data analysis such as principle component analysis (PCA) and projection to least squares discriminant analysis (PLS-DA). Metabolites responsible for separation in PLS-DA model were tested using independent sample t-test. Principal component analysis (PCA) and partial least- squares discriminant analysis (PLS-DA) models were capable of distinguishing LN patients from SLE patients. The metabolites responsible for separation of two groups were identified using loading plot and Variable importance in projection (VIP) score. The serum of LN patients was characterized by higher levels of lipoproteins (LDL and VLDL), lipids, as well as lower levels of acetate as compared to SLE patients. Our observed data reveals that the serum NMR-based metabolomics might have high sensitivity and specificity to distinguish LN from SLE patients and might have the potential to be a useful tool in diagnosis and monitoring of Lupus nephritis.

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Interaction between novel fluorescence probes and a dodecameric dsDNA: A 1H-NMR study

Sudakshina Ganguly1, Nagarjun Narayanaswamy2, Debasis Ghosh2, T. Govindaraju2, Gautam Basu1

1Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054; 2New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bengaluru 560064.

AT-specific hemicyanine-based thiazole coumarin1 (TC) and quinone cyanine–dithiazole2 (QCy–DT) based switch-on probes, red and NIR fluorescent, respectively, have been recently reported for DNA recognition. We have probed the interactions of TC and QCy-DT with a short dodecameric self-complementary sequence D1 (5‘-CGCGAATTCGCG-3‘) using 1H NMR spectroscopy. The aim was to identify specific interactions with DNA and determine the DNA-binding mode.

Figure 1. Structures of TC and QCy-DT, along with D1 imino protons resonances with increasing amounts of TC and QCy-DT.

Titration of TC to D1 resulted in differential peak shifts in D1 reflecting binding. Further analysis showed that TC specifically perturbs the central AATT base pairs of D1. However, upon reverse titration (D1 added to TC), TC resonances broadened and disappeared by 0.2:1 D1:TC molar ratio. This corroborated with the observation that TC resonances were undetectable when TC was added to D1. This suggests that TC binds DNA at the central AATT nucleotides but probably undergoes fast exchange between multiple binding modes leading to line broadening. Titration of QCy-DT to D1 (and the reverse) also resulted in differential peak shifts in D1 and QCy-DT due to binding, however, unlike TC, QCy-DT resonances were observable. A 1:1 complex of D1 and QCy-DT exhibited NOE cross-peaks between QCy-DT (H1/H12 and CH3 protons) and D1 (AATT), with a concomitant disappearance of some intra-D1 NOE peaks and appearance of some new intra-DNA NOE peaks. Although the results are preliminary, the results show that both TC and QCy-DT bind the self-complementary DNA sequence D1 at the AATT stretch using different binding 194 modes (modeling suggested TC to be an intercalator and QCy-DT to be a groove binder). Further experiments and analysis are in progress for elucidating the binding modes.

References:

1. Narayanaswamy N., Kumar M., Das S., Sharma R., Samanta P., Pati S.K., Dhar S.K., Kundu T.K., Govindaraju T. (2014) Sci. Rep, 4, 6476. 2. Narayanaswamy N., Das S., Samanta P.K., Banu K., Sharma G.P., Mondal N., Dhar S.K., Pati S.K., Govindaraju T. (2015) Nucleic Acids Res., 43, 8651.

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P55:

Fluorine chemical shift tensor and NMR cross correlation studies in fluorinated molecule

Rakesh Sharma and Kavita Dorai*

Department of Physical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, P.O. Manauli, Punjab-140306, India *[email protected]

Tryptophan is an important amino acid in biomolecules and proteins. Its 19F-labeled analogues are generally biocompatible and cause small perturbation in structure. So it can be used as an NMR probe to study the structure and dynamics of bigger biomolecules like proteins and oligonucleotides. Together, these considerations led us to study fluorine relaxation parameters in Tryptophan. Here we studied the Fluorine CSA tensor and cross- correlation effects in Tryptophan. We used 1D and 2D 19F-1H cross-correlated relaxation experiments to measure cross-correlation between the 19F chemical shift anisotropy (CSA) and its dipolar interaction with a scalar coupled proton. These cross-correlated CSA/dipolar relaxation rates were used to characterize the 19F CSA tensor.

References:

[1] J. W. Peng Journal of Magnetic Resonance, 2001, 153, 32-47. [2] K. Dorai & A. Kumar Chem. Phy. Letters, 2001, 335, 176-182. [3] S. B. Elavarasi & K. Dorai Chem. Phy. Letters 2010, 489, 248-253.

196

P56:

Entanglement dynamics of three qubits under intrinsic decoherence and protection using dynamical decoupling on an NMR quantum information processor

Harpreet Singh *, Kavita Dorai † and Arvind ‡

Department of Physical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81, SAS Nagar, Manauli PO 140306 Punjab India. *[email protected], † [email protected] , ‡ [email protected]

Quantum entanglement arises due to correlations between the two or more quantum systems [1]. Recent studies on two-qubit systems have showed that the decoherence of quantum entangled states leads to interesting phenomena such as entanglement sudden death i.e. finite- time destruction of quantum correlations. The class of entangled states which are long lived decoherence-free states and the existence of entangled states that lose coherence faster has also been studied [2]. Multiqubit entanglement behaviour is very different under various types of decohering channels [3]. We explore experimentally the entangled states of a three- qubit system, by preparing the different states belonging to the GHZ and W class of states, and observe their decay under intrinsic decoherence. We applied dynamical decoupling sequences to protect the entanglement of these states [4].

References

1. M. A. Nielsen and I. L. Chuang, Quantum Computation and Quantum Information (Cambridge University Press, Cambridge UK, 2000). 2. 2.S. Maniscalco, F. Francica, R. L. Zaffino, N. Lo Gullo, and F. Plastina, Phys. Rev. Lett. 100, 090503 (2008). 3. R. Chaves and L. Davidovich, Phys. Rev. A 82, 052308 (2010). 4. A. M. Souza, G. A. Alvarez, and D. Suter, Phys. Rev. Lett., 106, 240501 (2011).

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P57:

Sine-Square Shifted REDOR: A Non-Linear Version of REDOR

Mukul G. Jain a, G. Rajalakshmia, Vipin Agarwala, P. K. Madhu a,b

a TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research, Hyderabad b Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai

Rotational-Echo DOuble Resonance (REDOR) is one of the most used recoupling experiment to estimate the dipolar coupling between two spins in MAS solid-state NMR. Accurate distance estimation will depend on the ability to match simulated curves with experimental dephasing curves. This in turn necessitates acquiring modulations to the maximum extent which is difficult, if not impossible, in strongly dipolar coupled spin systems. Hence, it is necessary to scale the effective dipolar coupling in order to achieve maximum modulations and better fitting thereby making the experiment possible in strongly coupled spin systems, like N-H and C-H. Here, we introduce a distinct class of REDOR sequence which is inspired by a sine-squared function introduced by Uhrig in the context of dynamical decoupling, denoted as SS-REDOR. We present results with the standard REDOR, shifted-REDOR (that also has a scaling property), and the new scheme, the SS-REDOR. The results demonstrate that the SS-REDOR scheme, not only has the desired scaling property, but it is also fairly robust with respect to the experimental parameters, thus setting it apart from shifted-REDOR.

REFERENCES:

1) Gullion, T.; Schaefer, J. Rotational-echo double-resonance NMR. J. Magn. Reson. 1989, 81, 196-200. 2) Uhrig, G. S. Keeping a quantum bit alive by optimized pi-pulse sequences. Phys. Rev. Lett. 2007, 98, 100504. 3) Schanda, P.; Meier, B. H.; Ernst, M. Quantitative analysis of protein backbone dynamics in microcrystalline ubiquitin by solid-state NMR spectroscopy. J. Am. Chem. Soc. 2010, 132, 15957-15967.

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P58:

Structural Investigation of Syndecan Multimerization Mediated by Its Transmembrane Domain

aJae Hyun Park, aJi-Hye Yun, bMi-Jung Kwon, bEok-Soo Oh, aWeontae Lee* aStructural Biochemistry & Molecular Biophysics Laboratory, Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, 120-749, Republic of Korea bDepartment of Life Science, Research Center for Cellular Homeostasis, Ewha Womans University,Seoul 120-750, Korea

Syndecans are cell surface adhesion receptors that initiate intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domain (TMD). Syndecan multimerization regulated by its TMD modulates functions of syndecan family. For example, the wound healing process, the crucial function of Syn-2, is controlled by Syn-2 multimerization. Three constructs of syndecan are designed (Syn-2TMD, Syn-2eTC, Syn-2eTC-F167I) to study on its multimerization. Syn-2eTC has short additional extracellular and cytoplasmic residues on Syn-2TMD. NMR titration experiments are performed using these constructs, and we observe cross peaks changed after titration. Our results show that the cross peaks of the Syn-2TMD have no significant shift in Syn-2eTC titration experiment. On the other hand, titration of the Syn-2eTC-F167I on Syn-2TMD made dramatic shifts in several cross peaks. It is indicating that Syn-2TMD/Syn-2eTC-F167I titration makes huge conformational changes than Syn-2TMD/Syn-2eTC titration. In detail, F167 and F172 residue of Syn-2TMD showed dramatic chemical shifts in the Syn-2TMD / Syn-2eTC-F167I titration, whereas F169 residue of Syn-2TMD didn‘t changed not much. This means larger conformational changes are induced in F167 and F172 but not much in F169 residue. Also, our structural studies showed that a F167 residue plays an important role of interaction between the TMDs of the Syn-2 homo-dimer. UPLC is performed on Syn-2 homo-complex and Syn-2/Syn-2F167I hetero-complex. In results, Syn-2 homo-complex formed higher tetramer formation than Syn-2/Syn-2F167I hetero-complex. The presence of F167 on TMD was correlated with a higher tendency toward multimerization. Our study is offering new insights into the syndecan signaling mediated by the TMD.

199

P59:

Analytic Theory of Spin Diffusion in Solid State Nuclear Magnetic Resonance (SSNMR)

Shreyan Ganguly and Ramesh Ramachandran

Indian Institute of Science Education and Research, Mohali, India

Solid state NMR spectroscopy is a widely used tool for characterizing proteins and other biomolecules. Spin diffusion studies are one of the main set of experiments done for this purpose. In such experiments one observes the transfer of spin polarization between two different nuclei (of low ) in the absence of RF irradiation. The key interaction in this case is the dipolar interaction of the intermediate nuclei (usually protons) and it is important to elucidate their role in such transfer mechanisms using analytic theory. In our presentation, we consider the case of two carbon/nitrogen nuclei connected to each other and also to a so called ‗bath‘ of protons which act as the intermediate medium aiding the transfer of polarization. We obtain analytic expressions for the observed polarization of the low  nuclei using contact transformation methods. Signal expressions obtained using analytic theory, are compared with numerical simulations to show the accuracy of our results.

References:

1) Suter. D & Ernst R.R, Physical Review B, 1982, 25, p6038 2) Suter. D & Ernst R.R, Physical Review B, 1985, 32, p5608 3) Veshtort Mikhail & Griffin R.G, JCP, 2011, 135, p134509 4) R. Ramesh et al, JCP, 2001, 114, p5967

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P60:

Probing the dynamics of LiClO4 mixtures of PEG-based polymers in various molecular solvents: A PGSE NMR and relaxation approach

Satnam Singh1, Dr Raju Nanda2, Dr Kavita Dorai3

Department of Physical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81 SAS Nagar, Manauli PO 140306 Punjab India. [email protected],[email protected],[email protected]

NMR spectroscopic study has been performed to study the temperature dependent dynamics and relaxation behavior of LiClO4 mixtures of polyethylene glycols-based polymers with a wide variation in their molecular weight in the presence of three different molecular solvents such as water, dimethyl sulfoxide (DMSO), and acetonitrile (ACN). The LiClO4 doped polymeric mixtures have been selected because of its use as an electrolytic media in various electrochemical devices such as the lithium ion battery and double layer supercapacitors. Temperature dependent NMR self-diffusion coefficients, spin-lattice (T1), and spin-spin relaxation (T2) studies has been performed in order to understand the dynamics of these mixtures. The ethylene oxide unit (-OCH2CH2-, EO) of polymers are probed through the NMR spectroscopy and observed that an increase in its self-diffusion coefficient values with the increase temperature of the medium. However, different hydrodynamic behavior has been observed with the increase in the MW of polymer as obtained through the Stokes-Einstein equation. Both the T1/T2 relaxations of EO unit of polymer have also been increase with the increase temperature of the medium because of their increasing reorientational correlation motion. A substantial interaction between the lithium cation with the EO unit of the polymer has been observed as probed through the 1H-7Li 2D HOESY spectra. It is expected that the current study will show a new dimension to understand the dynamics of other polymer- lithium-solvent systems through these NMR spectroscopic methods for the better designing of novel electrolytic media for their potential use in various electrochemical devices.

References:

[1] Li, Y; Wang, X; Dong, S; Chen, X; Cui, G. Recent Advances in Non-Aqueous Electrolyte for Rechargeable Li–O2 Batteries. Adv. Energy Mater. 2016, DOI: 10.1002/aenm.201600751. [2] Xue, Z; Dan, H; Xie, X. Poly(ethylene oxide)-Based Electrolytes for Lithium-Ion Batteries. J. Mater. Chem. A, 2015, 3, 19218-19253. [3] Nanda, R. Thermal Dynamics of Lithium Salt Mixtures of Ionic Liquid in Water by PGSE NMR Spectroscopy. RSC Adv., 2016, 6, 36394-36406. [4] Shukla, M; Dorai, K. Resolving Overlaps in Diffusion Encoded Spectra Using Band- Selective Pulses in a 3D BEST-DOSY Experiment. J. Magn. Reson. 2011, 50, 69-75. [5] Shukla, M; Dorai, K. Disentangling Diffusion Information of Individual Components in a Mixture with a 3D COMPACT-IDOSY NMR Experiment. Magn. Reson. Chem. 2012, 50, 341-346.

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P61:

Single-shot construction of high-fidelity three-qubit quantum gates via genetic programming

Amit Devra*, Prithviraj PrabhuƗ, Harpreet Singh† and Kavita Dorai‡ Department of Physical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81 SAS Nagar, Manauli PO 140306 Punjab India. ƗSri Sivasubramaniya Nadar College of Engineering, Kalavakkam Chennai ,Tamil Nadu 603110 India.

*[email protected], Ɨ[email protected],†[email protected], ‡[email protected]

In quantum computing breaking up of a quantum gate unitary matrix into pulse sequence for higher qubits is not a simple task. Here we considered this problem as an optimization problem and used Genetic algorithms (GAs) [1],[2] to optimize desired unitary matrices. Genetic algorithms which is a global optimization technique based on the logic of natural process of evolution and biological genetics have been widely used for quantum computing applications. Here we employ this optimization technique to optimize the three qubits quantum gates namely the Toffoli(CCNot), Fredkin(CSWAP) and CCPhase gates[3]. We obtained the RF pulses corresponding to these gates with very high fidelity. These gates were then experimentally implemented and final fidelities were computed. We compare our results with equivalent gate decomposition using standard 2 qubit gates.

References

1. Manu V.S., Kumar A Singlet-state creation and universal quantum computation in NMR using a genetic algorithm; PRA 86, 022324 (2012). 2. Ajoy A., Kumar A.;A New Hierarchical Genetic Algorithm Approach to Determine Pulse Sequences in NMR; arXiv:0911.5465v2 [quant-ph] 2009. 3. Zahedinejad E., Ghosh J.,Sanders B.; High-Fidelity Single-Shot Toffoli Gate via Quantum Control; PRL 114, 200502 (2015).

202

P62:

Comparison of schemes to prepare pseudopure states on an NMR quantum information processor

Akanksha Gautam, Kavita Dorai, Arvind*

Department of Physical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, P.O. Manauli, Punjab-140306, India *[email protected]

Abstract: The preparation of pseudopure states play a central role in the implementation of quantum computing and information processing in high temperature ensemble systems such as NMR. It is created by rearranging the states of N-spin molecules (which are initially in thermal equilibrium) such that a portion forms a uniform background that does not contribute to the measured signal, and the remainder forms a deviation that does. This deviation will behave as a pure state (pseudopure state). We describe here the diﬀerent schemes available for the preparation of pseudopure states and advantages and disadvantages related to each method of pseudopure state preparation.

References: (1) Cory, D. G.; Fahmy, A. F.; Havel, T. F. Proc. Natl. Acad. Sci. USA 1997, 94, 1634-1639. (2) Kawamura, M.; Sakai, H.; Manmoto, Y. AIP Conf. Proc. 2009, 1110, 305. (3) Kawamura, M.; Rowland B.; Jones, J. A. Phys. Rev. A, 2010, 82, 0323315.

203

P63:

Metabolic analysis of liquid formulations of organic manures and its influence on growth and yield of Solanum lycopersicum L. (tomato) crop in field.

Dattatraya U. Ukale a, Rhohit V.Bhagwat b, Santosh Kumar Upadhyay c, Nivedita Cukkemane Nivedita d, Abhishek A. Cukkemane d *

a Department of Chemistry, Central Instrumentation Facility, Savitribai Phule Pune University (SPPU), Ganeshkhind Road, Pune411007, Maharashtra, India.b Department of Environmental Sciences, SPPU, Ganeshkhind Road, Pune 411007, Maharashtra, India.c CSIR-Institute of Genomics & Integrative Biology, New Delhi110020, India d Bijasu Agri Research Laboratory LLP,S.No.37, Oldno.33,Kondhawa Industrial Estate, Pune 411048, Maharashtra, India.

Abstract: We report biochemical analysis of two formulations of liquid Organic Manures (OMs), which were prepared using raw materials readily available to farmers in fields; thus presenting them with cost- effective organic preparations. Previously reported microbial analysis of these OMs indicated presence of rich microbial consortia, which enhance plant growth by making nutrient available to crop from soil. Likewise, we observed stimulatory effects of these two OMs on tomato plants in field trials that were marked byimprovedseedgerminationandgrowthby2-and3-folds,respectively.Metabolic profiling of the two preparations using Nuclear Magnetic Resonance (NMR) spectroscopy revealed presence of several plant-growth regulators, nucleo bases, vitamins, amino acids, sugars and organic acids. This study provides a vital biochemical link in understanding the positive effect of organic molecules of microbial origin in OMs. Nevertheless, along with these biomolecules, we also observed traces of fungicides and herbicides in all the preparations hinting at contaminated raw materials. This highlights the importance of robust analysis procedures in animal feeds and plant materials using analytical tools such as NMR spectroscopy based analysis that will aid farmers in not only preparing high quality nutrient management systems but with minimal residual pesticides. Moreover, cost effective preparations of OMs from animal wastes will open a window for organic Farming beneficial for human society.

Reference:

204

1) Gore, N., Sreenivasan, J. Agric. Sci, 2011, 24, 153–157. 2) Heeb, A., Lundegardh, B, Savage, G, Ericsson, T., J. Plant Nutr. Soil Sci. 2006, 169, 535– 541.

