Single crystal neutron diffraction is one of the powerful methods to obtain the structure information, including that of the hydrogen atoms. The IBARAKI biological crystal diffractometer called iBIX is a high-performance time-of-flight single crystal neutron diffractometer to elucidate the hydrogen, protonation and hydration structures of biological macromolecules in various life processes. Most notably merit of neutron diffraction is the observation of hydrogen atoms and protonation states. From the remarkable results by the iBIX, actual application of merit of neutron diffraction for protein science are as follows: (a) Finding tautomerization, (b) Observation of hydrogen bonds, (c) Determination of orientations of side chains and water solvents, (d) Observation of hydrogen around metal atom. Current status of the iBIX and interesting results with merit of neutron diffraction obtained by the iBIX are reported.

Incoherent neutron scattering is a powerful tool to investigate the dynamics of biomolecules and water. In this article, a new dynamical model called “Matryoshka model” is first described, which provides detailed information on the motions contained in the phospholipid molecules based on the scattering spectra. In the latter half of the article, a relationship between water activity and water mobility, and the effects of pressure and hydration on protein dynamics are discussed. These studies demonstrate the advancement of analytical methods which provide ever more detailed information for elucidating the mechanism of biological functions.

To elucidate, predict, and control the biological functions of proteins, it is important to clarify their solution structure and dynamics. Small-angle scattering is one of effective methods for examining the changes in their structures and dynamics due to their binding with target molecules under physiological conditions, although the spatial resolution of the structure revealed by this method is lower than that by crystallographic technique, which provides structural information at atomic resolution. In addition, neutron spin echo is the only technique that can measure the conformational dynamics of proteins in the Q-region, which is covered by small-angle scattering. In this paper, we will introduce some of our recent studies using small-angle scattering and neutron spin echo.

Small-angle X-ray/neutron scatterings (SAXS/SANS; collectively called SAS) offer the structure of a biomacromolecule under near a physiological condition. To reveal a structure of a single biomacromolecule with SAS, it is indispensable to acquire the scattering profile which does not include the contribution of aggregates and other components. In this manuscript, we introduce two state-of-the art methods, Size Exclusion Chromatography-SAS (SEC-SAS) and Analytical UltraCentrifugation-SAS (AUC-SAS), for realizing the selective observation of the concerned component in polydisperse and/or multi-component biomacromolecular solutions.

A hydrogen atom is one of the major components of biological molecules including proteins, and it is involved in hydrogen-bonds important for structure formation/recognition of molecules and in chemical reactions such as hydrolysis. BIX-3 and BIX-4 in JRR-3 are the neutron diffractometers for biological macromolecular crystallography. Many neutron crystal structures have been determined using BIX-3 and BIX-4 before the earthquake in 2011. Neutron crystal structures of rubredoxin and elastase were determined by using BIX-3. These structural studies indicated that the structural information, including hydrogen atoms, is essential for understanding the molecular mechanisms of the proteins. After the restart of JRR-3, neutron diffraction data with higher resolution than previous studies can be obtained by using a new monochromator installed in BIX-3. The high-resolution data will provide much useful information for structural biology.

Cyanobacteriochromes (CBCRs) are photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is an important signaling protein regulating chromatic acclimation of photosynthetic antenna in cyanobacteria, which is expected to be applied for optogenetic tool. We have determined a 1.63Å crystal structure of the GAF domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The PCB is buried within a “bucket” consisting of hydrophobic residues. We propose that the “leaky bucket” structure functions as a proton-exit/influx pathway upon photoconversion. NMR analysis using ¹⁵N direct detection demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Protonation states of ionizable amino acid residues (e.g., Asp, Glu, Lys) on a ligand-binding pocket are critically important information for structure-based drug design. However, in general, hydrogen atoms including protons on the amino acid residues are not determined by X-ray crystallography in PDB entries with resolution ca. 1.0~3.0Å. For example, we have generally treated Asp and Glu as deprotonated states at the physiological condition (pH 7.4). There are concerns that a ligand-binding mode depends on the wrong protonation states of amino acid residues in a protein. If we use a protein structure with the wrong protonation states, drug design would be misled by its incorrect ligand-binding mode. In this regard, we must appropriately assign such protonation and tautomerization states by pKa calculations and the visual inspection of X-ray crystal structures and their electron densities. In addition, we should confirm whether the predicted protonation states of complex are energetically stable. Thus, we will attempt to estimate the validity of the protonation states on protein by quantum chemical calculations based on fragment molecular orbital (FMO) method. Recently, our group has developed FMO database (https://drugdesign.riken.jp/FMODB/) to accumulate FMO data. Experimental researchers such as structural biologists and medicinal chemists can quantitatively understand molecular recognition and easily use FMO data to help in drug design.

Epigenetics is an emerging field of science that studies heritable changes caused by activation and deactivation of genes without any change in the genomic DNA information. In other words, epigenetics is the study to elucidate how environment and our behavior can cause changes that affect the way our genes function. Many types of regulatory mechanisms in epigenetics, such as DNA modification and numerous types of histone modification has been reported. Steady state recognition of histone modifications and their localizations on genome has been reported, however, it is still uncertain how the modification is dynamically recognized and contribute for establishing higher order structure in nuclei. In this article, we introduce our recent trials to analyze protein with chemical modifications and/or intrinsic disorder regions by combining biochemistry, organic chemistry and ESR, and also show future plan to utilize neutron scattering to further characterize the relevant proteins.