Grant‐in‐Aid for Scientific Research on Innovative Areas
(Research in a proposed research area)

Novel measurement techniques forvisualizing 'live' protein molecules at work

Achivements

The number of research papers published every year
5 in 2014 / 56 in 2015 / 82 in 2016 / 63 in 2017 / 84 in 2018 / 45 in 2019
335 in Total

Manegement activities

The General Meeting was held four times in order to promote joint researches within and among research groups. The steering committee was held at the same time as the General Meeting. In addition, a total of 14 technical workshops were held to promote the popularization of newly developed measurement techniques, which generated 16 joint researches and resulted in 20 coauthored papers. We organized three international meetings, and ten workshops/symposium sessions in the annual meetings of the scientific societies, as a place to introduce our aims and activities. The web site was released and 12 newsletters were issued for exchange and disclosure of information.

Support activities aimed at international network formation in the research area of dynamic structural life sciences

We aimed to expand the scope of the academic research activities conducted in our Grant‐in‐Aid for Scientific Research on Innovative Area, entitled “Novel measurement techniques for visualizing ‘live’ protein molecules at work” internationally. During the four-year period, five invited speakers, 13 joint researchers including postdoctoral fellows and students, and one sabbatical professor were accepted from abroad. Our four members stayed in a laboratory in Germany for a joint experiment. Three Japan-residing foreign students were dispatched to an international conference held in Japan. We organized an international version of the annual technical AFM workshops during the last three years and accepted 31 researchers and students from abroad. Finally, overseas travel expenses were supported for 11 members of our research activity.

Daisuke Kohda(A01)

We developed a fusion protein method to create crystal contact-free space (CCFS) in protein crystals and to place the mobile parts or ligands in the CCFS. The mobile parts/ligands appear as smeared electron densities in the CCFS in the difference electron density map. We applied the CCFS method to visualize the movement of a highly mobile presequence peptide as bound to the mitochondrial import receptor, Tom20, and to estimate the solution conformation of a flexible loop segment in another mitochondrial import protein, Tim21, which was distorted in the conventional crystals by the crystal contacts.

Toshio Ando(A01)

The aim of this study is largely classified into two: (1) Extensively exploring a new research field of“dynamic structural biology”that seeks to film protein molecules in dynamic action with high-speed AFM, not only by conducting the study by myself but also by collaborating with researchers of the project group as well as external researchers, and (2) enabling observation of new molecular phenomena that have been impossible with the current high-speed AFM, by advancing the capability of high-speed AFM. For the aim (1), our instruments were opened to these researchers. Through many collaborations, we succeeded in observing a variety of proteins, providing mechanistic insights into their molecular processes. For the aim (2), we succeeded in developing high-speed AFM combined with optical tweezers and super-resolution fluorescence microscopy based on high-speed AFM. The actual application studies of these new instruments are subjects to be done in the future.

Masahiro Shirakawa(A02)

Methods to create NV centers which are characterized by a crystal lattice vacancy and it adjustments are created. In addition, method targeting the NVC containing ND to biomolecules and technique to measure ODMR of the biomolecule-attached ND have been developed. For protein assemblies (ex. cytoskeltons), by labeling subunit of the assemblies with ND particles, it was possible to characterize the assembly states and mechanism. We also generated a method to measure NMR spectroscopy of proteins under the presence of rheologic forces --- rheology NMR. The new method is convenient, and applicable to modern NMR machine.

Noritaka Nishida (A02)

We have established the in-cell NMR methodology that enables the observation of the intracellular biological events in real-time manner. To observe the intracellular molecular response against oxidative stresses, we prepared the HeLa cells, in which both thioredoxin and glutathione were isotopically labeled, and monitored the redox status of Trx and glutathione by alternate in-cell NMR measurements. This study demonstrated the redox status of Trx is regulated not only by the intracellular redox potential but also by various endogenous regulatory molecules, such as Trx reductases. We also performed the in-cell NMR observation of small GTPase Ras and its oncogenic variants. We demonstrated that the GTP-bound ratio of Ras in the steady state is decreased in the cell for all Ras constructs, due to the increase of khy and the decrease of kex. We also succeeded in quantifying the contribution of the specific regulatory proteins by in-cell NMR experiments using the knockout cells.

Yuji Suigta(A03)

We developed new methods combining experimental information with simulation data to obtain reliable structure-dynamics relationship of proteins. Generally, experimental data tend to be low-resolution and low-dimension, whereas simulation can provide high-resolution structural information. In our methods, we combined cryo-EM with MD simulation in flexible fitting calculation and utilized machine learning for combining single-molecule analysis with MD simulation. The developed methods were installed into GENESIS software package and can be used together with enhanced sampling methods and multi-scale/multi-resolution molecular models. We applied these methods to understand structure-dynamics relationships of membrane proteins or protein/nucleic acid complexes.

Tomoya Tsukazaki(A03)

Protein secretion in bacteria is an essential mechanism, which is mediated by Sec proteins. However, the detailed molecular mechanism of how Sec proteins are transported still remains unclear. In this study, we performed X-ray crystallographic analysis and observation of Sec proteins using high-speed atomic force microscope to elucidate the mechanism. As the result, the system which could observe the protein secretion reaction by real-time single unit analysis was established, while the structures of Sec proteins in various forms was solved.


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