主なイベント

In solid-state NMR, 1H-13C cross polarization (CP) has been widely used to enhance the 13C signals. However, in the multiphase polymers containing both rigid and mobile components, the fast motion of mobile segments will significantly average the 1H-13C dipolar couplings, rending the 13C signals of mobile components only slightly enhanced or even completely lost. Herein, we proposed an efficient strategy, by fully utilizing the abundant 1H polarization and tactfully combine CP with NOE or RINEPT, to efficiently acquire the 1D 13C spectra and 2D 1H-13C HETCOR spectra of multiphase polymers with barely increasing the experimental time, accompanied with significant enhancement of the 13C signals of mobile components. [1-4] This could be particularly advantageous for the elucidation of structures and dynamics of polymers used as pharmaceutical excipients or in pharmaceutical drug delivery systems. In addition, abundant residual 1H polarization after the CP is typically discarded in most solid-state NMR experiments. Thus, we will also show how those 1H polarization can be tactfully utilized to obtain multiple 2D and 3D proton homonuclear correlation spectra in together with HETCOR spectra in a single experiment [5-7]. These techniques can offer atomic-level insights into the structural characteristics and molecular dynamics of complex pharmaceutical systems or biomaterials, elucidating critical properties such as polymorphism, amorphous stability, drug-polymer interactions as well as the interfacial behaviors in multi-component bioengineered materials.

Phase separation of RNA-binding proteins via multivalent interactions between aromatic/polar-rich disordered domains contributes to the formation of functional cytoplasmic granules and nuclear puncta. These domains have also been identified as players in cancer-causing fusion proteins as well as the nucleators of neuronal inclusions in amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and some forms of Alzheimer’s Disease (AD). We use solution NMR spectroscopy and biophysical methods combined with molecular simulation to see and quantify the residue-by-residue details of interactions along to the pathway from monomer, to liquid-liquid phase separated state, to static aggregates and hydrogels. Enhanced by collaborations, we demonstrate how these interactions play important roles in cell and organism models of disease. 1) We probe the sequence-specific contribution of residues in the disordered region of the archetypal phase separating protein FUS and find extensive contributions of arginine and polar residues in determining both phase separation and aggregation. 2) We examine the details of how RNA mediates contacts with the disordered domains of TDP-43 and FUS, as well as the ability of condensed phases to disrupt RNA G-quadruplex structure. 3) Through integrative modeling joining AlphaFold Multimer, molecular simulation, and experiment, we attain an atomic model of the dynamic helical oligomerization of the conserved region in the C-terminal domain of TDP-43, aggregates of which are the primary hallmark of ALS and some AD. Together, these data provide insight into the ability of disordered domains to form contacts mediating phase separation and protein aggregation.

I will overview solution NMR studies on RAS, which my laboratory has carried out over two decades with an emphasis on cancer therapeutic development. RAS is an oncogene product in which about 30% of all cancer patients carry mutations. A recent success in targeting KRAS resulted in very potent inhibitors against its G12C mutant and two compounds are now clinically available to treat patients with a tumour carrying the mutation. However, this type of inhibitors only works for a fraction of cancer patients, and drug resistance has been a major issue of this treatment. NMR-based studies done by our and other groups had played a major role in our mechanistic understanding of this cancer target as well as the development of KRAS-targeted therapeutics.

In this short presentation I will present my great memories of Professor Masatsune Kainosho with whom I enjoyed both professional and personal relationships over many years. Kai was an enthusiastic scientist and passionate food lover: He did everything with passion, and we had many common interests in both science and foods. I deeply miss him and the time we spent together.