205

P64:

pH induced structure and stability modulation of T7 endolysin

1 3 1,2 Meenakshi Sharma Dinesh Kumar and Krishna Mohan Poluri

1 2 Department of Biotechnology, Centre for Nanotechnlogy, Indian Institute of Technology Roorkee

Roorkee – 247667, Uttarakhand, India 3 Centre of Biomedical Research, SGPGIMS, Lucknow – 226014, Uttar Pradesh, India

Email: [email protected], [email protected]

T7 bacteriophage endolysin (T7L), also known as N-acetylmuramyl-L-alanine amidase or T7 Lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the L-alanine of the peptidoglycan layer. The majority of the endolysins originating from bacteriophages that infect Gram- negative bacteria adopt a single-domain structure consisting of catalytic domain such as an amidase domain of T7 family endolysins. Although, the mechanism of lysis is investigated for these endolysins, the molecular basis for their pH dependent differential activity is not yet known. Therefore, the current study aims to unravel the pH dependent structural and functional characteristics of T7L family members using bacteriophage T7 endolysin as a model protein. pH-induced conformational transition has been investigated by a combination of optical methods, size exclusion chromatography and NMR spectroscopy.

Our studies established reversible structural transition of T7L below pH 6 and the formation of a partially collapsed conformation at pH 3 characterized by the substantial secondary structure with exposed hydrophobic pockets. We experimentally confirmed the thermal stability and structure-function reversibility of partially denatured conformation. Furthermore, to unravel the residue level insights on the dynamic behavior of T7L, NMR relaxation measurements and hydrogen exchange analysis has been performed. The data established that T7L is highly dynamic in its native state and the repulsive interactions of the partially buried histidine residue protons facilitate the reversible pH dependent structural transition.

REFERENCE

Sharma M, Kumar D, Poluri KM. Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin. Biochemistry. 2016;55(33):4614-25.

206

P65:

Structural Dynamics in Hierarchy of Time-scale Probe the Amyloidogenic Nature of Transthyretin

Aritra Bej, Jitendra K. Das, Shyam S. Mall, and Sujoy Mukherjee*

Structural Biology and Bioinformatics Division, CSIR - Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal - 700032, India *E-mail: [email protected]

Transthyretin (TTR) is a 127-residue homotetrameric protein (~55 kDa) that forms amyloid fibrils in a large number of protein aggregation diseases like senile systemic and familial amyloidoses1. Previous studies have suggested that amyloid formation is preceded by tetramer dissociation, enhancing the formation partially unfolded intermediates2. Due to the transient nature and low population, these intermediates are difficult to characterize. In recent work, we have performed a quantitative and qualitative study of the backbone dynamics of the wild-type TTR and its clinically important four mutants (V122I, V30M, L55P, and T119M) over an extended timescale using NMR spectroscopy and MD simulations and observed a significant correlation in structural flexibility with their amyloidogenic propensity3. We have performed relaxation NMR techniques and atomistic MD simulations on five TTR proteins to show that the pathogenic mutants exhibit more structural perturbations than the wild-type. We further show that backbone dynamics in different hierarchy of time-scale is significantly higher in pathogenic mutants, energetically favouring their transition to a more stable non-native intermediates with respect to the wild-type. This study opens up novel insights into TTR aggregation process that may help to develop new therapeutics against TTR amyloidogenesis.

References

(1) Saraiva, M. J. Hum. Mutat. 1995, 5 (3), 191–196. (2) McCutchen, S. L.; Lai, Z.; Miroy, G. J.; Kelly, J. W.; Colón, W. Biochemistry 1995, 34 (41), 13527–13536. (3) Das, J. K.; Mall, S. S.; Bej, A.; Mukherjee, S. Angew. Chem. Int. Ed. Engl. 2014, 53 (47), 12781–12784.

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P66:

NMR Elucidation of Structural and Dynamic features of MIP2-

Khushboo Gulati1 Dinesh Kumar3 and Krishna Mohan Poluri1,2*

1Department of Biotechnology, 2CentreforNanotechnology Indian Institute of Technology Roorkee, Roorkee – 247667, Uttarakhand, India 3Center of Biomedical Research, SGPGIMS Campus, Lucknow-226014, Uttar Pradesh, India

Chemokines are chemotactic cytokines that exhibits manifold physiological and pathological functions in cells. Growth related Oncogene (Gro) chemokines, comprise of activating chemokines (NACs), that are recognized by their highly conserved ―ELR‖ motif and plays an essential role in orchestrating neutrophil recruitment to the site of infected tissue. During recruitment, chemokines forms oligomers to form gradients, interact with endothelial cell surface GAGs and CXCR2 receptors present on neutrophils. We explored the molecular level details of chemokine oligomerization and chemokine-GAG interactions using Gro------NMR spectroscopy (HNCA, HNCACB, CBCACONH, HNCO, NOESY, TOCSY etc). We compared the inh - - their GAG binding affinities using heparin binding assay. We further deciphered the residue level stabilities and dynamics of CXCL3 using native state hydrogen / deuterium exchange and 15N relaxation (longitudinal (R1), transverse (R2), and NOE) measurements. Our studies revealed that MIP2- to MIP2- inding affinities similar to MIP2- providing the structure of MIP2- oligomerization and GAG binding affinities in comparison with MIP2- differential functionalities.

208

P67:

NANOMATERIALS BASED CANCER DIAGNOSTICS, IMAGING AND THERAPIES

Zinia Mohanta, H S Atreya (NRC) and Chandan Srivastava(MatE)

This project involves development of graphene based nanomaterials for imaging/detection of cancer cells and targeted destruction of cancerous tumors. Magnetic resonance imaging (MRI) is the most popular tool for diagnosis in medical science today. Inspite of rendering an excellent imaging spatial resolution, MRI suffers from low sensitivity which often results in several diseases such as cancers remaining undetected in their early stages of development. This project work addresses this particular issue with the objective of developing and investigating nanoparticle-graphene oxide/graphene/carbon composites that will act not only as strong image contrast enhancing agents in MRI but also help in targeting the tumor cells. Targeting will be achieved by covalent coupling of the tri-peptide: RGD (known to bind to cancer cells) to graphene oxide along with a fluorescent probe (Au nanorods). Photothermal properties of gold nanorods will then be used to kill the cancer cells.

References:

1. J Shen et al, Synthesis of graphene oxide-based biocomposites through diimide-activated amidation, J. Colloid Interface Sci., 2011, 356, 543 –549

2. N Lee et al, Designed synthesis of uniformly sized iron oxide nanoparticles for efficient magnetic resonance imaging contrast agents Chem. Soc. Rev.,2012, 41, 2575–2589.

209

P68: 1H-[13C]-NMR Investigations of Energy Metabolism in Spinal Cord and Brain in Amyotrophic Lateral Sclerosis Madhuri Puvvada, Sampath Kumar and Anant Bahadur Patel NMR Microimaging and Spectroscopy, CSIR-Center for Cellular and Molecular Biology, Habshiguda, Uppal Road, Hyderabad 500007, India

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by selective degeneration of upper and lower motor neurons leading to progressive muscle weakness and eventual death as a result of respiratory failure [1]. The objective of the study was to evaluate neuronal metabolic activity in the brain and spinal cord of ALS affected mice using 1H-[13C]- NMR spectroscopy. G37R SOD1 mutant mice (17month old) were used as a model of ALS. Mice were genotyped for the presence of transgene using taqman qPCR method and subsequently assigned into ALS and control groups. Limb strength was assessed by paw grip strength meter and visual neurological scorings. Mice were anesthetized using urethane (1.5 g/kg, i.p) and infused with 13 13 [1,6- C2]glucose or [2- C]acetate to investigate neuronal or astroglial metabolic activity, respectively [2-3]. The 13C labeling of spinal cord and brain metabolites were measured ex vivo in tissue extracts using 1H-[13C]-NMR spectroscopy [4]. The cerebral metabolic rate of glucose oxidation by glutamatergic and GABAergic neurons was calculated as described earlier by Patel et al [5]. The ALS mice exhibited reduced grip strength and rigid paralysis as compared with the wild type controls. Decreased level of NAA and increased myo-inositol suggest neuronal death and inflammation in the spinal cord and brain stem of ALS mice. The decreased labeling of glutamate- C4, GABA-C2 and glutamine-C4 in ALS mice indicate reduced cerebral metabolic rates of glucose oxidation by glutamatergic and GABAergic neurons in the spinal cord of ALS mice. Surprisingly, the metabolic activity of glutamatergic and GABAergic neurons was found to be enhanced in the cerebral cortex and striatum suggesting increased excitatory and inhibitory neurotransmission in ALS brain. These data indicate contrasting changes in the neuroenergetics of spinal cord and non-motor region of the brain in ALS mice. References: 5. Robberecht, W. and Philips, T. Nat. Rev. Neuroscience 2013, 14, 248-164. 6. Patel et al. J. Cereb. Blood Flow Metab. 2004, 9, 972-985. 7. Patel et al. J. Cereb. Blood Flow Metab. 2010, 30, 1200-1213. 8. deGraaf et al. Magn. Res. Med. 2003, 49, 37-46. 9. Patel et al. Proc. Natl. Acad. Sci. USA 2005, 102, 5588-5593.

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Acknowledgements: The study was supported by funding from DST (CO/AB/013/2013) and network project (BSC0208).

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P69:

1H-[13C]-NMR Investigations of Brain Energy Metabolism in Alzheimer’s Disease following Riluzole Intervention Kamal Saba and Anant Bahadur Patel

NMR Microimaging and Spectroscopy, CSIR-Centre for Cellular and Molecular Biology, Habshiguda, Uppal Road, Hyderabad 500007, India Introduction: Alzheimer‘s disease (AD) is a neurodegenerative disorder characterized by a progressive and gradual decline in cognitive function1. Although different approaches have been used to combat the AD, there is very limited success. Most of the currently available drugs are used to maintain cognitive function and delay the symptoms of disease without affecting the underlying disease process. The objective of the present study is to evaluate the efficacy of riluzole on neuronal metabolic activity in AβPP-PS1 mouse model of AD. Materials and Methods: AβPP-PS1 (Tg) and wild type (Wt) mice (8 months) were divided into four groups: Group (i) WT + Carboxymethyl cellulose (CMC 1%) (n=6); (ii) Tg + CMC (n=6); (iii) WT + RZ (n=7); (iv) Tg + RZ (n=6). Mice in Group (iii) and (iv) were administered 30 doses of riluzole on alternate day while those in Group (i) and (ii) received same volume of CMC. For metabolic measurements, mice were anesthetized using urethane 13 (1.5 g/kg, ip) and [1,6- C2]glucose was infused through lateral tail vein2. Concentration and percentage 13C enrichment of brain amino acids were measured in 1H-[13C]-NMR spectra of tissue extracts acquired at 600 MHz spectrometer (Fig. 1)3. The metabolic rates of glucose oxidation were determined from the 13C labeling of brain amino acids from 13 2 [1,6- C2]glucose . Results and Discussion: The concentrations of 13C labeled amino acids were found to be significantly lower in AβPP-PS1 as compared with age matched controls suggesting glucose hypometabolism by glutamatergic neurons in the cerebral cortex (Fig. 2). The intervention of riluzole in AβPP-PS1 mice increased 13C labeling of brain amino suggesting improved cerebral metabolic rate of glucose oxidation to the control level. As rate of neuronal glucose oxidation is stochiometrically coupled with neurotransmitter cycling, finding of increased neuronal oxidation with riluzole intervention suggest improved neurotransmission in AD mice. These data suggest that riluzole has potential to manage cognitive function in Alzheimer's Disease.

212

Reference: 1. Martin et al, Eur. J. Pharmacol. 1993, 250, 473–476; 2. Patel et al, Proc. Natl. Acad. Sci. USA. 2005 102, 5588-5593; 3. de Graaf et al, Magn. Reson. 2005 49, 37-46. Acknowledgements: This study is supported by funding from (CO/AB/013/2013) and network project (BSC0208).

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P70:

NMR-based metabolomics studies of the effect of air pollution on cycling metabolites in plants grown on traffic dividers

Sumit Mishra1, Navdeep Gogna1, Rakesh Sharma1 and Kavita Dorai*1 *E-mail: [email protected]

1Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City Sector 81, Mohali PO Manauli, India

Abstract: NMR-based metabolomics is a widely used technique to investigate changes in plant metabolism due to environmental stress. We perform 1D and 2D NMR experiments for untargeted metabolite fingerprinting in several different plant species grown on traffic dividers. We use NMR spectroscopy in conjunction with univariate and multivariate statistical analysis to identify the cycling metabolites in the plants. We then study the effect of air pollution, including vehicular emissions, on the cycling metabolites in these plant species.

References: 1. Gogna, N., Krishna, M., Oommen, A. M., & Dorai, K. (2015). Investigating correlations in the altered metabolic profiles of obese and diabetic subjects in a South Indian Asian population using an NMR-based metabolomic approach.Molecular BioSystems, 11(2), 595-606. 2. Gogna, N., Hamid, N., & Dorai, K. (2015). Metabolomic profiling of the phytomedicinal constituents of Carica papaya L. leaves and seeds by 1 H NMR spectroscopy and multivariate statistical analysis. Journal of pharmaceutical and biomedical analysis, 115, 74-85. 3. Gogna, N., & Dorai, K. (2015). HR-MAS NMR-based metabolomic approach to study the effect of fungicidal stress on wheat seed germination. CURRENT SCIENCE, 108(9), 1694. 4. Gogna, N., Singh, V. J., Sheeba, V., & Dorai, K. (2015). NMR-based investigation of the Drosophila melanogaster metabolome under the influence of daily cycles of light and temperature. Molecular BioSystems, 11(12), 3305-3315. 5. Augustijn, D., Roy, U., van Schadewijk, R., de Groot, H. J. M., & Alia, A. (2016). Metabolic Profiling of Intact Arabidopsis thaliana Leaves during Circadian Cycle Using 1 H High Resolution Magic Angle Spinning NMR. PloS one, 11(9), e0163258.

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Selective and Efficient Quantum Process Tomography without Ancilla

Akshay Gaikwad, Kavita Dorai*

Department of Physical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, P.O. Manauli, Punjab-140306, India *[email protected]

Abstract: The characterization of quantum process is task of fundamental and practical importance. It allows us not only to study the dynamics of system but also helps to investigate the noisy channel affecting the quantum processor and helps to design good error correcting strategies. In general, quantum process tomography (QPT) is a challenging task due to exponential amount of parameters are involved as a function of no. qubits. In this study, we compare two different ways of doing QPT. The 1st one is ―Standard quantum process tomography (SQPT)‖ based on full state tomography of input and output states and other one is ―Selective and Efficient quantum process tomography (SEQPT) without ancilla‖ based on the fact that the elements of process matrix can be interpreted as average survival probability over something called ‗quantum 2-design state‘. Here, these two protocols are experimentally demonstrated on NMR for two qubit quantum gates.

References: (1) A. Bendersky, F. Pastawski, and J. P. Paz, Phys. Rev. A 80, 032116 (2009). (2) J. M. Renes, R. Blume-Kohout, A. J. Scott, and C. M. Caves, J. Math. Phys. (N.Y.) 45, 2171 (3) "On single qubit quantum process tomography", R.Bhandari, arXiv:1502.01016 (4) NMR quantum information processing by Ivan S. Oliveira

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P72:

Exploring Neurochemical Homeostasis under Anesthesia using 1H NMR Spectroscopy

Sreemantula Arun Kumar, Pooja Gautam and Anant Bahadur Patel

NMR Microimaging and Spectroscopy, CSIR-Center for Cellular and Molecular Biology, Habsiguda, Uppal Road, Hyderabad 500007, India.

Brain energy metabolism is highly regulated to attain proper neuronal function, and has been implicated in cognitive function, particularly in aging and neurodegenerative disorders. Anesthetics play significant role during surgical interventions. Though, different anesthetics have been used to study brain energy metabolism in rodents1,2, their impact on the neuronal function and homeostasis of different neurometabolites is not well understood. The objective of the current study is to evaluate effects of two anesthetics, urethane (injectable) and isoflurane (volatile), on the homeostasis of neurometabolites in brain. Male C57BL6/J mice (5 months old) were divided into three groups (n=6 each): Group A, awake mice; Group B, mice under isoflurane (1.2–1.5%); Group C, mice under urethane (1.5 g/kg i.p.). Mice in groups B and C were maintained under particular anesthetic for 45 min, and euthanized using focused beam microwave irradiation3. Neurometabolites were extracted from dissected brain tissues4. Concentration of brain metabolites were measured ex vivo in 1H NMR spectra of tissue extracts acquired at 600 MHz spectrometer5. Typical 1H NMR spectrum obtained from the cerebral cortex is shown in Fig. 1. Exposure with urethane and isoflurane exhibit significant reduction in the levels of glutamate, NAA, aspartate and creatine in the striatum, while that of aspartate and alanine was decreased in the cerebral cortex. Additionally, urethane decreased the level of cortical glutamate, NAA, taurine and creatine, while isoflurane lowered GABA only. Moreover, concentration of alanine was lowered in isoflurane group while myo-inositol and taurine were decreased under urethane in the striatum. This is the first study evaluating the impact of anesthetics on the neurometabolites homeostasis, which revealed a significant changes in level of key neurometabolites, emphasizing the role of anesthetics for regulating brain function. Reference: 1. de Graaf et al. Proc. Natl. Acad. Sci. USA 2004, 101, 12700-12705; 2. Tiwari et al. J. Cereb. Blood Flow Metab. 2013, 33, 1521-1531; 3. Epstein et al. J Neuroimmune Pharmacol. 2013, 8, 1224–1238; 4. Patel et al. Brain Res. 2001, 919, 207-220; 5. Bagga et al. J. Neurochem. 2013, 127, 365–377.

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Acknowledgements: The study was supported by funding from DST (CO/AB/013/2013) and network project (BSC0208).

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P73:

NMR based evaluation of chemosensory phytomarkers Aruna Singh, Rama Jayasundar*

Department of NMR, All India Institute of Medical Sciences, New Delhi, India Corresponding author ([email protected])

Taste is a widely known but poorly understood chemosensory property, associated with all physical substances including plants. Proton NMR spectroscopy is ideally suited to study the chemosensory characteristic of plants, since it allows simultaneous detection of both primary and secondary metabolites. The present study explores the use of NMR in the context of ayurvedic pharmacology, which has a chemosensory based classification of medicinal plants. Plants (n=24) from four taste categories (sweet, astringent, bitter and pungent) were subjected to chloroform-methanol/water extraction to separate the non-polar and polar fractions and followed by vacuum drying. For the NMR studies (1H and 13C) (700MHz Spectrometer,

Agilent, USA), dried polar extracts were dissolved in D2O and non-polar in CDCl3. Figure 1 shows representative spectra from a polar and a non-polar fraction. The NMR spectral data was binned and bucketed for multivariate analyses. Principle component analysis (PCA) of non-polar fractions showed distinct clustering between the sweet, astringent and bitter groups of plants (Fig. 2a) whereas the polar fractions showed distinct differences between all the taste groups (Fig. 2b). The study has important implications for ayurveda and its use of medicinal plants. Further in-depth studies are underway to explore the use of NMR to characterise the polar and non-polar chemosensory phytochemicals.

Fatty acids/lipids (a) (a)

cannabinoids

olefinic signals of fatty acids

acetate

lactate β-glucose β-glucose valine

α-glucose (b) formate sucrose alanine (b) fumarate leucine

citrate isoleucine

Figure 1: 1H NMR of Cannabis sativa (a) non-polar (b) Figure 2: PCA of the NMR spectral data polar fractions from (a) non-polar and (b) polar fractions

of plants from different taste categories 218

P74:

NMR Investigations on structural-dynamics-stability relationship of Interleukin-8 Krishnakant Gangele1 and Krishna Mohan Poluri1,2

1Department of Biotechnology, 2Center for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, India.

