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

Novel measurement techniques forvisualizing 'live' protein molecules at work

Novel Techniques

List of the novel measurement techniques

Crystal contact-free space for analyzing spatial distribution of protein internal motions

Daisuke Kohda(Kyushu University)

Contacts between molecules in protein crystals often restrict internal motions of proteins. The crystal-contact effect provides crystallographic snapshots of working proteins in crystal lattice. It is possible to collect many snapshots under different crystallization conditions, but the snapshots may not represent the true distribution because of non-random sampling. For full description of protein dynamics, we propose intentional creation of space in crystal lattice, and place a target region in the space created. For the realization, we used a fusion protein with maltose-binding protein (MBP). The key technique is the rigid connection to fix the relative orientation of the two proteins.

We applied this technique to the mitochondrial receptor Tom20, which recognizes diverse mitochondrial presequences with similar affinities. Our hypothesis is that the Tom20 recognizes different partial features of a presequence in each bound state, and the dynamic equilibrium between the multiple bound states enables the recognition of the full features of a presequence. The C-terminal helix in MBP and the N-terminal helix in Tom20 were directly connected. We obtained smeared, rod-shaped electron density in the crystal contact-effect free space in the Fo-Fc omit map. The electron density was fully consistent with the three poses of the crystallographic snapshots of the presequence peptide and the results of our previous molecular dynamics simulations.

Advanced high-speed AFM for wider biological applications

Toshio Ando(Kanazawa University)

Protein molecules undergo structural changes, bind to and dissociate from interaction partners, and traverse a range of energy and chemical states. Therefore, directly observing protein molecules in dynamic action has long been a holy grail for biological sciences. To meet this desire, Ando’s group developed high-speed atomic force microscopy (HS-AFM) that can visualize dynamic structure and processes of proteins at sub-molecular spatial and sub-100 ms temporal resolution, without disturbing their function. In fact, various proteins, including bacteriorhodopsin in response to light and myosin V walking on actin filaments, were successfully visualized. Importantly, these molecular movies provided not only corroborative “visual evidence” for previous inferences but also solved long-standing questions that had previously been difficult or impossible to address by other approaches.

Current HS-AFM employs the sample-stage scan mode, which restricts the size of the sample stage and hence specimens to be placed on it. To remove this restriction, Ando’s group has recently developed a tip-scan HS-AFM system. Importantly, this system allows us to use large sample stages that are fabricated for various purposes. Therefore, it is now possible to image, for example, membrane proteins that are activated depending on membrane voltage. Of note, various devices and techniques can be implemented to this tip-scan HS-AFM instrument. Ando group is now developing multifunctional HS-AFM systems by implementing a fluorescence microscope and optical tweezers.

Real-time background-free selective imaging of fluorescent nanodiamonds in vivo

Masahiro Shirakawa (Kyoto University)

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. We describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds (Nano Lett. 12, 5726-32, 2012). This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

In-cell NMR observation of the biological events within living cells

Noritaka Nishida (University of Tokyo)

In-cell NMR is a useful approach for obtaining structural information within living cells, but the rapid increase of the dead cells has limited the application of various NMR methods with a long experimental time. To overcome this problem, we developed a novel bioreactor system, in which fresh culture medium is continuously supplied to cells encapsulated in thermoreversible Mebiol gel inside the NMR tube (Angew Chem Int Ed (2013) 52, 1208-11). It was demonstrated that the intracellular ATP concentration, which was depleted within 30 min without the bioreactor, can be maintained more than 15 hours in the bioreactor. In addition, the population of the dead cell was significantly suppressed after 15 hours of incubation.

By using the bioreactor system, we were able to observe the intracellular protein-protein interaction between the exogenously introduced protein and the endogenous macromolecules by the transferred cross saturation method. In-cell NMR observation using the bioreactor system will enables us to apply various time-consuming NMR measurements, such as 3D NOESY and relaxation analyses. We will also try to observe the various intracellular events in a real time manner.

Free-energy calculations of protein conformational changes by multi-resolution simulations

Yuji Sugita (Riken)

Protein structures has been determined mainly by X-ray crystallography and NMR. This analysis enables us to investigate the average structures and the fluctuations in the crystal environments or in solution. However, it is rather difficult to investigate large-amplitude motions of proteins at the atomistic detailed resolution only by these high-resolution experiments. Our aim is to develop novel computational methods for observing large-amplitude motions of proteins. We then combine the simulation data with the recent experimental measurements such as single molecule (sm) FRET or small angle X-ray scattering (SAXS).

All-atom molecular dynamics (MD) simulations are quite useful to examine rather fast conformational changes or fluctuations in proteins on the time-scale of 100 ns or μs. To simulate large-amplitude motions in proteins, the simulations may not be sufficient due to the time-scale limitations. In the study, we combine all-atom MD with coarse-grained MD simulations and QM/MM hybrid simulations to investigate not only the fast motions but also the slow and large-amplitude motions. The potential drawback in the multi-resolution approach is the accuracy of coarse-grained models and parameters. To overcome it, we develop new coarse-grained models of protein conformational changes. In another approach, we incorporate dynamic trajectories observed by experiments like smFRET into MD simulations of protein conformational changes. Although this approach, so called ‘data assimilation’ has been utilized in earth sciences or climate predictions, this is the first application of the data assimilation methods in protein structural dynamics.

For structural determination of large protein complexes like ribosomes, electron micrograph (EM) has been one of the most important research tools. We also improve the structural refinement protocols by introducing multi-resolution models, normal mode analysis, and enhanced conformational sampling techniques. The new methods and techniques will be installed into an MD program package, GENESIS, developed by RIKEN Advanced Institute for Computational Sciences (AICS). GENESIS is freely available software under the license of GPLv2 from the RIKEN web site.

Visualization of Sec Translocon Machinery

Tomoya Tsukazaki (NAIST)

Translocation of secretory proteins through the membrane is one of the evolutionally conserved mechanisms. Biological lipid bilayers generally prevent the passage of ions and small molecules. In order to achieve the transport of large molecules such as proteins, all cells possess well-controlled, specialized machineries for the secretion. We perform structural biological analyses for elucidation of protein translocation via SecYEG translocon, a conserved protein-conducting channel. Because the SecYEG complex is itself a passive channel, the driving force is required. SecA ATPase, a SecYEG-associated cytosolic motor, repeatedly pushes the substrate protein into SecYEG channel using the energy of ATP hydrolysis (Fig. left). It is one of the most important issues to fully understand the essential protein translocation. Hence, lots of functional and structural analyses have been reported since 1970's. From around 2000, X-ray crystal structures of Sec components have been solved one after another. Our group also has been determined the crystal structures of the bacterial Sec factors, SecYEG, SecA and SecDF. Although several researchers have been analyzing the molecular mechanism based on the crystal structures, the conformational changes and interactions of Sec proteins during the protein translocation still remains unclear. Therefore, to visualize dynamic protein translocation reaction we reconstituted one unit of Sec machinery in vitro using Nanodisc and are trying to image it by high-speed atomic force microscopy (AFM) (Fig. right).