The isotope labeling strategies pioneered by Professor Masatsune Kainosho have revolutionized NMR studies of the structure and dynamics of ever more complex protein systems. In this talk, I will describe how isotope labeling enables NMR analysis of the structure and dynamics of eukaryotic proteins containing both intrinsically disordered and structured domains. Intrinsically disordered proteins (IDPs) and protein regions (IDRs) are highly abundant in eukaryotes and play a central role in cellular regulatory pathways. Approximately half of the proteins in the human proteome and more than 80% of transcription factors contain extended regions that are intrinsically disordered. Molecular level characterization of proteins that contain both structured and disordered domains represents an enormous challenge to traditional methods of structural biology. Most structural studies to date have relied upon a reductionist, divide-and-conquer approach, in which the ordered and disordered regions are expressed independently and investigated in isolation using X-ray crystallography, cryo-EM, NMR and other biophysical tools. While this approach provides valuable information on the structure, dynamics, and interactions of the isolated domains, it can provide only limited insight into the interplay between the structured and dynamically disordered regions of the full-length protein. Within the cell, the ordered and disordered regions of a given protein act synergistically to allow it to perform its biological function; a detailed understanding of the underlying molecular mechanism can only be achieved through analysis of the conformational ensemble of the intact, full-length protein molecule. NMR plays a major role in characterization of the structural propensities and dynamics of disordered protein regions. Such studies are facilitated by segmental isotope labeling strategies. By integrating NMR data with data from other biophysical methods such as single molecule FRET, SAXS, and X-ray crystallography, detailed molecular level insights can be derived into the dynamic intramolecular interactions between structured and disordered regions, providing new understanding of the factors that regulate biological function.

Masatsune Kainosho (Kai) and I were friends and colleagues for 49 years. We met in Tokyo in 1976 on the occasion of my first visit to Japan. We were brought together by Asao Nakamura, Kai’s coworker at the Ajinomoto Company who had been a postdoctoral fellow in the laboratory at Harvard where I began my graduate studies. We bonded immediately over our shared interest in stable isotope labeling as an approach to NMR studies of peptides and proteins. In the years before the internet and email, we interacted primarily through letters and face-to-face meetings at international conferences. Our scientific approaches were complementary, and led to fruitful exchanges of techniques and materials. I was able to see Kai on frequent visits to Japan, once on a mini sabbatical that he organized. Kai made extended visits to my laboratory, first at Purdue University and later at the University of Wisconsin-Madison. Kai and I had several joint publications. More often, we exchanged individual manuscripts in progress, with requests for comments and suggestions. In this way, we kept up with the work going on in our separate laboratories. We both found ways to scale up the yields of cell-free protein synthesis as a way to minimize the scrambling of labeled atoms that occurs in whole cells.

Kai’s curiosity and enthusiasm for science and the good things in life were infectious. He reveled in the flora and fauna and natural wonders of the world, good food, and travel. He was loyal to his friends and acquaintances, and they, in turn, revered him.

On the personal side, I became a honorary member of the Kainosho family and got to know his family members. Their house in Ogikubo was my home whenever I visited Tokyo. He, in turn, was an honorary member of my family and stayed with us during his visits. Kai and I made frequent excursions together, and these often included family members and mutual friends.

I was fortunate to have been counted among the friends of this remarkable human being and to have witnessed, first hand, his stellar scientific trajectory. Kai’s passing is most sorely mourned.

Late Professor Masatsune Kaisho had a profound impact on the NMR community in Japan and around the world in many ways. His academic contributions are highly regarded internationally. He played an important role in the founding and operation of the Nuclear Magnetic Resonance Society of Japan, serving as its second president. He also played a central role in organizing two International Conferences on Magnetic Resonance in Biological Systems (ICMRBS) held in Japan. I would like to express my deepest condolences on his loss and share some personal recollections to honor his memory.

I started my NMR carrier in 1971 when I got the first NMR spectrum under the guidance of Kainosho-sensei at Ajinomoto Central Institute. He was already an established NMR chemist at that time. His background was in organic chemistry. NMR had become indispensable for determining the structures of organic compounds, and many organic chemists actively participated in the NMR discussion meeting. What set him apart from other organic chemists was his strong interest in the structural and functional analysis of biological macromolecules such as nucleic acids and proteins by NMR, looking for the unique contributions that organic chemistry could make to this field. We often talked about the future prospects of our research when he visited the Institute for Protein Research, Osaka University for measurements. I was deeply impressed by his remark that what really matters was not the immediate measurement at hand, but searching for a lifelong research goal. The keyword, of course, was the stable isotopes. I witnessed how this later bore fruit in the development of the SAIL (stereo-array-isotope labeling) amino acid system.