Chemokines are 8-10 kDa disulphide bridged proteins that provide regulatory cues during leukocyte trafficking. Neutrophil activating chemokines (NACs) are subgroup of chemokines that are actively involved in the recruitment of neutrophils during various exalted inflammatory events at the site of infection. It is of great interest since many decades, to understand the chemokine functioning and their regulatory role in the immune surveillance. Interleukin-8 (IL-8) is a well-studied chemokine belongs to NACs subfamily. It interacts with cognate receptors (CXCR1/CXCR2) and the glycosaminoglycans which are involved in the processes of neutrophil recruitment, inflammation and pathophysiology of many more diseases. IL-8 exhibits monomer-dimer equilibrium at physiological pH. However, the structural fluctuations and the stability features of this molecule are not yet well understood. In the current study, we intend to dissect the structure-dynamics characteristics of IL-8 under various environmental perturbations. To accomplish this objective, we have cloned full length (77aa) gene into the pET 32-Tev vector, overexpressed in the E. coli Rosetta (DE3) cells and recombinant IL-8 protein was purified up to the maximum purity with the help of various chromatographic techniques. Backbone assignment of the IL-8 has been done using multidimensional 3D-NMR experiments. pH dependent 1H-15N HSQC experiments indicated that IL-8 exist in the different structural conformations at pH 7.0 (Neutral) and at pH 3.0 (Very acidic). Further these conformations have been characterized using biophysical techniques such as Circular Dichroism (CD), fluorescence life time and NMR relaxation measurements.

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P75:

The Chemo-Preventive Mechanisms of Cruciferous Vegetables Derived Phytochemicals on Ovarian Cancer Metabolism: An NMR-based Metabolomics Study

Purba Sarkar, Nawneet K Kurrey, Somashekar BS

Department of Biochemistry, CSIR-Central Food Technological Research Institute (CSIR- CFTRI), Mysuru-570020, Karnataka, INDIA

Dietary phytochemicals derived from cruciferous vegetables Indole-3-Carbinol (I3C) and 3,3ʹ-diindolylmethane (DIM) has been known to arrest the proliferation of various cancer types by targeting wide range of signaling pathways involved in cell-cycle progression and proliferation. However, despite substantial research, there are several questions that remain unknown including (1) which metabolic pathway(s) are these phytochemicals targeting for cancer prevention and (2) are there any differences in the mode of action between I3C and DIM? With this prospect, in the present study, we utilized ovarian cancer cell model (PA-1) to delineate the chemo-preventive mechanism of I3C and DIM. 1H NMR-based metabolic profiling were carried out on PA-1 untreated and treated cells with varying concentrations of I3C and DIM. Prior to metabolomics study, the cytotoxic potential of I3C and DIM were analyzed using MTT assay, which revealed that both the indole phytochemicals inhibit PA-1 cell growth with concentration ranging from 25µM-200 µM. 1H NMR spectra of polar extracts were used to identify more than thirty metabolites. Further, Principal Component Analysis (PCA) on NMR data revealed clear group separation among untreated and, I3C and DIM treated cells. The group separation among I3C and DIM revealed that these two indole phytochemicals have differential mode of chemo-preventive mechanism. I3C and DIM treated PA-1 cells exhibited significantly altered levels of various metabolites involved in hexosamine pathway, energy metabolism, glutaminolysis, membrane choline phospholipid metabolism and osmo-regulatory mechanism. In summary, to the best of our knowledge, this is the first report in revealing differential chemo-preventive mechanisms of I3C and DIM on cancer metabolism. Further, this report clearly indicates that appropriate dose of these phytochemicals could be formulated as functional foods for ovarian cancer management as well as prevention.

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P76:

NMR Dynamics Study on the Formation of Z-RNA Induced by the Zα Domain of Human RNA Editing Enzyme

Ae-Ree Lee and Joon-Hwa Lee

Department of Chemistry, Gyeongsang National University, 501 Jinju-daero, Jinju-si, 52828, Republic of Korea

Double-stranded RNA deaminase I (ADAR1) deaminates adenine in pre-mRNA to yield inosine (I), which codes as a guanine residue in mRNA. These A-to-I conversions can lead to functional changes in the resulting proteins. At its NH2-terminus, ADAR1 has two left- handed Z-DNA binding domains and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The main difference between DNA and RNA is the presence of the ribose 2‘-OH groups; however, this difference makes the two macromolecules very different with regard to their biochemical behavior as well as the structures they adopt as double helices. Both B-DNA and A-RNA can undergo a transition to left-handed double-helical structures, referred to as Z-DNA and Z-RNA. The crystal structural study of the Z-DNA binding domain of ADAR1 complexed to a r(CG)3 duplex RNA found that the Z-RNA helix is associated with a unique solvent pattern that distinguishes it from the otherwise similar conformation of Z-DNA.

In order to characterize the molecular recognition of Z-RNA by ADAR1, we performed NMR experiments with complexes of ADAR1 bound to r(CG)3 and these results were compared with those of Z-DNA, d(CG)3, induced by ADAR1 previously reported. We compared to the binding affinity of ZαADAR1 for both A-DNA and Z-DNA and characterized its A–Z transition activity.

References

[1] Kang, Y.-M.; Bang, J.; Lee, E.-H.; Ahn, H.-C.; Seo, Y.-J.; Kim, K. K.; Kim, Y.-G.; Choi, B.-S.; Lee, J.-H. J. Am. Chem. Soc. 2009 ,131,11485.

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P77:

NMR study of the antifreeze activities of active and inactive isoforms of type III antifreeze protein

Seo-Ree Choi, Yeo-Jin Seo and Joon-Hwa Lee

Department of Chemistry, Gyeongsang National University, 501 Jinju-daero, Jinju-si, 52828, Republic of Korea

Antifreeze proteins (AFPs) are found in a variety of cold-adapted organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. One of most widely studied classes of AFPs is the type III AFP from arctic fishes such as the ocean pout and Japanese notched-fin eel pout. The type III AFPs have been categorized into two subgroups, quaternary-amino-ethyl (QAE) and sulfopropyl-Sephadex-binding (SP) isoforms, based on their isoelectric points. The QAE isoforms can be further divided into two subgroups, QAE1 and QAE2 isoforms. The QAE1 isoform binds both pyramidal and primary prism planes and is able to halt the growth of ice, whereas the QAE2 and SP isoforms are unable to stop, the growth of ice crystals.

We determined the antifreeze activities of the wild-type of the Japanese notched-fin eelpout AFP (nfeAFP8) and four mutants that converted it to the inactive QAE2-like isoform: two single mutants, V20A and V20G; one double mutant, L19V/ V20G (nfeAFP8_di); and one triple mutant, Q9V/L19V/V20G (nfeAFP8_tri). We also characterized the structural and dynamics properties of their ice-binding surface using NMR. We found that the three constructs containing mutation were incapable the V20G of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 310 helix (residues 18 – 22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane-binding surface.

References

[1] Nishimiya, Y.; Sato, R.; Miura, A.; Tsuda, S. FEBS J. 2005, 272, 482. [2] Kumeta, H.; Ogura, K.; Nishimiya, Y.; Inagaki, F.; Tsuda, S. J. Biomol. NMR. 2013, 55, 225. [3] Kondo, H.; Hanada, Y.; Sugimoto, H.; Hoshino, T.; Garnham, C.P.; Davies, P.L.; Tsuda, S. PNAS. 2012, 109, 9360

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P78:

Interaction of anthracycline daunomycin bound to Tetrahymena G quadruplex sequence d-TTGGGGT

Zia Tariq, Ritu Barthwal

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India

Telomere shortening at every cell division is the characteristic of normal cells resulting in cell death. This shortening is hampered in cancerous cells with the action of telomerase enzyme which maintains telomere length. However elongation by telomerase is inhibited with the formation and stabilization of G quadruplex structure in telomeric DNA which has since evolved as an important anti cancer strategy. Several ligands have been evaluated for stabilization of G quartet but lack of structural data of ligand-G-quadruplex complexes has restricted therapeutic applications. We report here the solution structure of anthracycline daunomycin complexed to parallel stranded G-quadruplex d-(TTGGGGT)4 containing sequence TTGGGG from Tetrahymena based on NMR techniques. On binding to daunomycin, the imino protons show that G-quadruplex DNA gets thermally stabilized by ~10-15 K. Progressive addition of daunomycin (D) to DNA (N) at D/N molar ratio 4.0 results in upfield shifts up to ~0.34 ppm in GNH protons and downfield shift of 0.21 ppm in

TCH3 protons. Significant chemical shift changes, up to ~0.41 ppm, have been observed in ring A aromatic and daunosamine sugar protons-1H, 2H, 3H, 4OCH3, 5‘CH3, 1‘H and 5‘H. About thirty intermolecular short contacts between drug and DNA protons have been used to build a possible model of the complex. The structural data obtained is a step forward in development of novel anticancer agents.

Reference:

Scaglioni L, Mondelli R, Artali R, Sirtori FR, Mazzini S. Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22. Biochim. Biophys. Acta Gen. Subj. 2016, 1860(6), 1129-1138.

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P79:

Mesoporous 3D Carbon Framework Encapsulated Janus Nanoparticles as Novel Biocompatible Dual MR Imaging Probe Kashmiri Deka and Pritam Deb Advanced Functional Material Laboratory, Tezpur University (Central University), Tezpur, India E-mail: [email protected] Conventional single mode MRI contrast agents encounter several ambiguities. Dual mode

(both T1 and T2) MRI contrast agents can address these challenges. MnFe2O4@MnO Janus nanosystem showing both T1 and T2 relaxations can be explored for this purpose. ―Janus‖ particle is a particle which is combination of two distinct sides with differences in chemical nature on each face. Researchers prefer Mn based systems like MnO for T1 contrast due to its 3d5 electronic configuration, suitable electronic relaxation time and labile water exchange. Besides, Mn plays an essential role in human body as co-factor in enzymatic reactions. Since, Mn is involved in mitochondrial function, it can be proved to be an excellent contrast agent for the mitochondria rich organs. On the other hand, MnFe2O4 nanocrystals are potential T2 contrast agent candidate since they show higher magnetization than other contemporary T2 contrast agents. To avoid possible toxicity of metals, the MnFe2O4@MnO Janus nanosystem can be encapsulated within a bio-compatible shell. Mesoporous shell is more relevant as it allows easy access of surrounding water protons to the magnetic core leading to enhanced relaxation.

Nanodimensional Janus system of MnFe2O4@MnO encapsulated in mesoporous 3D carbon framework was synthesized with controlled microstructure and morphology. Various characterizations (microstructure, morphology, composition, stability, magnetic property etc.) were performed to ensure high quality product. Cell viability studies established the applicability of the nanosystem for biomedical purpose. Distinct enhancement of relaxivity and contrast of MR images were observed by using this engineered contrast agent.

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P80:

Structure, Toxicity and Membrane Interaction of two Synthetic Antimicrobial Peptides using Solution and Solid State NMR and Microscopy

Nutan Agadi1 & Prof. Ashutosh Kumar2

1 Centre for Research in Nanotechnology and Science (CRNTS), IIT Bombay 2 Department of Biosciences and Bioengineering, IIT Bombay

Antimicrobial peptides are an important part of the innate immune system and are considered to be the first line of defense to any living organism. Natural antimicrobial peptides have sub- optimal biological activities and very few of them have been able to successfully replace the conventional antibiotics. Efforts in the development of antimicrobials have focused on relatively small peptides that are more potent and cost-effective.

In this context, we have characterized two synthetic antimicrobial peptides BMAP-28(1-18) and mutant of wtBMAP-28(1-18) belonging to the cathelicidin family. Solution State 2D NMR experiments were used to determine the structure of the two peptides in aqueous and SDS micellar environment. Scanning and cryoTransmission Electron Microscopy techniques were used for visualizing the impact of these antimicrobial peptides on the bacterial membranes and membrane mimetics. The results were in good agreement with the minimum inhibitory concentration values determined. 31P Solid State NMR experiments are also being performed to determine the changes in the lipid bilayer structure upon addition of the two peptides. These studies could give an insight into the mechanism of action of these peptides.

The structural and functional investigations can further assist in improving the accuracy of prediction algorithms and in de-novo design of potentially active antimicrobials.

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P81:

N-back working memory in normative population: A comparison between visual and auditory tasks

Shefali Chaudhary1, Sunita Gudwani1, S. Senthil Kumaran1, Rajesh Sagar2 and Alok Thakar3

1Departments of NMR& MRI Facility, 2Psychiatry, 3E.N.T, All India Institute of Medical Sciences, New Delhi

Introduction: Working memory retains information temporarily for processing cognitive tasks. The mostly recognized Baddley-Hitch model1 proposes working memory to consist of central executive, and an attention controller aided by-phonological loop and the visuospatial sketchpad. We studied the brain activation pattern during verbal-n-back and visuospatial working memory tasks in normal population to understand the underlying neural basis targeting the phonological and visuospatial domains separately. Methodology: Verbal 1, 2 back, and visuospatial tasks were presented to eight subjects (3M, 5F, mean age: 28 years) in 3T MRI, with 32 channel head coil using EPI to observe the BOLD effects. Results and Discussion: Middle/superior temporal gyrus, insula, parahippocampal gyrus, posterior cingulate were more active in verbal-1-back compared to verbal-2-back task (Figure 1). Middle/superior frontal and temporal gyri, precentral gyrus, postcentral gyrus, declive, uvula were found to be more active in verbal 1-back as compared to the visuospatial task, whereas superior parietal lobule, cingulate (posterior and anterior) gyrus, precuneus, fusiform gyrus were contributing more in visuospatial task as compared to verbal-1-back task (Figure 2).

Figure 1: Brain areas activated during (A) verbal-1-back task, (B) verbal- Figure 2: surface rendered image for brain areas activated 2-back task and (C) verbal-1-back compared to verbal-2-back surface during verbal-1-back task (A) and visuo-spatial task (B) rendered on standard template. Brain areas active during verbal 1-back compared to verbal 2-back task are involved in memory encoding, depicting similar neural pathway underlying both tasks. Frontal and temporal cortex are involved more in complex memory encoding auditory processing and parietal lobe is attributed to visuospatial memory pathway2.

References 1. Baddeley, AD, Hitch, G. (1974). Working memory. Psychology of learning and motivation, 8, 47-89.

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2. Crottaz-Herbette, S, Anagnoson, RT, Menon, V. (2004). Modality effects in verbal working memory: differential prefrontal and parietal responses to auditory and visual stimuli. Neuroimage, 21, 340-351.

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P82:

Model Free analysis of relaxation parameters of NMR in various biomolecules.

Akshay Kumar , Rakesh Sharma and Kavita Dorai*

Department of Physical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, P.O. Manauli, Punjab-140306, India *[email protected]

NMR relaxation experiments provides wealth of information about molecular dynamics in macromolecules and fluids. To get the meaningful explanation of NMR relaxation data, Model free approach for analysis of the data is used. We analyse the spin- relaxation experimental data within the model free formalism (Clore et al.1990;Lipari and Sizabo,1982) to study and analyse molecular dynamics with atomic resolution of biomolecules like ubiquitin, Rnase using the program FAST-Model free for the fully automated, high throughout analysis of NMR spin relaxation data.

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P83:

Studying chemical exchange in NMR using Singlet states

Balvinder Singh and Narayanan D. Kurur*

Department of Chemistry, Indian Institute of Technology Delhi, New Delhi 110016

Key words: Long- lived states, longitudinal Relaxation, Chemical Exchange, Conformations

H H H H O H hgfedb Clca N H Cl N O H H H H H H

A B

[Figure: Two conformations of p-Chloroformanilide.]

Slow dynamic processes occurring at millisecond to second scale are amenable for study by NMR1, 2. The maximum time for which these processes could be studied depends on the 3 longitudinal relaxation time constant value (T1) . Singlet states or long-lived states (LLS) are nuclear spin states, which have slower than normal relaxation rate of the molecule 4, 5. To detect exchange information in a system showing slow dynamics, Sarkar et al. proposed the use of singlet state two dimensional exchange spectroscopy methods 6. Following the idea, we exploited the long lifetime of LLS to extract kinetic information of such processes. We demonstrate with p-Chloroformanilide the use of LLS for the study of chemical exchange. The molecule shows restricted rotation around the C-N bond which has partial double bond character. Two extreme conformational isomers could be represented by structures A and B as shown above. The rate of exchange of A to B was studied by classical selective inversion and non classical long lived state methods. We found that LLS provide opportunity to capture kinetic information for very slow processes which could not be monitored by existing NMR techniques.

References:

1. Bain, A. D. Prog. Nucl. Magn. Reson. Spectrosc. 2003, 43, 63. 2. Williams, T. J.; Kershaw, A. D.; Li, V.; Wu, X. J. Chem. Educ. 2011, 88, 665. 3. Perrin, C. L.; Dwyer, T. J.; Chem. Rev. 1990, 90, 935. 4. Carravetta, M.; Johannessen O. G.; Levitt M. H. Phys. Rev. Lett.2004, 92, 153003(4). 5. Carravetta, M.; Levitt, M. H. J. Am. Chem. Soc. 2004, 126, 6228. 6. Sarkar, R.; Vasos, P. R.; Bodenhausen, G. J. Am. Chem. Soc. 2007, 129, 328.

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P84:

Evaluation of various 2D J-resolved NMR on Bio-fluids Ajay Verma, Bikash Baishya

Centre of Biomedical Research (Formerly Centre of Biomedical Magnetic Resonance), SGPGIMS Campus, RaeBareli Road, Lucknow, 226014, India

2D J-resolved NMR is a valuable tool for multiplet analysis as it dispalys the multiplet information along F1 and pure shift spectrum along F2. It is particularly useful in metabolomics for assignment of metabolites in bio-fluids. In the present work, we have evaluated the performance of regular magnitude mode 2D J-resolved NMR1, PSYCHE-2D J NMR2, and Pell-Keeler 2D J-resolved NMR3 on lyophilized human urine samples, and tissue extract from goat leg samples. PSYCHE-2D J NMR and Pell-Keeler 2D J-resolved NMR are absorptive mode J-spectroscopy with pure shift spectrum in direct dimension and hence expected to produce better resolved spectrum compared to regular magnitude mode 2D J- spectrum which retaines the problematic broad dispersive signals. However, we observed that in terms of resolution the performance of PSYCHE 2D J NMR and Pell-Keeler 2D J-resolved NMR were marginally better than regular magnitude mode 2D J-resolved NMR on such biological samples. In fact the much higher sensitivity of regular magnitude mode 2D J-resolved NMR underscores a great advantage in metabolite identification when compared to the pure shift based absorptive J-resolved spectra even when signal averaging for the later were higher by a factor of ten.

References: 1). Aue, W. P.; Karhan, J.; Ernst, R. R. J. Chem. Phys., 1976, 64, 4226-4227. 2). Foroozandeh, M.; Adams, R. W.; Kiraly, P.; Nilsson, M.; Morris, G. A. Chem. Commun. 2015, 51, 15410-15413. 3). Pell, J.; and Keeler, J. J. Magn. Reson. 2007, 189, 293-299.

