Toshiyuki Chatake

Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X‐ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy. 1 In this article, the hydration structures of three proteins are described— myoglobin, wild‐type rubredoxin, and a mutant rubredoxin—the structures of which were solved at 1.5‐ or 1.6‐Å resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments. Proteins 2003;50:516–523.

Hydrogen and hydration in proteins

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins. High-resolution neutron diffractometers dedicated to biological macromolecules (BIX-type diffractometer) have been constructed at the Japan Atomic Energy Research Institute and they have been used in the 1.5Å-resolution crystal structure analyses of several proteins. Interesting topics relevant to hydrogen and hydration in proteins, such as (1) the detailed geometry of hydrogen bonds; (2) information regarding hydrogen/deuterium exchange behavior; (3) the acidities of certain H atoms; (4) the role of hydrogen atoms in enzyme mechanisms and thermostability; (5) the location methyl hydrogen atoms; and (6) dynamical behavior of hydration structures that include H positions have been extracted from these structural results. In addition, a method for the systematic growth of large single crystals based on phase diagrams has been introduced and will be briefly described in this article.

Neutron diffraction study of hydrous phase G: Hydrogen in the lower mantle hydrous silicate, phase G

A neutron powder diffraction study was performed to determine the sites occupied by hydrogen in the structure of DHMS phase G, which is stable at the pressure-temperature conditions of the transition zone and lower mantle. The diffraction data from small samples (about 3 mg) were collected by using a highly sensitive imaging plate detector at the BIX-3 beamline at JRR-3M nuclear plant in JAERI Tokai Laboratory, Japan. The present neutron diffraction data reveal that hydrogen (and deuterium) is located in the 6k site of Wykoff letter in the MO 6 layer of the phase G structure with the space group P31m. The bond lengths of O-H and O-D are 1.1(4) and 0.8(3) A, respectively. This result is consistent with previous Raman spectroscopic studies of this phase.

Crystallization of a large single crystal of a B-DNA decamer for a neutron diffraction experiment by the phase-diagram technique

Crystallization of a large single crystal of a B-DNA decamer, d(CCATTAATGG), for a neutron-diffraction experiment has been accomplished by an analysis of its solubility phase diagram and a large single crystal was successfully crystallized at around the minimum solubility point of the oligonucleotide: 30%(v/v) MPD, 100 mM MgCl(2) pD 6.6 using 0.4 ml D(2)O solutions of the DNA (sample concentration 1.5 mM). It is confirmed that the resulting crystal (dimensions: 1.7 x 1.3 x 0.6 mm) diffracts sufficiently well for neutron data collection.

Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.

More rapid evaluation of biomacromolecular crystals for diffraction experiments

The parameters used to evaluate biomacromolecular crystal quality [Rmerge, I/sigma(I), maximum resolution and mosaicity] strongly depend on the experimental diffraction conditions. In this paper, the distinctive features of the relative Wilson plot method are described and it is shown that the overall B factor obtained from this plot is more appropriate for the characterization of protein crystals. The relative Wilson plot has been applied to the characterization of crystals of the B-DNA decamer d(CCATTAATGG) and crystals of the proteins DsrD (dissimilatory sulfite reductase D) and hen egg-white lysozyme (HEWL), which were studied by neutron diffraction. It was found that the crystal quality of the B-DNA decamer and DsrD depended significantly on the regions of the crystallization phase diagram from which the samples were taken. However, in the case of HEWL crystal quality appears to be independent of the region of the crystallization phase diagram.

ATP Binding and Hydration State Analyses of DAPK: Steps toward Neutron Protein Crystallography Studies

Graduate School of Sci. and Eng., Ibaraki Univ., Hitachi, Ibaraki 316-8511, Japan Frontier Center for Appl. Atomic Sci., Ibaraki Univ., Tokai, Ibaraki 319-1106, Japan Department of Bioengineering, Nagaoka Univ. of Tech., Nagaoka, Niigata 940-2188, Japan Research Reactor Institute, Kyoto Univ., Kumatori, Osaka 590-0494, Japan Faculty of Pharmaceutical Sciences, Univ. of Toyama, Toyama 930-0914, Japan 5 College of Eng., Ibaraki Univ., Hitachi, Ibaraki 316-8511, Japan