Thanks to Kainosho-sensei, the application of stable-isotopes to NMR research began relatively early in Japan. His 13C, 15N-double labeling method in peptide bonds opened new frontiers. The SAIL system was a quite powerful technique and was widely used in solution NMR worldwide. His contributions to the field of biological solid-state NMR were also relevant. Since it was difficult to obtain high resolution 1H-NMR spectra, stable-isotope labeling was inevitable in high-resolution solid-state NMR. We have collaborated on the complete signal assignment of solid adenosine, the ATP structure in the 1H-ATP synthase b-subunit, and the structural and functional analysis of the c-subunit rotor ring of the 1H-ATP synthase embedded in membranes, and on other projects. To commemorate his contributions to this field, I would like to discuss some of them in this session.

Gタンパク質共役受容体、イオンチャネルなどの膜タンパク質は様々な生命現象に関与する生理学的に重要なタンパク質であり、同時に創薬の標的タンパク質でもある。膜タンパク質の機能を明らかにするためには、X線結晶構造解析およびクライオ電子顕微鏡から得られる精密な立体構造情報に加えて、膜タンパク質の動的構造情報も必要となる。核磁気共鳴法は溶液中のタンパク質の構造平衡など動的構造をとらえることのできる構造生物学的手法である。しかしながら、膜タンパク質のような高分子量タンパク質をNMRにより解析するためにはNMRの分子量制限を乗り越える必要がある。本講演では我々の取り組みおよびその成果に関して概説する。1）

Membrane proteins, including G protein coupled receptors and ion channels, play fundamental roles in many physiological processes and are target proteins for drug development. For better understanding of the functions of the membrane proteins, not only precise static three-dimensional structures determined by X-ray crystallography and cryo-electron microscopy methodologies, but also dynamical nature are required. NMR (nuclear magnetic resonance spectroscopy) provides us information about membrane proteins dynamics, including conformation equilibrium related to functions. However, it is frequently difficult to obtain information about the membrane protein dynamics related to the functions, due to the molecular weight limitation in NMR. We have recently developed novel NMR methods for characterizing protein dynamics utilizing multiple quantum relaxation rates of side-chain methyl groups, which can be sensitively observed in high molecular weight proteins. In this paper, we will show our recent results of function-related dynamics of membrane proteins.1)

機能性核酸の構造、ダイナミクス及び相互作用をNMR法によって解析し、機能発現機構の解明とその応用を目指した研究を紹介します。

荒田洋治先生のご指導により，ラマン分光法からNMRへと転向した時期が，安定同位体利用NMRの黎明期であった．同時期には，ETH Wuthrich教授がタンパク質構造解析技術の開発を進める一方で，NIHのBax博士らは華麗な安定同位体利用多次元NMR技術を次々と開発して，まさにNMRに無限の可能性を感じる時期だった．甲斐荘正恒教授の研究室に参加する機会を得て，甲斐荘先生が開発する最先端の安定同位体標識技術と，異種核スピン相互作用に関する甲斐荘先生の深い知識と洞察に牽引されて黎明期のNMRを甲斐荘先生と一緒に楽しんだ．

安定同位体利用NMRも一定の完成を見る時期になると，NMRはタンパク質構造解析ツールとして確立する一方で，NMRで分かる情報はタンパク質科学として必要な情報の一つにしか過ぎなくなり，自ずと「NMRの研究」から「NMRによる研究」へと研究の焦点が変わってきた．講演では，安定同位体利用NMRの黎明期から完成期における状況と，完成期以後に私が進めた研究の概要を報告する．この間，直接ご指導を頂いた，荒田洋治先生先生，稲垣冬彦先生，甲斐荘正恒先生，神田大輔先生は，いずれも故人となってしまわれた．講演では，哀悼の意もこめて偉大な先生方との関わりについても触れたい．