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P85:

Determining hidden coupling constants in multiple spin systems using LLC

Rituraj Mishra and Narayanan D. Kurur*

Department of Chemistry, Indian Institute of Technology Delhi, New Delhi 110016

Key words: Long- lived coherences, Coupling Constant

Recently, Long-Lived Coherences (LLCs)1 and Long-Lived States (LLSs)2,3 have shown its potential in the field of NMR due to longevity of their relaxation times. In addition to having longer relaxation times LLC also has the information of coupling constant which may be useful in conformational and structural analysis of molecules. Unfortunately, determination of coupling constant through LLC is limited to two and three coupled spin systems4, is time consuming, and involves tedious mathematical calculation particularly in three spin systems. A further complication in three spin system is smaller lifetime of LLCs than in two spin case. The involvement of the third spin affects the lifetime to such an extent that it become too difficult to probe small coupling constants. Here we study the effect of the third spin on the LLC lifetime in three spin systems experimentally and theoretically. To extend the use of LLC to multi spin system, which must be independent of relaxation time of LLC, an alternative and easy way is proposed. This is used to find out the coupling constant. In this process spin locking pulse delay is systematically varied to search an optimized delay which converts initial density operator I1x-I2x to –I1x+I2x. In LLC pulse sequence spin locking delay is directly related to the coupling constant, hence the coupling constant for different pairs can be determined. The technique is tested on 6-bromoindole, 6-methoxyindole-2-al and pyrrole- 2-al and coupling constant between –NH proton and its coupled partner is successfully determined.

References:

1. Sarkar, R.; Ahuja, P.; Vasos, P. R.; Bodenhausen, G. Phys. Rev. Lett. 2010, 104, 053001. 2. Carravetta, M.;Johannessen O. G.;Levitt M. H. Phys. Rev. Lett.2004, 92, 153003(4). 3. Carravetta, M.; Levitt M. H. J. Am. Chem. Soc. 2004, 126, 6228. 4. Mishra, R.; Singh, B.; Kurur, N. D., Long-Lived Coherences in Multi-spin Systems, 21st Conference of Nuclear Magnetic Resonance Society, GNDU Amritsar, March 6- 9, 2015, PP-65,141.

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P86:

A Cost Effective Low Field MRI Wrist Coil

Darshan Shivaramu Keelara1, ThejasVishnu Ramesh1, Syed Saad Siddiq1, Padma Chennagiri1, Samarth Singh1, Shivaprasad Ashok Chikop1, Shreyas Indurkar1, and Sairam Geethanath1

1Medical Imaging Research Center, Dayananda Sagar Institutions, Bangalore, India

Obtaining good resolution and Signal to noise ratio is challenging in low field brain1,2 and wrist3 MR. This work demonstrates a single channel transmit coil and a 4-channel receive coil with an efficient B1-field and SNR for wrist imaging at 9.5mT.

Figure 1: Block diagram of Tx-Rx chain.

The block diagram of the setup is depicted in figure 1. An Arduino Uno controlled Direct Digital Synthesizer produces a 405KHz sine wave which was sent to the relay and then to the solenoid transmitter coil at predetermined time intervals via an RF amplifier of 5W and a 37dB gain. Copper wire (18SWG) was wound on a PVC pipe of 11cm diameter to construct the transmitting coil whose inductance=866.4µH along with tuning/ matching capacitances of121.1pF /142.5pF respectively, resonated at 405KHz with Q=14. Baluns were designed to perform filtering due to impedance mismatch whose design and placement is depicted in figure 2 and figure1 respectively. The four channel receive coil was constructed using copper wire (18SWG) wound in three turns of 35mm diameter each and placed strategically on a 3-D printed flexible, cylindrical wrist band to improve sensitivity and gain of the coils at 405KHz as shown in figure 3A. The output of each coil is connected to an independent Low Noise

Amplifier and processed separately. The measured S21 which ranges from -10dB to -23dB between adjacent coils of the receiver array is depicted in figure 3B. Further work involves integration of the system with the magnet and implementing the above system for wrist and brain imaging.

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Figure 2: Schematic of Balun Figure 3A: 4 - channel receive array loaded with human hand

Figure 3B: Measured S21of adjacent coils of receive array

References: [1] Sarracanie, M.; Lapierre, C. D.; Salameh, N.; Waddington, D. E. J.; Witzel, T.; Rosen, M. S. Sci. Rep. Scientific Reports2015, 5, 15177. [2]Cooley, C. Z.; Stockmann, J. P.; Armstrong, B. D.; Sarracanie, M.; Lev, M. H.; Rosen, M. S.; Wald, L. L. Magnetic Resonance in Medicine2014, 73 (2), 872–883. [3] I. Savukov, T. Karaulanov, C.J.V. Wurden, L. Schultz, Non-cryogenic ultra-low field MRI of wrist–forearm area, Journal of Magnetic Resonance, Volume 233, August 2013, Pages 103-106, ISSN 1090-7807, http://dx.doi.org/10.1016/j.jmr.2013.05.012.

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A cost-effective, light weight Head Gradient System for very low field (VLF) (<10mT) imaging applications using three Maxwell coil pairs

Ashwini Kumnoor1, Aparna N V1, Sai Abitha Srinivas1, Vineet V Bhombore1, Anusha R Chandra1 , and Sairam Geethanath1

1Medical Imaging Research Centre, Dayananda Sagar Institution, Bangalore, India

Brain imaging at Very Low Field (VLF) MRI (<10mT) allows for accessible MR solutions for specific pathologies such as stroke1,2. The aim of this work is to develop a head gradient coil for VLF MRI with the following features (a) Cost effective (<$350) (b) Portable (<16kg) (c) Ease of building (d) Convenient for claustrophobic patients. The head gradient coil was designed on 123D AutodeskTM in three directions with 15cm radius shown in figure 1(b). Maxwell coils were hand-wound and built with the coil parameters as described in figure 1(a), using 12 AWG copper wire and industrial grade super glue to stay affixed on an acrylic base. The coil was characterized by measuring 3D gradient field over a FOV of 26cm for the coils using a gauss meter (Lutron emf detector). A gradient pipeline from an analog input to the coils was designed as shown in figure 2(a). The coil setup shown in figure 2(b) is to illustrate relief from claustrophobia only. A Tektronix AFG1022 function generator was used to generate a 1 kHz varying amplitude (0.5-4V) square wave test input. This was fed to a Gradient Power Amplifier (GPA) 3, a current driver which was biased with a single +12V, 15A supply. The voltage across a 0.1Ω current sense resistor was used to ensure that the output current tracks the input signal. The complete setup of the head gradient system was characterized for input voltage, current delivered and gradient field produced which is depicted in figure 3(a) and figure 3(b). From these, we can see that the fields vary linearly.

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References

[1] Zimmerman Cooley.C; stockman.J P ; Armstrong. B.D.; Sarracanie.M.; Lev.M.H.; Rosen M; L.Wald.L. : Two-dimensional imaging in a lightweight portable MRI scanner without gradient coils, ; Magnetic Resonance in Medicine 2014 [2] Sarracanie M.; LaPierre C.; Salameh N.; David E. J.; Waddington D.; Witzel T, Rosen M.: Low-Cost High-Performance MRI. Sci. Rep..1994-2016 2015; 5:15177 [3] Nick Arango: Open-source, low-cost, flexible, current feedback- controlled driver circuit for local B0 shim coils and other applications http://cds.ismrm.org/protected/16MPresentations/abstracts/1157.html ISMRM 2015

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P88:

Binding interaction of 2, 6 Difluorobenzoic acid (DFBA)-Bovine Serum Albumin (BSA): A solution state 19F NMR transverse relaxation and diffusion analysis

Bhawna Chaubey, Deepak Kumar, Samanwita Pal*

Department of Chemistry, Indian Institute of Technology Jodhpur, 342011, India.

Abstract: Outstanding advancement of NMR hardware along with recent development of fluorine chemistry allowing synthesis of myriad of fluorine containing small molecules have made 19F NMR a compelling technique to unravel molecular interactions involving fluorinated molecules relevant in the fields of macromolecules, biomedicine, pharmaceutics, drug discovery and agrochemicals [1-2]. In the present study, we aim to characterize the intermolecular interaction of 19F containing small molecule 2, 6-difluorobenzoic acid (DFBA) with serum albumin by employing 19F relaxation and diffusion measurements. DFBA is a starting reagent as well as a metabolite of various pesticides of benzoylphenylurea family. Therefore analysis of the binding interaction of DFBA-serum albumin turns out to be important. 19F NMR of DFBA (1mM) in presence of bovine serum albumin (BSA, 1µM) exhibits a single broad triplet; the broadening being dependent on protein concentration reflects effect of chemical exchange between a free and a bound state of DFBA on the transverse relaxation rate. On the other hand a single 19F line is also evident of a single binding site. Further from the competitive 19F NMR binding experiments using Warfarin (Site-I ligand) and L-tryptophan (Site-II ligand) as site markers for BSA [3], it is found that

DFBA binds to site I of BSA. To quantify the DFBA-protein dissociation constant (Kd), transverse relaxation rate (R2) is measured using CPMG pulse sequence. To eliminate the effect of chemical exchange, CPMG experiments are recorded with fast interpulse spacing [4]. It is found that for a protein to ligand ratio of 1:1000, the bound state transverse relaxation time (T2p=318ms) changes approximately by 86% relative to that of the free DFBA (T2= 2.30s). A series of diffusion measurements are also performed to validate the ligand- protein dissociation constant and number of binding sites [5].

Refrences:

1. Fujiwara, T.; Hagan, D.O. Successful fluorine-containing herbicide agrochemicals. J. Fluorine Chem., 2014, 167, 16–29. 2. Chen, H.; Viel, S.; Ziarellic, F.; Peng, L. 19F NMR: a valuable tool for studying biological events. Chem. Soc. Rev., 2013, 42, 7971. 3. Kitamura, K. et. al. 19F NMR spectroscopic study on the binding of triflupromazine to bovine and human serum albumins. J. Pharm. Biomed. Anal. 2004, 36, 411–414.

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4. Dubois, R.; Evers, A.S. 19F- NMR Spin-Spin Relaxation (T2) Method for Characterizing Volatile Anaesthetic Binding to Proteins. Analysis of Isoflurane Binding to Serum Albumin. Biochemistry, 1992, 31, 7069-7076. 5. Price, W.S.; Elwinger, F.; Vigouroux, C.; Stilbs, P. PGSE-WATERGATE, a new tool for NMR diffusion-based studies of ligand–macromolecule binding. Magn. Reson. Chem., 2002, 40, 391–395.

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P89:

1H and 19F NMR Investigation of 5-Fluorouracil (FU) entrapped Poly Lactic-co-Glycolic Acid (PLGA) microsphere

Deepak Kumar1, Samanwita Pal1* *E-mail: [email protected] 1Department of Chemistry, Indian Institute of Technology Jodhpur 342011, India

Poly Lactic-co-Glycolic Acid (PLGA) microspheres are described in literature as effective drug carriers for the encapsulation of various anti-cancer agents such as paclitaxel, 9- nitrocamptothecin, cisplatin, doxorubicin (DOX), 5-fluorouracil etc.1, due to its biocompatibility, biodegradability and its approval by the US FDA and European Medicine Agency (EMA)2. In the present study, we aim to characterize microsphere encapsulated model drug 5-fluorouracil employing NMR relaxation analysis. A standard oil-in-water (o/w) emulsion solvent evaporation method is used for preparation of the 5-FU loaded PLGA microspheres. 1H NMR chemical shift and 19F NMR relaxation measurement are used to probe the molecular interaction and event of drug loading between PLGA and 5-FU. 1D 1H NMR spectra of 5-FU in presence of PLGA microspheres revealed that the microspheres are hydrated leading to slow exchange of –NH protons of 5-FU within the cavity. A significant change in linewidth and loss of coupling pattern in 19F NMR of 5-FU in presence of PLGA confirmed the event of encapsulation. Further 1H and 19F relaxation measurements are carried 1 out. In case of H, the change in longitudinal (T1) and transverse (T2) relaxation time is 19 minute; however a very substantial change is observed for F T1 and T2 relaxation time. It is well known from literature that in small molecules or mobile fluids, transverse relaxation time (T2) is nearly equal to longitudinal relaxation time (T1), whereas in large molecules or 3 slowly tumbling systems, T2 becomes significantly shorter than T1 . In the present case for 19 free 5-FU, the F T2 value is approximately 1.5 seconds and the T1/T2 ratio is 1.2, while the 19 F T2 value of 5-FU loaded in PLGA is approximately 0.13 seconds and T1/T2 ratio increases to 14. Hence in case of PLGA microsphere, 19F relaxation data are far more powerful compared to 1H relaxation measurements.

References

1. Danhier, F. et al. PLGA-based nanoparticles: An overview of biomedical applications. J. Control. Release 2012, 161, 505–522. 2. Blakney, A. K. et al. Rapidly Biodegrading PLGA-Polyurethane Fibers for Sustained Release of Physicochemically Diverse Drugs. ACS Biomater. Sci. Eng. 2016, 2, 1595−1607. 3. Becker, E. D. In High Resolution NMR Theory and Chemical Application, Academic Press, California (USA), Third, 2000, 206-224.

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P90:

Nucleic acid binding mechanism of RNA-binding motif, single-stranded- interacting protein 1 Priyanka Aggarwal and Neel Sarovar Bhavesh

Transcription Regulation group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, India.

The RNA Recognition Motif (RRM), commonly also known as RNA Binding Domain (RBD) or Ribonucleprotein domain (RNP), is the most abundantly present nucleic acid binding domain in higher vertebrates, which are very structurally versatile; evident from their varied types of interactions with proteins/nucleic acids. Consequently, they carry out diverse functions in the cell by virtue of those interactions.

RNA-binding motif, single-stranded-interacting protein 1 (RBMS1) or Single-stranded DNA- binding protein (MSSP-1) is a human nucleic acid binding protein that contains two RRM domains and binds both single and double stranded DNA. It specifically binds in the region upstream of the c-myc gene, a proto-oncogene where exists an origin of cellular DNA replication (ori) and a transcriptional enhancer. c-MYC has a pivotal function in growth control, differentiation and apoptosis, and its abnormal expression is associated with many tumors. Many studies have shown that the level of c-Myc is controlled by RBMS1, with a decline in RBMS1 levels in tumor cells. We have carried our solution-state NMR spectroscopy studies to understand the molecular mechanism of interaction between RBMS1 and c-Myc DNA sequence, which will be presented.

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P91:

Serum metabolic perturbations of mild and moderate TBI: An 800MHz NMR study

Richa Trivedi1, Kavita Singh1, Ajay Verma2, Bikash Baishya2 Subash Khushu1

1NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences, Delhi, India 2Centre for Biomedical Magnetic Resonance (CBMR), SGPGIMS Campus, Lucknow, Uttar Pradesh, India

Traumatic brain injury (TBI) has shown to affect a plethora of metabolic processes in both mild (miTBI) and moderate TBI (moTBI) which can be non-invasively assessed in serum. This study attempts to investigate metabolic alterations in miTBI and moTBI based on time and severity.

Animal model was developed by freely dropping 450 gm (1cm diameter, blunt tip) brass rod from a height of 25 cm (mild TBI) and 50 cm (moderate TBI) above the sagittal midway of the rat brain1. 41 rats were used in 3 groups of (i) miTBI (n=15), moTBI(n=21 out of which 15 survived) and controls (n=5). Serum was extracted at 4hrs, day1 and day5 post injury (PI). All NMR experiments were done at 800MHz Bruker NMR Spectrometer. 450ul of serum with co-axial TSP was taken and acquired using CPMG sequence with total spin echo (2nτ) of 200ms, relaxation delay(RD) of 2.5s, 128 scans, 32K data points, spectral width of 12820.51 Hz, acquisition time of 9.29 mins. Data analysis was done using Topsin 3.1 and Metaboanalyst 3.0. ANOVA results of 15 metabolites in serum showed decease in pyruvate, phosphocholine and lactate as early as 4hrs. At D1, glucose, lactate and taurine decreased while TMAO-Betaine, pyruvate and phosphocholine decreased at D5 in miTBI as compared to control. moTBI showed different metabolic upheaval with lactate, TMAO-Betaine and alanine decreased and glucose increased at all timepoints PI as compared to control. Alongwith these, taurine also increased at 4hr. LDL/VLDL decreased at D1 however glycine and glutamine increased at D1 as compared to control. Taurine and acetate increased at D5 as compared to control. Maximal changes were observed at D5, so comparative analysis at D5 was done. ANOVA results indicate taurine and glucose were significantly altered in both miTBI and moTBI however lactate was altered only in moTBI and LDL/VLDL in miTBI only. VIP scores also showed lactate, taurine, glucose and LDL/VLDL as major metabolites separating these two groups.Brain cells release organic osmolytes like taurine maintain homeostatis which is observed in our study as well. Reduced level of lactate suggesting towards increased glucose production via gluconeogenesis which is released from liver into blood stream seen as elevated serum glucose level. Due to non-availability of glucose for glycolysis, pyruvate and lactate has been found to decrease. Interestingly, moTBI shows increased glucose level in serum as reported in other TBI studies while miTBI shows transient reduction in glucose level. Authors speculate that ratios of glucose to lactate or pyruvate may serve to distinguish miTBI from moTBI.

References: 1. Singh et al. 2016 ; 2. Pasantes-Morales et al. 1997 240

P92:

Quantitative evaluation of macrophages infiltration after experimental traumatic brain injury in mice: A magnetic resonance imaging assessment

Sushanta Kumar Mishraa,b, Subash Khushua,* and Gurudutta Gangenahallib,*

aNMR Research Centre, bDivision of Stem Cell and Gene Therapy Research Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Delhi

Traumatic brain injury (TBI) is a significant health problem in victims of sports, accidents and military combatants1. Recent research suggests that infiltrating macrophages may play a pivotal role at the interface between early detrimental and delayed beneficial effects of inflammation2. Therefore, inhibition of infiltrating macrophages is considered as an attractive therapeutic strategy. The present study was to monitor the early effect of monocytes/phagocytic accumulation and further to explore its kinetics in TBI mice. Localized macrophage population was monitored using USPIO nanoparticles enhanced in vivo serial magnetic resonance imaging (MRI). ADC map revealed an increase in signal intensity after induction of injury, which is due to accumulation of water content after neuroinflammation. T2* images and FA maps showed a decrease in signal intensity at later time points (post-TBI) after administration of USPIO. Flow cytometry based gating study was performed to discriminate between resident microglia (Ly6GˉˉCD11b+CD45low) and infiltrating macrophages (Ly6GˉCD11b+CD45high). Approximately 7.2 and 6.6 fold increase in infiltrating population of macrophage was observed at 48 h and 72 h post-USPIO administration (72 h and 96 h post-injury). Imaging and flow cytometric analysis revealed that maximum macrophage infiltration occurs between 66-72 h post injury (42-48 h post administration of USPIO) at the site of inflammation. We are first to analyze the serial in vivo T2* images quantitatively, correlate with flow cytometric data in TBI at different time points after administration of USPIO and confirm the correct time window using 7 T MRI and gating based flow cytometric analysis. This study may help in setting an optimal time window to intervene and prevent damage due to inflammation and to increase the therapeutic efficacy.

References:

1.Mishra S K, Rana P, Khushu S, Gangenahalli G. Therapeutic prospective of infused allogenic cultured MSCs in traumatic brain injury mice: A longitudinal proton magnetic resonance spectroscopy assessment. Stem Cells Transl Med. 2016; 5: 1-16. 2.Gensel J C and Zhang B. Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. 2015; 14: 01752-01761.

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Voxel-Based Morphometric (VBM) Analysis in Patients with Major Depressive Disorder using DARTEL approach

B S Hemanth Kumar1, Ankit Saxena2, Pawan Kumar1, Prabjot Kaur1, Smita NDeshpande2, Subash Khushu1

1NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. SK Mazumdar Marg, Timarpur, New Delhi, India 2Post Graduate Institute of Medical Education and Research (PGIMER), RML Hospital, New Delhi, India

Major depressive Disorder (MDD) is a mental disorder characterized by pervasive and persistent low mood, accompanied by low self-esteem and loss of interest or pleasure in normally enjoyable activities. The diagnosis of major depressive disorder is based on the patient's self-reported experiences, behavior reported by relatives or friends, and a mental status examination. The etiology of Major Depressive disorder is complex and involves interplay of genetic and environmental factors. To investigate whether brain morphological differences exist between MDD patients and controls, we performed Voxel-Based Morphometry (VBM) with Diffeomorphic Anatomic Registration through Exponentiated Lie algebra algorithm (DARTEL) approach studies. High resolution structural MR images were taken in healthy controls and MDD patients (n=28 in each). The analysis was carried out using SPM8 software. The study revealed grey matter volume reduction in the left precentral gyrus, right postcentral gyrus and right superior frontal gyrus in MDD patients as compared to healthy controls. A significant white matter volume reduction was also found in the left fusiform gyrus, left inferior occipital gyrus and left middle temporal gyrus in MDD patients as compared with healthy controls. The results suggest that as compared to controls, this disorder is associated with differences in brain morphology in areas corresponding to known functional deficits in memory, visuospatial processing and executive functions in patients with MDD.

References:

1. Taki Y et. al., J Affect Disord. 2005 Nov;88(3):313-20. 2. Ashburner J. VBM Tutorial. www.fil.ion.ucl.ac.uk/~john/misc/VBMclass10.pdf

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P94:

Radiation induced temporal changes in hippocampus: A successive MRS, DTI and behavioral study

Poonam Rana1, Mamta Gupta1, Apurva Watwe1, Seenu Haridas2, Kailash Manda2, , Subash Khushu1

1NMR Research Centre, INMAS, DRDO, Delhi, India, 2Division of Radiation Biosciences, INMAS, DRDO, Delhi, India

Introduction: Nuclear accidents or terrorist activities may lead to moderate dose whole body radiation exposure to a mass population. Ionizing radiation is known to influence brain and behaviour. Of all the brain regions, hippocampus is known to be highly sensitive to radiation injury. Radiation induced acute, early delayed and late delayed brain injury is often observed after fractionated partial or whole brain irradiation. However, effect of whole body radiation on brain is still a problem of research to work upon. A few recent studies have observed whole body radiation induced acute changes in brain1-3, yet a comprehensive study is required to understand whole body radiation induced changes in brain at all the phases of injury like early delayed or late delayed. Therefore, the present study was planned to study whole radiation induced cognitive, metabolic and microstructural changes in hippocampus at different phases of radiation injury in animals employing two approaches, behavioural and advanced MR techniques. Material and Methods: Study was conducted on 120 C57 male mice (8 to 10 weeks old). Out of 120 animals, 60 animals were irradiated with 5Gy of whole body radiation dose. The remaining 60 animals served as sham irradiated controls. The Behavioral, DTI and MRS experiments were carried out on 20 animals each at 1,3, 5, 8, 10 and 12 months (n=10 irradiated and n=10 sham) post-irradiation. Independent students t test was performed to evaluate the differences among age matched control and irradiated groups. Results and Discussion: The data for MRS, DTI and behavioural parameters were acquired at sub acute (1 month), early delayed (3, 5 and 8 months) and late delayed phases (10 and 12 months) of radiation injury. The data showed non-significant changes in behavioural as well as DTI parameters at sub acute phase of radiation injury. During early delayed phase, there was a significant decrease in cognitive index/discrimination ratio indicating impairment in hippocampal dependent recognition memory. However, MRS based metabolic changes, in particular myoinositol level was observed from one month post-irradiation and continued till 8 months time point. There was no significant change observed in FA values at any time point studied but MD values were significantly decreased mainly during early delayed phase. At later time points (10 and 12 month post-irradiation), no profound effect of radiation exposure was seen at behavioural, metabolic and microstructural level. It indicates that whole body radiation induced changes do not persist in late delayed phase and are reversible as no significant change in cognitive function observed as compared to placebo group. Conclusion: Whole body radiation induced behavioral, metabolic and microstructual changes were

243 observed till 8 months post-irradiation only. It is concluded from the study that whole body radiation of 5 Gy dose exposure does not have late delayed radiation injury. References: 1. Gupta, P Rana, R Trivedi, BSH Kumar, AR Khan, R Soni, RK Rathore, S Khushu (2013) Comparative evaluation of brain neurometabolites and DTI indices following whole body and cranial irradiation: a magnetic resonance imaging and spectroscopy study. NMR in Biomedicine 26:1733-41. 2. R Trivedi, A R Khan, P Rana, S Haridas, BS H Kumar, K Manda, R K Rathore, R P Tripathi, S Khushu (2012) Radiation-Induced Early Changes in the Brain and Behavior: Serial Diffusion Tensor Imaging and Behavioral Evaluation after Graded Dose of Radiation. Journal of Neuroscience Research, 90:2009-19. 3. M Gupta, SK Mishra, BS Hemanth Kumar, S Khushu, P Rana (2016). Early detection of radiation induced microstructural and neuroinflammatory changes in hippocampus: A DTI and gene expression study. Journal of Neuroscience Research (Accepted) DOI: 10.1002/jnr.23833

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P95:

Metabolic alterations of DLPFC in thyroid dysfunction: A 3T 1H MRS study

1 2 1 2 1 1* M. Kumar , R. Kanwar , S. Khushu , T. Sekhri , M. M D’souza , P. Rana

1NMR Research Centre, 2Thyroid Research Centre, Institute of Nuclear Medicine and Allied Sciences,, Brig.SK Mazumdar Marg, Timarpur, Delhi, India – 110054

Introduction: Thyroid dysfunction is among the most prevalent endocrine disorder worldwide. The human brain is one of the target areas affected by endocrine disorder (both hypothyroidism and thyrotoxicosis) and may affect cognitive function such as memory and attention. Earlier studies have shown thyroid dysfunction associated altered cognitive function mainly memory in thyroid patients as compared to control subjects1,2,3. DLPFC (Dorso-Lateral Pre-Frontal Cortex) is the seed region for memory associated function. MR spectroscopy is an in vivo technique which can identify changes at metabolic level for a very small region of interest in brain. The present study aimed to investigate the metabolic changes occurring in DLPFC due to thyroidism and to find any correlation with altered memory function. Materials & Methods: 26 healthy subjects, 33 hypothyroidism, 23 thyrotoxicosis and 23 subclinical hypothyroid (SCH) subjects (age 20-45year) were recruited for the study. The subjects chosen for the study were age, sex & education matched. Spectra were acquired with a circularly polarized Head coil using 3T whole body MR system (Magnetom Skyra; Siemens, Erfurt, Germany). Single voxel size (12x12x12mm) proton MRS (PRESS) was carried out with TR 2000ms and TE 30 ms of DLPFC (Dorso-Lateral Pre-Frontal Cortex). Spectra were post-processed and concentration was estimated using LC model software. Data was further statistically analysed using Multivariate Analysis of Covariance (MANCOVA). Results & Discussion: Our result revealed significant changes in metabolites in choline- containing compounds and creatine predominantly in thyrotoxicosis. Choline showed a significant decrease in thyrotoxicosis compared to hypothyroid (p<.001) and healthy control (p=.0004) groups. However, choline was significantly increased (p=.04) in hypothyroid group as compared to subclinical hypothyroid group. Whereas, decreased creatine was observed only in thyrotoxicosis as compared to hypothyroid group (p=.035). The results suggest that metabolic changes associated with membrane turnover and energy metabolism are present mainly in thyrotoxicosis4. Conclusion: Present study suggests an altered membrane lipid turnover in the DLPFC, thereby, indicating an altered metabolism that may be associated with memory impairment in thyroid dysfunction. Reference: 1. Zhu DF1, Wang ZX, Zhang DR, Pan ZL, He S, Hu XP, Chen XC, Zhou JN. fMRI revealed neural substrate for reversible working memory dysfunction in subclinical hypothyroidism. Brain. 2006 Nov;129. 2. Matsunaga A1, Yoneda M. Dementia due to Endocrine Diseases. Brain Nerve. 2016 Apr;68(4):399-405. doi: 10.11477. 3.Singh S1, Rana P1, Kumar P1, Shankar LR2, Khushu S3. Hippocampal Neurometabolite

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Changes in Hypothyroidism: An In Vivo (1) H Magnetic Resonance Spectroscopy Study Before and After Thyroxine Treatment. J Neuroendocrinol. 2016 Sep; 28(9). doi: 10.1111/jne.12399. 4. Bruce L. Miller. A Review of Chemical Issues in 'H NMR Spectroscopy: N-Acetyl-L- aspartate, Creatine and Choline. NMR IN BIOMEDICINE,VOL. 4, 47-52 (1991).

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Phase Composition−Property Correlation in Fast Ion Conductor Na- SrSiO3 Studied by Solid-state NMR

P. Lokeswara Rao,†,₶ Bholanath Pahari,§ Shivanand M,¶ Tukaram Shet, ¶ and K. V. Ramanathan†

†NMR Research Center & ₶Physics Department, Indian Institute of Science, Bangalore- 560012, India §Department of Physics, Goa University, Taleigao Plateau, Goa-403206, India ¶Materials Research Centre, Indian Institute of Science, Bangalore-560012, India email – [email protected]

Recent report of a new family of ionic conductors bearing a generic formula of Sr1-xNaxSiO3- -1 0.5x has received intense attention due to their high ionic conductivity of 0.01 S cm only at 525 °C [1]. Subsequent studies showed that Na hardly substitute Sr sites rather a mixed phase, consisting of a crystalline SrSiO3 phase and an amorphous Na2Si2O5 phase (AM‒

Na2Si2O5), is formed [2-4]. The crystalline SrSiO3 phase is an electrical insulator while the + AM‒Na2Si2O5 phase is a good Na -ionic conductor. Therefore, high conductivity observed in

Sr1-xNaxSiO3-0.5x family is essentially caused by the amorphous Na2Si2O5 phase. The AM‒

Na2Si2O5 is an unstable phase at elevated temperatures and often transforms into complex mixture of amorphous and/or several crystalline phases. This alters the conducting property of the material. Here we show 23Na and 29Si magic-angle spinning (MAS) nuclear magnetic resonance (NMR) results combined with XRD and ionic conductivity results for the samples of Sr0.55Na0.45SiO2.775 and AM-Na2Si2O5 together with their respective devitrified forms prepared after treating at various temperatures. This helped us to identify the devitrified products and their role in demonstrating the conducting property of Sr0.55Na0.45SiO2.775 and

AM-Na2Si2O5 ion conductors.

P97:

Organic Fluorine involved bifurcated intramolecular hydrogen bond in the derivatives of dibenzoyloxalamide : NMR evidence and DFT calculations

P. Dhanishta, Sandeep Kumar Mishra, N. Suryaprakash*

NMR Research Centre, Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India

Existence of bifurcated intramolecular hydrogen bond involving organic fluorine in the derivatives of dibenzoyloxalamide, is convincingly established by extensive NMR studies, viz., temperature perturbation, solvent titrations and two dimensional Techniques, such as, 15N-1H HSQC and 19F-1H HOESY. The conformational evidence for the experimentally derived results were obtained by Density Functional Theory (DFT) based theoretical calculations such as Quantum theory of atoms in molecules (QTAIM) and Non-covalent interaction (NCI).

References:

1) S. K. Mishra and N. Suryaprakash, RSC Adv., 2015, 5, 86013–86022.

2) A. Lakshmipriya and N. Suryaprakash, J. Phys. Chem. A, 2016, 120, 7810–7816.

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P98:

Unraveling the origin of 5- fold coordinated Al in Al20Te80 chalcogenide glass

P.T. Wilson1, R. Ramanna2, K. Ramesh2

1NMR Research Centre,

2Department of Physics, Indian Institute of Science, Bangalore-560012, India

Chalcogenide glasses are known for their wide range of application in many areas. Properties of these glasses can be altered to a larger extent by the addition of metal atoms as they enter into the chalcogenide glass network in a special way. For example, local structure of Al-Te glasses studied by 27Al Magic Angle Spinning (MAS) NMR shows 4- and 6- fold coordinations for Al [1,2]. Recent reports on Al- Te glasses [3] show 5- fold coordination for Al along with 4- and 6- fold coordination states. Correspondingly, the electrical properties, particularly, the electrical switching is found to be completely different. Unlike in oxide glasses, observation of 5- fold coordination is unusual in chalcogenide glasses. Hence, it is necessary to unravel the origin of 5- fold coordinated Al and its influence on electrical switching and glass formation. There may be a favourable condition, at which the 5- fold coordinated Al can form. Hence, 27Al MAS NMR studies were

undertaken on Al20Te80 glass prepared at different conditions. The obtained results very well explain the origin of 5- fold coordinated Al and glass formation in Al-Te glasses.

References: 1. S. Murugavel and S. Asokan, J. Mater. Res 13 (1998) 2982. 2. S. Murugavel and S. Asokan, Phys. Rev. B 58 (1998) 3022. 3. Pumlianmunga, K. Ramesh, Mater. Res. Bull. 86 (2017) 88.

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P99:

Two- and Three-Centered Hydrogen Bonds Involving Organic Fluorine Stabilize Conformations of Hydrazide and Hydrazone Halo Derivatives: NMR and Theoretical Evidence

A. Lakshmipriya and N. Suryaprakash*

NMR Research Centre, Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India

The presence of two- and three-centered hydrogen bonds (HB) of the type H(N)•••X-C and C=O•••H(N)•••X-C, respectively, involving organic fluorine in the synthesized hydrazide and hydrazone halo derivatives have been convincingly established by extensive multidimensional NMR studies and contributed their existence for the stability of proposed conformation. The stabilized conformation of the molecules involving two- and three- centered HBs derived by NMR studies have been further confirmed by density functional theory (DFT)-based calculations, such as quantum theory of atoms in molecules (QTAIM), noncovalent interaction (NCI), and relaxed potential energy scan.

Fig. 1. Two dimensional 1H-19F HOESY spectrum

References

[1] A. Lakshmipriya and N. Suryaprakash, J. Phys. Chem. A, 2016, 120, 7810–7816.

[2] A. Lakshmipriya, S. Rama Chaudhari, A. Shahi, E. Arunan and N. Suryaprakash, Phys. Chem. Chem. Phys., 2015, 17, 7528–7536. 250

P100:

Intramolecular Hydrogen Bonding in the Derivatives of N’- Benzylidenebenzohydrazide: NMR Investigations Corroborated with DFT based Theoretical Calculations

Neeru, Sandeep Kumar Mishra and N. Suryaprakash*

NMR Research Centre, Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India

It is well known that the organic fluorine hardly ever accepts intramolecular hydrogen bond. The rare occurrence of such type of intramolecular Hydrogen bonds (HBs) and their control on conformations of a molecule are detected in the derivatives of N‘- Benzylidenebenzohydrazide (NBB) by employing multi-dimensional and multinuclear NMR techniques in a low polarity solvent. The obtained experimental results are further confirmed by Density Function Theoretical (DFT) based Non Covalent Interaction (NCI) and Quantum Theory of Atoms in Molecules (QTAIM) that are in close agreement with the NMR experimental findings.

References

1) S. K. Mishra and N. Suryaprakash, RSC Adv., 2015, 5, 86013–86022. 2) A. Lakshmipriya and N. Suryaprakash, J. Phys. Chem. A, 2016, 120, 7810–7816.

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P101:

Low field NMR in Hyperpolarized noble gases Shashank Yadav, Vineeth Francis T J, Naresh Kumar, G. Rajalakshmi and P. K. Madhu Tata Institute of Fundamental Research Centre for Interdisciplinary Sciences, 21, Brundavan Colony, Narsingi, Hyderabad-500075, India; email: [email protected]

Conventional NMR signal sensitivity is limited by the low spin polarization of the sample even in the high magnetic fields of the NMR apparatus. Noble gases such as 129Xe can be spin polarised by spin exchange optical pumping (SEOP) with alkali atoms, leading to improved NMR sensitivity. Magnetic Resonance Imaging exploiting hyperpolarised noble gases is fast becoming an important diagnostic tool in various areas of biomedical imaging. We present here, implementation of techniques for creating hyperpolarized 129Xe and detecting its NMR signals at low fields in our laboratory. References 1. Goodson, B. M.. Nuclear magnetic resonance of laser-polarized noble gases in molecules, materials, and organisms. J Magn. Reson., 2002, 155, 157–216. 2. Albert, M. S., & Balamore, D. Development of hyperpolarized noble gas MRI. Nuclear Instruments and Methods in Physics Research Section A, 1998, 402, 441–453

252

P102: -peptide sequences: Analysis of NOE Spin Diffusion Effects.

H. Yamanappa1, B. Vasantha2, S. Raghothama1, P. Balaram2.

1NMR Research Centre, 2Molecular Biophysics Unit, Indian Institute of Science,

Bangalore-560012, India.

NMR is a very useful tool for characterization of secondary and tertiary structures in

peptides and protein sequences. Sequential dNN (NHi ↔NHi+1) and d iNHi+1) are diagnostic NOE connectivities for identifying helical and extended sheet structures. Since last two decades incorporation of higher - amino acid into peptide -amino acids proved to be valuable as they offer more resistance to proteolytic cleavage and hence quite useful for drug development. Further novel helical structures which have no precedence - peptide sequences were reported1-2. Notable among them are 12- helix and 14-helix which differ in the nature of their hydrogen bond directionality3-4. Much of the earlier studies were done with conformationally constrained cyclic -amino acids.

3 In the current study we focus on unconstrained Boc- (Val)12-OMe oligomer sequence. In apolar solvents like CDCl3 they form aggregates because of association of sheet structures. Upon addition of DMSO, aggregation is broken and they tend to form expanded

(C12, C14) helical structures. Aggregate dissociation and formation of helical structure was followed by systematically titrating the peptide sample in CDCl3 with DMSO.d6, yielding

titrative curves that confirm disruption of aggregates at concentration  20% DMSO / CDCl3 (v/v). DOSY experiments allow an estimate of the self diffusion coefficient of these peptides as a function of chain length and concentration.

Enhanced correlation times due to self-association leads to observation of negative NOEs and spin diffusion effects in the 9 and 12 residue peptides. Relayed NOEs by limited spin diffusion permit sequence specific assignment in these sequences where there is extreme resonance overlap. Results of both 2D and 1D NOE experiments are presented.

References:

1. Horne, W. S.; Gellman, S. H. Acc Chem Res 2008, 41, 1399-1408. 2. Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem Rev 2011, 111, 657-687. 3. Choi, S. H.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. J Am Chem Soc 2010, 132, 13879. 4. Vasantha, B.; Gijo G; Raghothama, S; Balaram, P. Biopolymers (peptide science) 2016 (in press)

253

P103:

Study of human metabolism by integrating high throughput Omics data with in silico model

Sumant Indurkhya, Debnath Pal, Hanudatta S. Atreya

IISc Bangalore

Constraint based metabolic models [1] are emerging mathematical representation [2] of biochemical network of cells of different organisms, which are being used to simulate, analyse and predict metabolic phenotypes of these cells under certain conditions. Mathematically, solution space of these in silico models represents all metabolic state a cell can achieve [3], but only select few of these state are biologically feasible or are of interest to researchers. Moreover, availability of omics data had proved to be useful tool for reconstructing and analysing these models under specific conditions [1]. On one hand transcriptomic data can be used to identify active reactions [4] with in the cells while on the other hand metabolomics gives insight into rates at which metabolites are consumed or released [5] and metabolite pool required to maintain homeostasis. Our goal is to reduce the solution space of in silico human metabolic model by constraining it with integration of high throughput transcriptomic and metabolomics data to reconstruct a context specific model Further we wish to analyse context-specific model with omics data to identify biomarkers and target pathways to cancer.

References:

1. R.P. Vivek-Ananth, Areejit Samal, Advances in the integration of transcriptional regulatory information into genome=scale metabolic models. BioSystems 147, 1- 10(2016). 2. Christophe H. Schilling and Bernhard O. Palsson, The underlying pathway structure of biochemical networks. Proc. Natl. Acad. Sci. 95, 4193-4198(1998). 3. Christophe H. Schilling, David Letscher and Bernhard O. Palsson, Theory of the systemic definition of metabolic pathways and their use in interpreting metabolic functions from a pathway-oriented perspective. J. theor. Biol. 203, 229-248(2000). 4. Hadas Zur, Eytan Ruppin and Tomer Shlomi, iMAT: An integrative metabolic analysis tool. Bioinformatics 26, 3140-3142(2010). 5. Mohit Jain, Roland Nilsson, Sonia Sharma, Nikhil Madhusudhan, Toshimori Kitami, Amanda L. Souza, Ran Kafri, Marc W. Kirschner, Clary B. Clish and Vamsi K. Mootha, Metabolite Profiling Identifies a Key Role for Glycine in Rapid Cancer Cell Proliferation. Science 336, 1040-1044(2012) .

254

P104:

Optimized peptide based inhibitors targeting the Dihydrofolate Reductase pathway in Cancer Amrinder Singh, Hanudatta S. Atreya NMR Research Centre, Indian Institute of Science, Bangalore-560012, India

Abstract: In recent years, peptide-based therapeutics have emerged as effective alternatives to small chemical entity drugs.1 On the basis of structural analysis of dihydrofolate reductase (DHFR) (cocrystallized separately with dihydrofolate and NADPH, methotrexate),2 peptide 2 was optimized for inhibition of DHFR. Evaluation of compound 2 in human lung adenocarcinoma cell line A549 showed a significant reduction of cell proliferation and IC50 of 72.54 µM was obtained. Although the IC50 of peptide 2 is comparable to the standard drug MTX but its binding energy is more favourable determined using prime MM-GBSA based on molecular mechanics calculation. The interactions of peptide 2 with DHFR were supported −1 by isothermal calorimetry experiments showing a Kd of 0.7 µM and ΔG of −67.6 kJ mol . Hence, we suggest peptide 2 as a highly potent and promising candidate for further development into an anti-cancer agent.

Figure 1. 2D diagram of the binding pose of compound 2 in the active site of DHFR.

References 1. (a) Cloutheir, C. M.; Pelleteir, J. N. Chem. Soc. Rev. 2012, 41, 1585-1605; (b) Kaur, N.; Lu, X.; Gerhengorn, M. C.; Jain, R. J. Med. Chem. 2005, 48, 6162-6165; (c)

255

Rivier, J.; Vale, W.; Monahan, M.; Ling, N.; Burgus, R. J. Med. Chem. 1972, 15, 479- 482. 2. Bhabha, G.; Ekiert, D.C.; Jennewein, M.; Zmasek, C.M.; Tuttle, L.M., Kroon, G.; Dyson, H.J.; Godzik, A.; Wilson, I.A.; Wright, P.E. Nat. Struct. Mol. Biol. 2013, 20, 1243-1249.

256

P105: Solvation and Ion-pairing in non-aqueous propylene carbonate electrolyte by dynamic 7Li NMR

Ravikanth Reddya, Vikas Kumarb, B.V.N. Phani Kumara, C.V. Avadhanib, S. Ganapathyb and S. Sivaramc

aNMR Laboratory, Central Leather Research Institute, Adayar, Chennai 600020 bPolymer Science and Engineering Division, National Chemical Laboratory, Pune 411008 cIndian Institute of Science Education and Research, Pune 411008 email id: [email protected]

Lithium ion batteries (LIBs) power our digital daily life and are extensively used in electronic devices and tools. LIBs consist of electrodes, electrolyte and a separator. The most widely used electrolyte for LIBS is LiPF6 in nonaqueous carbonate solvent mixture of a cyclic carbonates, e.g, ethylene carbonate (EC), with one or more linear carbonates, eg, dimethyl carbonate (DMC). The key to the battery performance is the Li-ion conduction in the liquid electrolyte medium and this is crucially determined by the kind of Li species that exist and the various factors governing these species. Much of the research efforts in the last few years are focused on studying the effects of solvation. While valuable insights are provided from observed chemical shifts leading to the determination of the solvent coordination number,3-5 no information about the non-solvated cation environments, such as the contact ion pairs (CIP), is provided as such studies are invariably done in the solvent-rich regime (< 1.4 M). The direct observation of Li-cation using 7Li NMR spectroscopy offers new opportunities to probe the cationic environments in the electrolyte at the atomic level. 7Li NMR offers high detection sensitivity (n = 92.6 %) and, despite being quadrupolar (I= 3/2), a high spectral resolution is guaranteed due to vanishing electric field gradients in solution. 7Li observed NMR thus would allow the inspection of all the Li cation environments present, namely, dissociated, solvated and ion-paired Li species, and enable their complete characterization under high resolution conditions. To this end, we have carried out a study of the electrolytic dissociation of lithium hexafluorophosphate (LiPF6) in non-aqueous cyclic propylene carbonate in a large 0.2 – 3.5 M concentration range by 7Li solution state dynamic NMR via relaxation and diffusion measurements. These measurements have enabled us to infer that solvated Li species are the ones to dominate in the beginning, and ion-paired Li species do emerge at mid concentration levels. The identification of these two major species has enabled us to study their NMR relaxation and diffusion behavior. The results of our preliminary studies will be presented and discussed. References 1. Seo, D. M.; Reininger, S.; Kutcher, M.; Redmond, K.; Euler, W. B.; Lucht, B. L. J. Phys. Chem. C 2015, 119, 14038. 2. Yang, L.; Xiao, A.; Lucht, B. L. J. Mol. Liq. 2010, 154, 131. 3. Bogle, X.; Vazquez, R.; Greenbaum, S.; Cresce, A. v. W.; Xu, K. J. Phys. Chem. Lett. 2013, 4, 1664. 257

P106:

Steric crowding of the turn region alters the tertiary fold of Amyloid-β18-35 and makes it soluble*

Muralidharan Chandrakesan‡@, Debanjan Bhowmik†@, Bidyut Sarkar†∩, Rajiv Abhyankar†, Harwinder Singh€, Mamata Kallianpur†, Sucheta P. Dandekar‡, Perunthiruthy K. Madhu†$, Sudipta Maiti†, and Venus Singh Mithu€*

‡Department of Biochemistry, Seth G.S. Medical College and KEM Hospital, A.D. Marg, Parel, Mumbai 400012, India. †Department of Chemical Sciences, Tata Institute of Fundamental Research, HomiBhabha Road, Colaba, Mumbai 400005, India. ∩Present address: Molecular Spectroscopy Laboratory, RIKEN, 2-1, Hirosawa, Wako 351- 0198, Japan. €Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India. $TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsinghi, Hyderabad 500 075, India. @ These authors contributed equally to this work

To whom correspondence should be addressed: Venus Singh Mithu, Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India, Tel: +91 183 2258802-09 (Ext) 3199, Email: [email protected]

Aβ self-assembles into parallel cross-β fibrillar aggregates, which is associated with Alzheimer‘s disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aβ in its aggregated state. Major biophysical properties of Aβ, including this turn, remain unaltered in the central fragment Aβ18-35. Here, we synthesize a single deletion mutant ΔG25 with the aim of sterically hindering the hairpin turn in Aβ18-35. We find that the solubility of the peptide goes up by more than twenty fold. Though some oligomeric structures do form, solution-state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration, but have widths about twice that of Aβ18-35, suggesting an opening of the hairpin bend. Surprisingly, 2D solid-state NMR shows that the contact between F19 and L34 residues, observed in full length Aβ and Aβ18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel β-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel β-sheet. We conclude that the self-assembly of Aβ is critically dependent on the hairpin turn, and on the contact between the F19 and L34 regions, making them potentially sensitive targets for Alzheimer‘s therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by non-specific hydrophobic interactions.

258

P107:

Dihedral angle study in Hesperidin using NMR Spectroscopy Yashwantsinh S Jadeja, Khushal M Kapadiya, Hetal J Jebaliya, Anamik K Shah and Ranjan C Khunt* Centre of Excellence, NFDD Complex, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India. *Correspondence to: Dr. Ranjan C Khunt, Department of Chemistry, Saurashtra University, Rajkot-360 005 (Gujarat), India. E-mail: [email protected]

Hesperidin is flavonoid molecule found in citrus fruits, especially difficult to extract, classify and characterise. Present work is study the unresolved relative configuration of Hesperidin through the combination of different two -dimensional NMR techniques, coupling constant and Karplus equation. The Karplus equation and its modifications have been originated from the valence bond theory, and associated with coupling constant and dihedral angle. The result data set of calculated dihedral angle can probe significant assignment of the virtual configuration of natural products.

References:-

1. M. Karplus, The Journal of chemical physics, 1959, 30, 11-15. 2. M. Karplus, Journal of the American Chemical Society, 1963, 85, 2870-2871 3. C. A. Haasnoot, F. A. de Leeuw, H. P. de Leeuw,C. Altona, Recueil des Travaux Chimiques des Pays-bas, 1979, 98, 576-577

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P108:

Conformational Appraisal of Oxalohydrazine and Oxalohydrazide Derivatives:

NMR and Theoretical Evidences

C. Madhusudan, Sumana Gaonkar, A. Lakshmipriya and N. Suryaprakash*

NMR Research Centre, Indian Institute of Science, Bangalore-560012, India

The stable conformation of Oxalohydrazine and Oxalohydrazide halo derivatives have been studied by derived by 2D HOESY experiment. The stabilized conformation of the molecules derived by NMR studies have been further confirmed by density functional theory (DFT)- based calculations, such as quantum theory of atoms in molecules (QTAIM), noncovalent interaction (NCI), and relaxed potential energy scan.

References:

1 A. Lakshmipriya and N. Suryaprakash, J. Phys. Chem. A, 2016, 120, 7810–7816.

P109:

260

Optimization of Conditions of Smad3-MH1 Protein Preparation and Purification for NMR Studies

Sarita Tripathi1, Himani Rawat1, Shilpy Sharma3 and Jeetender Chugh1,2,*

1Department of Chemistry, 2Department of Biology, Indian Institute of Science Education and Research (IISER), Pune-411008, India 3Department of Biotechnology, Savitribai Phule Pune University, Ganeshkhind, Pune- 411007, India *[email protected]

Smad proteins act as intracellular transducers of TGF-β/BMP/Activin signaling pathway which orchestrates several biological phenomena including cell growth & differentiation, development and maintaining the overall homeostasis.1,2 Smad family of proteins have been categorized as DNA and RNA binding proteins where they bind to nucleic acids in a sequence-specific manner.2,4 They also play a vital role in modulation of miRNA expression by controlling the Drosha-mediated cleavage of pre-miRNA to mature miRNA.2 These proteins are ~500 amino acids long consisting of two domains. The N-terminal MH1 domain binds to DNA/RNA and C-terminal MH2 domain binds to interacting protein partners. Further these proteins can be grouped into three classes- Receptor regulated smads or R- smads (Smad1, 2, 3, 5 and 8); Common smad or Co-smad (Smad4) and Inhibitory smad or I- smads (-6 and 7).4 Recombinant Smad3-MH1 domain, chosen as a model system to investigate Smad-RNA interactions, was expressed in E.coli BL21 (DE3) cells and purified using affinity, ion-exchange followed by size exclusion chromatography. MALDI-TOF spectra of purified protein showed the presence of multiple peaks indicating multimerization of protein. The structural refinement of Smad3-MH1 domain using NMR spectroscopy demands the protein to be in monomeric form. The conformational stability and aggregation of proteins in aqueous solution have been shown to be affected by the presence of different salts from the Hofmeister series, co-solvents and osmolytes.3,4 In this study, we examine the influence of these additives on thermostability and oligomerization of Smad3-MH1 using CD spectroscopy, MALDI and NMR. The results from these experiments will be presented.

References:

1. Heldin, Carl-Henrik, Kohei Miyazono, and Peter Ten Dijke. Nature 1997, 390.6659: 465-471. 2. Davis-Dusenbery, Brandi N., and Akiko Hata. RNA biology 2011, 8.1: 71-76. 3. Davis, Brandi N., et al Molecular cell 2010, 39.3: 373-384. 4. Liliana Attisano and Si Tuen Lee-Hoeflich. Genome Biology 2001, 3010: 1-8

P110:

261

Visualizing the ensemble structures of phosphorylated ubiquitin

Xu Dong1, Zhu Liu2, Zhou Gong1, Meng-Lin Ran1, Wei-Ping Zhang2 and Chun Tang1,2,3,4

1CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China. 2Department of Pharmacology, Institute of Neuroscience, Key Laboratory of Medical Neurobiology of Ministry of Health of China, and Zhejiang Province Key Laboratory of Mental Disorder's Management, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310058. 3Wuhan National Laboratory for Optoelectronics, Wuhan, Hubei Province 430074, China. 4Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China. Correspondence to: [email protected]

Ubiquitin (Ub) is an important signaling protein, which functions by covalent attachment to substrate proteins and by noncovalent interactions with target proteins. In 2014, phosphorylation at S65 of ubiquitin by PINK1 has been discovered, and this interesting finding thus interwove the two most important post-translational modifications - phosphorylation and ubiquitination. Whereas, how phosphorylation regulates the structure and functions of ubiquitin is not well studied so far. Previous study showed that phosphorylation altered the ubiquitin structure, generating two conformations. We further investigated the structure of phosphorylated ubiquitin with the joint use of traditional and newly developed NMR methods. Our results show that Conformation 1 of phosphorylated ubiquitin resembles the structure of unphosphorylated ubiquitin, and Conformation 2 shows low structural similarity to unphosphorylated ubiquitin. The comparison between the structures of Conformation 2 and unphosphorylated ubiquitin reveals that the phosphoryl group at S65 perturbs the region of β5-sheet. Besides the perturbation of β5-sheet, a newly formed helix around S65 is observed in Conformation 2. Although these conformational changes induced by phosphorylation do not dramatically alter the tertiary structure of ubiquitin, these local structural changes might influence the surface charge and recognition site, leading to regulation of the Ub-protein interactions.

262

P111:

Mechanistic insights into the interactions of Proteins with Silver Nanoparticles and Graphene oxide.

Shahid Malik, Somnath Mondal, Hanudatta S. Atreya.

NMR Research Centre, Solid state and Structural Chemistry Unit, IISc,Banglore.

The rapid development of novel nanoscale materials for applications in biomedicine requires the characterization of nano-bio interfaces. Nanoparticles and other nanomaterials exhibit unique structures and properties, often different from the corresponding bulk materials1. The properties of these nanomaterials are dependent on their morphology2,3. From a biological and medical applications point of view, due to their varying sizes, these nanomaterials can reach previously inaccessible targets and can therefore interact with the various cellular components4. The interaction between nanomaterials and biomolecules results in the formation of a biological corona on the nanomaterial surface that is quite dramatically different from that adsorbed on a flat surface of the same bulk material in the same experimental conditions5. When nanomaterials interact with proteins, they can alter the protein conformation, expose new epitopes on the protein surface, or perturb the normal protein function, which could induce unexpected biological reactions and lead to toxicity. For this reason, it is important to study the interaction of protein with some potent nanomaterial. Up to now the information about protein structural changes upon binding to nanomaterials has been based mainly on infrared spectroscopy, circular dichroism, fluorescence, and other methods that can monitor changes in the secondary structure of proteins but give limited information at the amino acid level. NMR can provide high resolution information on protein structural changes in protein-nano complexes in aqueous solution, thus in an environment very close to that found in physiological conditions. Also, NMR is particularly well suited to detect detailed information in the case of weak protein-target interactions. Towards this end, we have chosen two proteins viz. ubiquitin (globular protein) and disordered linker domain of the human Insulin-like Growth Factor Binding Protein-2 (L-hIGFBP2) in order to understand the interactions of the nanomaterials with the proteins. The interaction of proteins has potent implication in drug delivery and biomedicine and here we have chosen Graphene Oxide and three different sizes of AgNPs, 10 nm, 30 nm and 50 nm to study the interaction with Ubiquitin and L-hIGFBP2 using solution state NMR spectroscopy.

References:

(1) Dujardin,E.;Mann,S.Adv.Mater.2002,11,775.

(2) Wang,Y.Acc.Chem.Res.1991,24,133.

(3) Steigerwald,M.L.;Brus,L.E.Acc.Chem.Res.1990,23,183.

(4) Mahmoudi, M.; Sahraian, M. A.; Shokrgozar, M. A.; Laurent, S. ACS Chem. Neurosci. 2011, 2, 118. 263

P112:

Mechanistic insight into photacatalytic degradation of highly refractive phenolic polymer as revealed by Electron Spin Resonance (ESR) and Solid-State NMR spectroscopy

Sridevi Ja1, S. Sundarapandiyan;a*, T.S. Shiny Renithaa, , P. Saravanana, B. Chandrasekarana, G. Bhaskar Rajub a 1NMR Lab, Inorganic & Physical Chemistry, Council of Scientific & Industrial Research-

Central Leather Research Institute, New Delhi, Adyar, Chennai 600 020, India

Advanced Oxidation Technologies (AOTs), which involve the in situ generation of highly potent chemical oxidants such as oOH have emerged as an important class of technologies for accelerating the oxidation of a wide range of organic contaminants in water. The degradation of phenolic syntan by photocatalytic process was studied in a re-circulated batch reactor. The adsorption of phenolic syntan on TiO2 surface was also considered. The generation of hydroxyl free radicals during photo-oxidation was investigated by ESR spectroscopy (1) and found that above pH 9.0 is more congenial for hydroxyl radical generation. The samples of syntan solution collected during the course of photocatalysis were analyzed by Solid-State 13C NMR spectroscopy to understand the degradation pathway (2). In addition, electro- oxidation (3) was attempted to achieve the complete mineralization of phenolic syntan.

References

1. Schwarz, P.F.; Turro, N.J.; Bossmann, S.H.; Braun, A.M.; Wahab, A.; Durr, H.; A new

method to determine the generation of hydroxyl radicals in illuminated TiO2 suspensions. J. Phys. Chem. B. 1997, 101, 7127-7134. 2. Grabowska, E.; Reszczynska, J.; Zaleska, A.; Mechanism of phenol

photodegradation in the presence of pure and modified-TiO2: A review, Wat. Res. 2012, 46, 5453-5471. 3. Sundarapandiyan, S.; , Renitha, T.S.; Sridevi, J.; Chandrasekaran, B.; Saravanan, P.; Raju, G.B.; Mechanistic insight into active chlorine species mediated electrochemical degradation of recalcitrant phenolic polymers, RSC Adv. 2014, 4, 59821-59830.

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P113:

Isolation and Structure Elucidation of an Oleanane

Triterpene from Verbascum Cederiti

Mahmood Al-Azwani *, Hisham A. Abdullkhader Department of Chemistry, College of Science, Sultan Qaboos University, Box 36, Al-Khod 123, Oman

* Corresponding author: e-mail: [email protected]

The crude powder of the inflorescence of Verbascum cederiti plant have used as a fish poison in Oman. It was powdered (500g) and extracted repeatedly with methanol (MeOH) to get viscous crude material (50 g). Crude material was first partitioned between hexane/H2O, then between EtOAc / H2O and finally partitioned with n-butanol to get a butanol extract. Butanol fraction was chromatographed through gravity-driven column with increasing solvent polarities. Two compounds were isolated and one of them identified for the first time from this plant. Using 1D and 2D NMR technique, structure of compound B was assigned as 3,4,2,8-Trihydroxy –11,13-oleanadiene .The carbon skeleton of this molecule is found to be identical to one of the a glycone part in ―saikosaponions‖ found in Verbascum specie1. However, considerable difference in the 1H and 13C NMR shifts for atoms in position 3 indicates that compound B could be a C-3 epimer of the above sapogenin.

Reference:

1- Miyase, T.; Horikoshi, C.; Yabe, S.; Miyasaka, S.; Melek, F. R.; Kusano, G. Saikosaponin homologues from Verbascum spp. The structures of mulleinsaponins I-VII. Chem Pharm Bull., 1997, 45(12), 2029-2033.

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P114:

NMR and computational approaches of APP intracellular Domain (AICD) reveals crucial heavy metal binding motifs: Implications in Alzheimer’s disease

Jagadeesh Kumar D3, Priya Narayan3, Venkatesha M. A1, H S Atreya1, H G Nagendra3, KRK Easwaran2 and S Raghothama1

1 NMR Research Center, IISc Bangalore 2 Molecular Biophysics Unit, IISc Bangalore 3 Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bangalore

ABSTRACT

The pathogenesis of Alzheimer's disease (AD) is extremely complex and involves the formation of neuropathological lesions, amyloid plaques (Aβ) and neurofibrillary tangles (NFTs). Recent evidences reveal the controlled intramembrane proteolysis of the C-terminal fragment of amyloid precursor-protein (APP) by Gamma-secretase yielding an additional catabolite called APP intracellular domain (AICD). The AICD is known to be involved in AD pathology and appears to be a potential drug candidate and a major molecule of interest in understanding the pathology of AD. However, the full length 3D structure of AICD (57aa) is not exist in PDB and poorly understood. Therefore, much structural data remains to be deciphered.

Hence, the objectives of the present study involves in obtaining the structure of AICD Motifs and to find the determinants of ligand binding. Likewise, present existing data, except the central region, partial structural information is available on N- and C-terminus part of AICD sequence (TVIVITLVMLKKKQY-TSIHHGVVEVDA- AVTPEERHLSKMQQNGYENPTYKFFEQ-MQN). The segments –TPEE- and –NPTY- is known to form Type-I turn motif and propagate helices. We have collected NMR data on these motifs and in the process of collecting more data on their extended fragment parts. Similarly, Extensive insilico sequence, molecular modeling and structural analysis of whole AICD is carried out. Preliminary efforts were successful in generating full length Model of AICD. The curated model was further used for docking analysis with heavy metals. Docking results display interesting evidence that, the metals bind to the key residues like Asparagine, Glutamine, Histidine and tyrosine, similar to the Aβ indicating the possibility of aggregation of AICD hence neuronal death. These results need to be validated by NMR and we are in the process of collecting such data.

References:

1. Müller T, Meyer HE, Egensperger R, Marcus K. The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeleton dynamics-relevance for Alzheimer's disease. Prog Neurobiol. 2008 Aug;85(4):393-406. 266

2. Kenneth S. Kosik The Alzheimer's Disease Sphinx:A Riddle with Plaques and Tangles.The Journal of Cell Biology, Volume 127, Number 6, Part l, December 1994 1501-1504

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P115:

NMR and Insilco docking analysis of Aβ(1-16) with polyphenols suggest their exploration as potential disruptors of aggregation events in AD

Priya Narayan3, Jagadeesh Kumar D3, Dilip Senapati1, H G Nagendra3, KRK Easwaran2 and S Raghothama1

1 NMR Research Center, IISc Bangalore

2 Molecular Biophysics Unit, IISc Bangalore

3 Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bangalore

It is well known that one of the reasons for the plaque formation in Alzheimer‘s Disease (AD) is the metal induced aggregation of Amyloid peptide Aβ(1-42). It is also known that the C-terminal residues being hydrophobic are generally found inside the membrane, and hence the exposed regions of 1-28 residues are responsible for aggregation leading to plaque formation. Recent evidences have indicated that the smaller fragments of Aβ like (17-28), (1-16) and (1-10) are produced in presence of secretases and elastase.

With a view to decipher the determinants of binding, docking studies were carried out with polyphenols like Rosmarinic acid, Quercetin, Kaempferol, Oleocanthal, Mangostin, Miricetin, NDGA, EGCG, Resveretol and Curcumin, respectively. Our studies reveal a common set of amino acids in the Aβ(1-16) peptide, which potentially interact with these ligands, at the metal-binding sites. The set of residues forming strong hydrogen bonds within 5 Å with the ligand atoms include D1, Y10, E11, H13, H14 and Q15. Amongst these, the residues that form critical interactions (most probable in most cases) are H13, H14 and Q15 respectively. Based on the frequency of interactions with ligands, these three residues appear to be critical for ligand association. Solution NMR studies also revealed perturbations in charged residues on interaction with Rosmarinic acid. Further NMR analysis is in progress.

These results clearly suggest the plausible design of suitable therapeutics in order to prevent the progression of aggregation events in AD.

References

1. D. J. Selkoe, Alzheimer‘s Disease: Genes, Proteins, and Therapy,Physiol Rev, vol. 81, no. 2, Apr. 2001, pp. 741–66. 2. C. M. Hasler and J. B. Blumberg, Symposium on Phytochemicals: Biochemistry and Physiology, J. Nutr, vol. 129, 1999, pp. 756–757. 3. Mamta Saxena, Jyoti Saxena, Rajeev Nema, Dharmendra Singh and Abhishek Gupta., Phytochemistry and medicinal plants, Journal of Pharmacognosy and Phytochemistry, vol. 1, no. 6, Feb. 2013, pp. 168–182. 4. Davinelli S, Sapere N, Zella D, Bracale R, Intrieri M, Scapagnini G., Pleiotropic protective effects of phytochemicals in Alzheimer‘s disease, Oxid Med Cell Longev., vol. 2012, Jan. 2012, p. 386527.

268

5. Queen BL, Tollefsbol TO., Polyphenols and Aging, Curr Aging Sci., vol. 3, no. 1, Feb.2010, pp. 34–42.

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P116:

Structural and Biophysical Investigations on Sigma4 Domain to Reveal How RNA Polymerase Holoenzyme is Recruited to PmrA Box Promoters

Yuan-Chao Lou1, Yi-Fen Kao1, Tsai-Hsuan Weng2, Yi-Chuan Li2, Chwan-Deng Hsiao2 and Chinpan Chen1

1Institute of Biomedical Sciences, 2Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC

PmrA is a two-component response regulator that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. Recently, we reported the 3.2 Å resolution - crystal structure of phosphoryl analog BeF3 -activated PmrA in complex with the promoter DNA, revealing that activation of PmrA induces the formation of a two-fold symmetric dimer in the N-terminal receive domain (REC), promoting 2 C-terminal DNA-binding domains (DBDs) to recognize the PmrA box located at the -35 position of the promoter. NMR dynamics experiments suggested that the REC and DBD domains tumble separately and have diverse orientations, which together with the DBD-DBD interface may facilitate PmrA searching best interactions with RNA polymerase holoenzyme (RNAPH) for transcription activation (1). The primary sigma factor, controls the transcription by directing RNAPH to promoters composed of ―-10‖ and ―-35‖ elements recognized by sigma2 and sigma4 domains, respectively. It is hence possible that PmrA transcription activation is achieved by the interactions between PmrA and the sigma4 domain in the promoters, where the -35 sigma70- recognition element is replaced by the PmrA box. In this poster, I will show you how we -flap tip helix through an artificial linker to improve its solubility and stability. Solution structure of the sigma4 chimera shows that it adopts a similar conformation as within RNAPH. The interactions - between the sigma4 chimera protein, the PmrA box promoter DNA and the BeF3 -activated PmrA are investigated to reveal how RNAPH is recruited to PmrA box promoters by PmrA.

270

References

1. Lou, Y. C.; Weng, T. H.; Li, Y. C.; Kao, Y. F.; Lin, W. F.; Peng, H. L.; Chou, S. H.; Hsiao, C. D.; Chen, C. Nat Commun. 2015, 6:8838.

271

P117:

A NEW CLASS OF MRI CONTRASTING COMPLEX: CURCUMIN CONJUGATE BASED

Harshit Gupta, Varsha Yadav, Soma Ghorai, Meenu Shrivastava, Neha Kapoor Hindu College University of Delhi

Magnetic Resonance Imaging or MRI enables the acquisition of high resolution three dimensional images of the distribution of water in vivo. These MR images are enhanced by the use of contrast agents that catalytically decrease the relaxation time of protons of water coordinated to a paramagnetic metal center. These contrast agents are used to improve the visibility of internal body structure. Gadolinium(III) or Gd(III), with seven unpaired electrons and the long electronic relaxation time, is paramagnetic and its chelates are ideally suited for such contrast agents. Curcumin-conjugate when chelated to gadolinium(III) forms a completely different class of the contrast agents. Curcumin, also known as magic substance, has effective anti-disease effect against cancer, gastrointestinal diseases, cognitive disorders, and psychiatric conditions. From ancient times, curcumin is used to heal wounds, bruises and sprains. The use of such contrast agents leads the chemistry of MRI to proceed in a safer way.

Keywords: MRI, Contrast agents, Curcumin.

272

P118:

Exploring the Proton-Coupled Transport Mechanism of E. coli Multidrug Transporter EmrE using NMR

Anindita Gayen

CSIR Pool Scientist, Department of Chemistry, IIT Kanpur, Kanpur 208016

Despite the fact that membrane transporters are key contributors of bacterial resistance to multiple antibiotics, minimum is known about the molecular details of their transport process. This work elucidates how protonation of a single membrane-embedded glutamic acid residue (Glu14) in Escherichia coli small multidrug efflux pump EmrE modulates its transport mechanism through global conformational changes that take place in an allosteric manner. A ‗latch on off‘ model is proposed where the loop between transmembrane helices 2 and 3 acts as a latch to stabilize the cytoplasm-facing open conformation of EmrE. Methyl-TROSY NMR has been used as the main probe tool in this work in combination with biophysical and biochemical experiments.

Reference:

Gayen, A, Leninger M, and Traaseth NJ. Protonation of a Glutamate Residue Modulates the Dynamics of the Drug Transporter EmrE. Nat. Chem. Biol. 2016, 12, 141–145.

273

P119:

keto-Enol Tatumarizm Prevention Study for 5-methyl-3(2H)- Furanone by Using 400 MHz NMR Spectrometer.

Khalid AL Maqbli, Saleh AL Busafi, Fakhr Eldin Suliman

Sultan Qaboos University, College of Science (Chemistry Department, Muscat, Sultanate of Oman)

Inotilone is an effective medicinal molecule that is strongly defends the Arthritis and

inflammation of the Human boon‘s joints (1). Obviously, it had been synthesized by myriad techniques. One of the most short, effective, and economical synthetic mood has an

intermediate molecule is called 5-methyl-3(2H)-Furanone that chemically is unstable (2). The reason for that because, it behaves a keto-enol tatumarizme. Exactly, The 5-methyl-3(2H)- Furanone interchanges with its tatumer 5-methyl-Furane-3-ol at room temperature 25 Co. Screening of tatumarizme behavior is highly essential to maintain the concerned molecule under stability condition. By future industrial and medicinal importance of the Inotilone to be intended for diagnostic and therapeutic substance, the physical& chemical studies are highly necessary to aid researchers and manufacturers to synthesis this pharmaceutical molecule efficiently under secured conditions. The 5-methyl-3(2H)-Furanone was mixed with

a trace amount of saturated NaHCO3. Then, it was reacted with acetic acid into deuterated

chloroform (CDCl3) media to validate NaHCO3 maintaining efficiency. Thereby, by applying a quantitative method with internal standard analytical mode by using 400 MHz BRUKER NMR spectrometer, the concentration of 5-methyl-3(2H)-Furanone rose from 52.0 mM to 54.0 mM through 3 days to yield reaction rate: 0.67 mM/day. Then, the stability of 5- methyl-3(2H)-Furanone was invstigated under variable heat medias at 15,25 ,35,45, 50,70 and 90 Co to conclude that 5-methyl-Furane-3-ol did not been generated. In conclusion, the

NaHCO3 is powerful component to preserve the of 5-methyl-3(2H)-Furanone in long terms. ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ

1- AL Balushi M., AL Busafi S., AL Maqbali K., Synthetic Communication, pp: 1088- 1092,2010. 2- Saleh Albusafi, Khalid AL-Maqbali, Synthesis of Inotilone& their Derivatives, Sultan Qaboos University, 2009.

274

P120: Structure and Glass Transition Temperature of Phosphate Glasses studied by Solid State NMR

Sabarinathan Venkatachalam1,2, Holger Kirchhain1, Zhongqing Liu1, Leo van Wüllen1 1 Institute of Physics, Augsburg University, Universiätsstr. 1, D86159 Augsburg, Germany 2 Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627012, India

Abstract:

In glass chemistry, it is important to study the structural changes during glass transition and phase transition. Solid State Nuclear Magnetic Resonance (SSNMR) is a good tool to study the glass structure by employing in-situ MAS NMR spectroscopy. This study deals with structure network of Sodium alumino-phosphate and its evolution upon heating and annealing using in-situ MAS NMR spectroscopy. The temperature used for MAS NMR is 700 oC to evaluate the structure evolution upon heating. The results from in-situ MAS NMR techniques have also been discussed, wiz. Doty HT-MAS, LASER and Inductive heating at temperature above 650 oC with a spinning speeds of up to 10 khz.

Ref.:

1. Phys.Chem. Glasses: Eur. J. Glass Sci. Technol. B 57(4) (2016) 173 2. Phys.Chem. Glasses: Eur. J. Glass Sci. Technol. B 55(6) (2014) 280 3. Solid state NMR 78 (2016) 37

275

P121:

In silico and experimental characterization of 3-Methyladenine DNA glycosylase I of MDR A. baumannii

Jyoti Singh Tomar and Ramakrishna V. Hosur* Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-400005, Maharashtra, India E-mail: [email protected] , [email protected].

ABSTRACT: The rise of multiple-drug resistance in bacterial pathogens imposes a serious public health concern and has led to increased interest in studying various pathways as well as enzymes. Different DNA glycosylases collaborate during bacterial infection and disease by overcoming the effects of ROS- and RNS-mediated host innate immunity response. Function and structure of 3-Methyladenine DNA glycosylase I (TAG) enzyme in the pathogenic bacterium A. baumannii and its closely related species are not well characterized. Inhibition of TAG enzyme is a promising drug design strategy against A. baumannii due to its structural variation from its human homologue. Here, optimized molecular modeling approaches used to provide a structural scaffold of TAG and screening of zinc library was done to find the potent inhibitors. The recombinant protein was expressed and purified as monomer (mwt ~21 kDa). Secondary structure and substrate binding were analysed using CD are in good agreement with the in silico predictions. Near UV-CD spectrum shows the involvement of Tyr residues in substrate recognition. Binding studies with 3mA and Zn2+ indicated that the activity of TAG is an enthalpy-driven process. Fluorescence thermal denaturation studies described that the denaturation of TAG is a two-step process. Modified RP-HPLC-based glycosylase assay attested that the heterologously expressed and purified TAG enzyme is active and catalyses the removal of 3mA. Structural elucidation of TAG enzyme is in process using NMR to understand the molecular recognition interactions involved in enzyme catalysis and this knowledge can be used to screen the inhibitors obtained from the virtual screening. Residues crucial for DNA holding and enzyme catalysis are planned to reconfirm by NMR studies.

References: Tomar et al., Mol. BioSyst., 2016, 12, 3259–1365. Tomar et al., Int. J. Biol. Macromol., 2016, 88, 102–112.

276

P122:

Inhibition of formation of -synuclein amyloid fibrils and their dissolution by Triphala, a herbal preparation

Mandar D. Bopardikar1, Kakita Veera Mohan Rao2, Kavitha Rachineni2, Anusri 2 2 1,2 Bhattacharya , Sinjan Choudhary , and Ramakrishna V. Hosur

1Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005. 2UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai, Mumbai-400098.

Neurodegenerative diseases such as Alzheimer, Parkinson, Huntington, etc. represent a significant and increasing threat to human health and well-being. Parkinson‘s disease (PD) belongs to the group of diseases known as Lewy body diseases (LBDs) which also includes PD with dementia (PDD) and dementia with Lewy bodies (DLB). The pathological characteristic of LBDs is progressive accumulation of the presynaptic protein -synuclein (- syn) in dopaminergic neurons in the substantia nigra1 and therefore they are often referred to as synucleinopathies. The oligomerization and fibrillation of -syn are central processes in the pathogenesis of synucleinopathies and hence represent significant therapeutic target. In this context, the effect of several ayurvedic preparations such as Ashwagandha, Triphala, etc. on the process of fibrillation of -syn was studied. It was observed that ThT fluorescence decreased systematically with increase in Triphala concentration indicating inhibition of - syn fibrillation. Further, TEM images showed confirmative evidence that Triphala prevented -syn fibrillation. However, the chemical composition of Triphala is not known completely2,3. Therefore, the chemical characterization of Triphala by heteronuclear 2D NMR experiments is under progress. Also our fascinating results on -syn fibrillation inhibition by Triphala throw up several interesting questions such as following:

 Which chemical constituents of Triphala are responsible for -syn fibrillation inhibition?  What is the mechanism by which these molecules inhibit -syn fibrillation?  Does there exist some kind of synergy in their action?

We are currently working towards addressing the above questions by NMR and other biophysical techniques.

References

1. Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Nature 1997, 388, 839-840. 2. Krishnamachary, B.; Purushothaman, A. K.; Pemiah, B.; Krishnaswamy, S.; Krishnan, U. M.; Sethuraman, S.; Sekar, R. K. Journal of Chemistry 2013, Article ID 951951, 8 pages, 2013. doi:10.1155/2013/951951. 3. Singh, D. P.; Govindarajan, R.; Rawat, A. K. Phytochem Anal. 2008, 19, 164–168.

277

P123:

Viral manipulation of the host sumoylation pathway

D. Shiba Sankar Hembram1 and Ranabir Das1

1National Centre for Biological Sciences,

Tata Institute of Fundamental Research,

GKVK Campus, Bellary Road,

Bengaluru-56, INDIA.

Like ubiquitin, small ubiquitin like modifier (SUMO) proteins (SUMO 1-3) can be covalently attached to lysine residue of the target proteins through posttranslational modification called sumoylation. It is reported that many cellular process like DNA-repair, viral defense, cellular cycle etc. are regulated via sumoylation. Promyelocytic leukemia (PML) proteins undergo sumoylation process, a critical step for forming PML-Nbs and is responsible for silencing the viral genome. In order to overcome this anti-viral defense, ICP0 (Infected cell protein 0) which is expressed during the early stage of Herpes Simplex Virus – 1 (HSV-1) infection, hijacks the host cell's ubiquitin-proteasome pathway to disrupt PML- Nbs by targeting sumoylated PML. ICP0 is part of a class of ubiquitin ligases (E3s) that harbors a catalytic RING (really interesting new gene)-finger domain and SUMO interacting motif (SIM). The E3-activity of ICP0 was found to be essential for viral genome replication. The mechanism of how ICP0 targets its substrates like PML proteins is poorly understood. In this work, we investigate the structure-based mechanism of how ICP0 manipulates the components of PML-Nbs.

Keywords: Protein structure, NMR spectroscopy, HSV-1, SUMO.

278

P124:

Unfolding of CPR3 Gets Initiated At The Active Site And Proceeds Via Two Intermediates

Vaibhav Kumar Shukla1, Jai Shankar Singh2, Neha vispute1, Basir Ahmad1, Ashutosh Kumar2*, Ramakrishna V. Hosur1, 3*

1 UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Mumbai - 400098, India

2 Department of Biosciences and Bioengineering, Indian Institute of Technology, Mumbai- 400076, India

3Department of Chemical Sciences, Tata Institute of Fundamental research, Mumbai- 400005, India

ABSTRACT Cyclophilin catalyzes the ubiquitous process, ―peptidyl-prolyl cis-trans isomerization‖ which plays a key role in protein folding, regulation and function [1]. Cyclophilins are expressed in all organisms including prokaryotes and eukaryotes in various varieties which are different in their size and localization [1]. Even though, several structures of the cyclophilins from different organisms are available in both ligand bound and free forms, however the folding and unfolding pathways of cylophilins are poorly understood. We report here detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native like secondary structure and shows strong ANS binding due to increased exposure of solvent accessible cluster of non-polar groups. Thus, it has some features of molten globule. The I2 state is more unfolded, but retains some residual secondary structure, and shows weak ANS binding. Chemical shift perturbation analysis (CSP) by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e. N-I1 transition. Regions showing high CSPs in the N-I1 transition match with the regions showing high RMSD in the NMR structure of protein, which showed the presence of two states (open and closed) in the ensemble [2]. This indicates that I1 state has some structural similarity with the functionally relevant ―open state‖. Both the intermediates, I1 and I2 showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

References

1. Wang P, Heitman J. The cyclophilins. Genome Biol.; 6(7):226, (2005). 2. Chi CN, Vögeli B, Bibow S, Strotz D, Orts J, Güntert P, Riek R. A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution. Angew Chem Int Ed Engl. 54(40):11657-61, (2015). 279

P125: SYNTHESIS and CHARACTERIZATION of (E)-3-(2- BENZYLIDENEHYDRAZINYL)-N-(5-METHYLISOXAZOL-3-YL)-3- OXOPROPANAMIDE DERIVATIVES

Ramu Kakkerla*1, Srinivas Marri 2 and M.P.S.Murali Krishna 3 1Department of Chemistry, Satavahana University Karimnagar-505001, Telangana, India. 2Department of Chemistry, Siddhartha Degree and P.G College ,Narsampet-506132, Warangal,Telangana, India 3Department of Chemistry, Andhra Polytechnic College, Kakinada-533003, Andhrapradesh, India. Hydrazones are active pharmacophore which possesses azomethine –NH-N=CH- proton constituting an important class of compounds for new drug development. They form a significant class of compounds in medicinal and pharmaceutical chemistry with several biological applications that include antibacterial1 and antioxidant activities2. The chemistry of isoxazole derivatives continues to draw attention of synthetic organic chemists due to their varied biological activities. As a sequel to our work on screening for new biologically active molecules possessing isoxazol moiety, we report the synthesis and characterization of isoxazolyl hydrazones.

Ethyl 2-(5-methylisoxazol-3-ylcarbamoyl)acetate was prepared, and it is converted into 3-hydrazinyl-N-(5-methylisoxazol-3-yl)-3-oxopropanamide with hydrazine hydrate in ethanol solvent. The hydrazones were obtained by reacting with different aromatic aldehydes in methanol. The structures of all the newly synthesized compounds are confirmed by their spectral data. The compounds having arylidene-hydrazide structure may exist as E/Z geometrical isomers about –C=N double bond and cis-trans amide conformers. R

O O O O O O HN OC H HN NHNH N C 2 5 2 HN NH H N2H4 H2O R-CHO N N N O O H3C H3C O H3C

1 2 3

R = C6H5 ; 4-ClC6H4 ; 4-OCH3C6H4 ; 4-NO2C6H4 ; 4-CH3C6H4 ; 4-OHC6H4 ; 3-OCH3C6H4 ;

2- CH3C6H4 1. Mahamoud A, Chevalier J, Davin-Regali A, Barbe J, Pages J M, Curr Drug Targets,7(7), 2006, 843 2. Belkheiri N, Bouguerne B, Bedos-Belval F, Duran H, Bernis C, Salvayre R, Negre- Salvayr A, Baltas M, Eur Med Chem, 45, 2010, 3019.

280

P126: Classification and molecular level structural insight into clarified oil of Indian origin by NMR spectroscopy: estimation of hydrocarbon types and average structural parameters

Sujit Mondal*, Anil Yadav, Ravindra Kumar, Veena Bansal, S. K. Das, J. Christopher & G. S. Kapur Indian Oil Corporation Limited, R&D Center, Sector-13, Faridabad-121007 *Corresponding author: [email protected]

Abstract A direct and easy to grasp methodology based on combination of quantitative 1H and 13C- NMR has been developed for the estimation of total aromatics, saturates and several important structural parameters like aromaticity, average number of aromatic rings per molecule, average number of aromatic carbon atoms per molecule, average molecular weight, degree of aromatic substitution, degree of aromatic condensation, nature of condensation and substitution to aromatic ring etc. for clarified oil (CLO) from Indian Oil refineries. These parameters along with HPLC analysis data for di- to penta-ring aromatics provide molecular level understanding of this potentially valuable feedstock, which can thus be correlated with process parameters for needle coke production from CLO. The method exploits the concept of group molecular weight (GMWt) and uses three empirical equations governing the nature of aromatic condensation. The various kinds of CLO originated from Indian Oil refineries have been classified into three major classes by virtue of their differential nature and composition. 2D HSQC NMR has been extensively studied for accurate assignment of different classes of protons in 1H NMR spectra of CLOs. The method was validated by SARA analysis using TLC-FID (IP-469) and by open column chromatography (ASTM D- 2549) for hydrocarbon types. References: 1. Gillet, S.; Delpuech, J.J.; Valentin, P.; Escalier,J.C. Anal. Chem. 1980, 52. 813. 2. Andre, H.; Faisal, A. H.; Hassan, A-R.; Mamun Absi, H. Energy & Fuels 2014, 28(7), 4321. 3. Bansal, V.; Kumar, R.; Krishna, G.J.; Patel, M.B.; Sarpal, A.S.; Basu, B. Fuel 2014, 118, 148 and the references thereof. 4. Rodriguez, J.; Tierney, J. W.; Wender, I. Fuel 1994, 73(12), 1870.

281

P127:

Derivatization for NMR spectroscopy Avik Mazumder, Ajeet Kumar Defence Research and Development Establishment, Jhansi Road, Gwalior, India. Phone number: +91751 2390183, E-mail: [email protected]

The ability of NMR spectroscopy to detect analytes is limited by absolute NMR sensitivity of observed nuclei, concentration of the analytes and nature of background chemicals1. Several sample preparation methods are adopted to enrich the analytes and/or remove interfering background. These steps often lead to loss of stable analytes, degradation of reactive analytes and incomplete removal of background. Introduction of NMR active sensitive hetero-nuclei can aid in the in-situ NMR analysis of such analytes2. Derivatization can also be helpful when other analytical techniques can be used in addition to NMR spectroscopy for conclusive identification of analytes. Since, different analytical techniques require different sample formats; specific sample preparation is required for each analytical technique. This increases the analysis time and has severe limitations for volume limited precious samples. A single derivatization reaction can be used to render the analyte amenable to multiple analytical techniques 3-7 allowing the analytical chemist to correlate data from different analytical techniques. In this presentation the utility of derivatization reactions for the detection and identification of chemicals will be discussed. References: 1. Mazumder, A.; Dubey, D. K., Nuclear Magnetic Resonance (NMR) Spectroscopy. In: Reedijk, J. (Ed.) Elsevier Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Elsevier: Waltham, MA, USA. doi:10.1016/B978-0-12-409547- 2.05891-1, 2013. 2. Mazumder, A.; Kumar, A.; Purohit, A. K.; Dubey, D. K., A high-resolution phosphorus- 31 nuclear magnetic resonance (NMR) spectroscopic method for the non-phosphorus markers of chemical warfare agents. Anal. Bioanal. Chem. 2012, 402 (4), 1643-1652. 3. Mazumder, A.; Kumar, A.; Purohit, A. K.; Dubey, D. K., Application of high performance liquid chromatography coupled to on-line solid-phase extraction-nuclear magnetic resonance spectroscopy for the analysis of degradation products of V-class nerve agents and nitrogen mustard. J. Chromatogr. A 2010, 1217 (17), 2887-2894. 4. Mazumder, A.; Kumar, A.; Dubey, D. K., High resolution 19F{1H}-nuclear magnetic resonance spectroscopy and liquid chromatography-solid phase extraction-offline1H- nuclear magnetic resonance spectroscopy for conclusive detection and identification of cyanide in water samples. J. Chromatogr. A 2013, 1284, 88-99. 5. Mazumder, A.; Gutch, P. K.; Dubey, D. K., A derivatization strategy for the detection and identification of volatile trialkylphosphites using liquid chromatography-online solid phase extraction and offline nuclear magnetic resonance spectroscopy. J. Chromatogr. A 2015, 1393, 26-36.

282

6. Mazumder, A.; Gupta, H. K.; Garg, P.; Jain, R.; Dubey, D. K., On-line high-performance liquid chromatography–ultraviolet–nuclear magnetic resonance method of the markers of nerve agents for verification of the Chemical Weapons Convention. J. Chromatogr. A 2009, 1216 (27), 5228-5234. 7. Mazumder, A.; Garg, P.; Pardasani, D.; Kumar, A.; Purohit, A. K.; Dubey, D. K., Polymer supported N-benzyl-N-nitroso-4-toluenesulfonamide: An improved reagent for the derivatization of the acidic markers of nerve agents. Anal. Methods 2011, 3 (7), 1574- 1581.

P128: 283

Characterization of the deamination activity of APOBEC3B by real-time NMR, which is distinct from that of APOBEC3G

Li Wan 1,2, Takashi Nagata1,2, Ryo Morishita3, Akifumi Takaori-Kondo4, Masato Katahira1,2

1 Grad. Sch. of Energy Sci., Kyoto Univ. 2 Inst. of Adv. Energy, Kyoto Univ. 3 CellFree Sciences Co.,Ltd. 4 Dept.of Hematology and Oncology,Grad.Sch.of Med.,Kyoto Univ.

Abstract Human APOBEC3B(A3B) deaminates cytosine to uracil in single-stranded DNA(ssDNA), which does not only lead to the blocking of virus pathogens and endogenous retrotransposons,but also causes DNA lesion in human genome which is a source of several cancers. A3B's deamination activity and specifity for the 5'-TC-3' motif are conferred by its carboxyl-terminal domain(CTD). However, little is known about the deamination mechanism of A3B. To elucidate the deamination properties of A3B CTD, we firstly used uracil DNA glycosylase (UDG) assay to study the activity properties of A3B CTD, then we applied real- time NMR method to further confirm the results obtained by UDG assay. We find that the way of deamination by A3B CTD toward to the target motif in ssDNA is quite different from that of well-studied A3G. To elucidate the origin of the distinct features between A3B CTD and A3G CTD, further analysis is underway using ssDNA substrates having multiple target motifs at different positions by means of real-time NMR method.

Name Page Number A.Lakshmipriya 250,P99

284

Aditya Mishra 174,P37 Ae Ree Lee 221,P76 Ajay Verma 230,P84 Akanksha Gautam 203,P62 Akhila Viswan 116 Akira Naito 39 Akshay Gaikwad 215,P71 Akshay Kumar 228,P82 Alex Smirnov 16 Amandeep Singh 126 Amardeep 192,P52 Amir Goldbourt 15 Amit Devra 202,P61 Amrinder singh 255,P104 Amrita Bhattacharya 185,P47 Anant Bahadur Patel 77 Ananthakrishnan R 161,P26 Anindita Gayen 273,P118 Anirban Bhunia 90 Anusha R. Chandra 234,P87 Arisu SHIGETA 171,P34 Aritra Bej 207,P65 Arnab Dey 190,P51 Aruna Singh 218,P73

Ashish Arora 106 Ashutosh Kumar 53 Atul Rawat P1,133 Avik Mazumder 289,P127 B V N Phani Kumar 68 B. S. Hemanth Kumar 242,P93 Balvinder Singh 229,P83 Bhagyawanti Chomal 158,P23 Bharathwaj Sathyamoorthy 44 Bhawna Chaubey 236,P88 Bhisma Narayan Ratha 178,P41 Bholanath Pahari 42 Bikash Baishya 108 Bipul Sarma 111 Biswaranjan Mohanty 74 Bolaji Fatai OYEYEMI 138,P5 Bong-Jin Lee 107 C.Madhusudan 260,P108 285

CHINAR PATHAK 109 Chojiro Kojima 18 Christian Griesinger 80 Chun Tang 51 Chunyang Cao 97 D. Shiba Sankar Hembram 278, P123 D.Adilakshmi 128 Deepak Kumar 238,P89 Deepshikha Verma 125 Dennis Hwang 27 Dinesh Kumar 79 DIPITA BHATTACHARYYA 167,P31 Dr. Junfeng Wang 81 DR.RANJAN C KHUNT 259, P107 DU Jiangfeng 3 Durgesh Dubey 193,P53 Eriks Kupče 20 G K S Prameela 183,P45

Garima Jaipuria 112 Garima Verma 124 Gunnar Jeschke 83 H.Yamanappa 253,P102 Haribabu Arthanari 19 Harindranath Kadavath 86 Harpreet Singh 197,P56 Harshad Paithankar 149,P15 Harshit Gupta 209,P67 Hartmut Oschkinat 100 Himani Rawat 153,P19 Hitendra Negi 137,P4 Humaira Ilyas 169,P32 Indrani Pal 122 Jae Hyun Park 199,P58 Jagadeesh Kumar 266, P114 Jai Shankar Singh 186,P48 Jayasubba Reddy Yarava 110 Jeong Yong Suh 54 JERRIPOTHULA K LAKSHMI 134,P2 Jierong huang 95 JITENDRA KUMAR DAS 121 Jithender Reddy Gurrala 25 Joon Hwa Lee 87 286

Jooyoung Lee 96 JUHI AUGUSTA 159,P24 JYOTHY G VIJAYAN 151,P17 Jyoti Tomar 276,P121 Jyotsana ojha 177,P40 K. V. R. Chary 6 Kalyan Chakrabarti 45 Kamal Saba 212,P69 KANAKARAJU MARUMUDI 160,P25 Kashmiri Deka 224,P79 Kavita Dorai 65 Ke Ruan 73 Keisuke Kamba 48 Khalid AL Maqbali 274,P119 Khushboo Gulati 208,P66 KIRAN SANKAR 144,P10 CHATTERJEE Koh Takeuchi 8 KOICHI KATO 72 Krishna Mohan Poluri 38 Krishnakant Gangele 219,P74 Krishnendu Kundu 62 Kshama Sharma 189,P50 Li Wan 291,P128 Lucio Frydman 1 Madhuri Puvvada 210,P68 Mahmood AlAzwani 265, P113 Mandar Bopardikar 277,P122 Mandar V. Deshmukh 47 Manish Kumar 172,P35 Manju Pandey 152,P18 Marc Antoine Sani 94 Martin Scanlon 70 Masato KATAHIRA 46 Matthias Ernst 11 Medha Karnik S R 140,P7 Meenakshi Sharma 206,P64 Mehdi Mobli 43 Mitsuhiko Ikura 5 Mukesh Kumar 34 Mukul Jain 198,P57 N. Chandrakumar 58 Nancy Jaiswal 114 287

Navin Khaneja 61 Neel Sarovar Bhavesh 88 Neeraj Sinha 13 Neeru 251,P100 Neha Kapoor 272,P117 Nikita Malik 175,P38 Nilamoni Nath 69 Nitin Nathubhai Kachariya 162,P27 Nitin Prakash Lobo 105 Nutan Agadi 225,P80 Oc Hee Han 101 P.Dhanishta 248,P97 P.Lokeswara Rao 247,P96 P.T.Wilson 249,P98 Palwinder Singh 78 Parag Surana 179,P42 PARVATHY JAYAN 154,P20 Patrick GIRAUDEAU 22 Paul Coote 23 Paul Gooley 37 Petrik Galvosas 64 Phani Babu 131 Poonam Rana 243,P94 PRINCY S WADHWANIYA 165,P29 Priya Narayan 268, P115 Priyanka Aggarwal 239,P90 PRIYATOSH RANJAN 135,P3 Prof. Ho Sup Yoon 7 Purba Sarkar 220,P75 R Ravikanth Reddy 181,P44 R. V. Hosur 2 raj kumar sharma 139,P6 Rajat Garg 145,P11 Rajiv Kumar Kar 92 Rajlaxmi Panigrahi 173,P36 Rakesh Sharma 196,P55 Ramu Kakkerla 280,P125 Ranabir Das 9 Rashmi Rao R 170,P33 Ravi Sankar Ampapathi 98 Ravikanth reddy 257,P105 RENUKA RANJAN 141,P8 Richa dubey 120 288

Richa Trivedi 240,P91 Rituraj Mishra 231,P85 S. Jayanthi 17 S. Khushu 28 S. Sendhil Velan 26 Sabarinathan Venkatachalam 275,P120 Sachin Chaudhari 59 Sadhana Kumari 127 Sairam Geethanath 29 saleem yousf 148,P14 Sandeep Kumar Mishra 123 Sarath Chandra Dantu 180,P43 sarita tripathi 261, P109 Sarita Tripathi 261,P109 Satnam Singh 201,P60 Scott Anthony Robson 67 Senthil Kumaran 30 SeoRee Choi 222,P77 Shahid Malik 263,P111 Shefali Chaudhary 226,P81 Shenlin Wang 103 Shine A 147,P13 Shin-ichi Tate 84 shivanand Pudakalakatti 32 Shreyan Ganguly 200,P59 SIRISHA KATUKURI 146,P12 Sk Abdul Mohid 166,P30 Soumya S. Roy 63 Sreemantula Arun Kumar 216,P72 Sridevi.J 264, P112 SRINIVAS MARRI 176,P39 Stanley Opella 49 SUDAKSHINA GANGULY 194,P54 Suhas Bhate 143,P9 Shashank Yadav 252,P101 Sujoy Mukherjee 55 Sulakhshana Mukherjee 89 Sumant Indurkhya 254,P103 Sumit Mishra 214,P70 Sunghyouk park 76 Sujeesh K. S 150,P16 Surajit Bhattacharyya 52 Sujit Mondal 281,P126 289

Sushanta Kumar Mishra 241,P92 Susmitha Ambadipudi 10 T. G. Ajithkumar 102 T. Narasimhaswamy 41 T. S. Mahesh 66 TAHSEEN RAZA 164,P28 Tatyana Smirnov 85 Tesmine Martin 119 ThejasVishnu Ramesh 232,P86 Toshimichi Fujiwara 57 TsyrYan Yu 71 Ukale Dattatreya Uttam 204,P63 Upasana Rai 118 Vaibhav Shukla 279,P124 Veera Mohan 91 Venus Singh Mithu 258, P106 Viji Sarojini 82 Vinay Ganapathy 130 Vineet V Bhombore 234,P87 Vinesh 56 Vipin Agarwal 104 Virender Singh 155,P21 Weontae Lee 50 Xu Dong 262, P110 Yangmee Kim 36 Yashwantsinh Jadeja 157,P22 Yuan-Chao Lou 270,P116 Yusuke Nishiyama 14 Zeyu Jin 187,P49 Zia Tariq 223,P78 Zinia mohanta 209,P67

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