Kazuo Kurihara

A novel approach to the solubility measurement of protein crystals by two-beam interferometry

Abstract A novel technique was developed for the rapid solubility measurement of protein crystals (lysozyme) by two-beam interferometry. The merits of this technique are: (1) time saving (less than 2 h to obtain one experimental point), (2) the small amount of the sample (∼66 μl), (3) high accuracy, (4) applicable to the solubility measurement of a metastable phase and (5) insensitive to impurities. The limitations of the present technique are also discussed.

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.

A new neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-4)

A new neutron single-crystal diffractometer (BIX-4) has been constructed at 1G-B port of JRR-3M in JAERI. Since at 1G-B port another diffractometer for biology, BIX-3, and a high-resolution powder diffractometer (HRPD) coexist, the monochromator house needed to be reconstructed. The main architecture of BIX-4 is based on that of BIX-3. BIX-4 uses an elastically-bent perfect-Si crystal monochromator and neutron imaging plates as BIX-3. In addition, several optimizations of the monochromator and modifications from previous BIX-3 are carried out. The final gain of the neutron intensity at the detector position is estimated to be 2.5 times larger than previous BIX-3. That higher performance increases the opportunities to apply neutron crystallography to biological macromolecules which give only weak reflections and/or small crystals.

Recent results on hydrogen and hydration in biology studied by neutron macromolecular crystallography

Abstract.Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, a technique complimentary to ultra-high-resolution [1, 2] X-ray diffraction. Three different types of neutron diffractometers for biological macromolecules have been constructed in Japan, France and the United States, and they have been used to determine the crystal structures of proteins up to resolution limits of 1.5–2.5 Å. Results relating to hydrogen positions and hydration patterns in proteins have been obtained from these studies. Examples include the geometrical details of hydrogen bonds, H/D exchange in proteins and oligonucleotides, the role of hydrogen atoms in enzymatic activity and thermostability, and the dynamical behavior of hydration structures, all of which have been extracted from these structural results and reviewed. Other techniques, such as the growth of large single crystals, the preparation of fully deuterated proteins, the use of cryogenic techniques, and a data base of hydrogen and hydration in proteins, will be described.

Interferometric study on the crystal growth of tetragonal lysozyme crystal

Abstract Using a Michelson interferometer, the concentration-gradient layers around a growing hen-egg-white lysozyme, tetragonal crystal were observed. By measuring the concentration distribution and the normal growth rate, the growth mode and the diffusion coefficient of lysozyme molecules were evaluated. The experimental results indicate that the growth is regulated by two-dimensional nucleation in the supersaturation range of 6 ≤ σ surf ≤ 20. The diffusion coefficient was nearly constant, 4.9 ± 0.7 × 10 −11 m 2 s −1 over a wide supersaturation range, differing from that of supersaturated bulk solutions.

Incorporation of impurity to a tetragonal lysozyme crystal

Abstract Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton–Prim–Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.

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.

Single Crystal Pulsed Neutron Diffractometer for Biologically Important Materials Crystallography No.1 (BIX-P1) at Material and Life Science Facility in J-PARC

Japan proton accelerator research complex (J-PARC) with the power of 1 MW have been constructing since 2001 in JAERI, Japan. And the first beam will come by 2007. As one important day-one neutron instruments at Material and Life Science Facility in J-PARC, a single crystal diffractometer No.1 for structural analysis of biological macromolecules and organic compounds with unit cell dimensions less than about 135 A (BIX-P1) has been proposed, considering the international competition in biological science and the expansion of the application to the industrial fields. It is expected to gain efficiency more than 50 times larger than neutron biological diffractometers BIX-3 and BIX-4 installed at JRR-3 reactor also in JAERI. In this paper, evaluation of moderator, items of necessary R & D and the current specification of BIX-P1 are shown.

Evaluation of the Resolvable Capacity of Bragg Reflections for a New Diffractometer at J-PARC/MLF Designed for Protein Crystals With Large Unit Cells

We aim to build a high-resolution neutron time-of-flight diffractometer for biomacromolecules at the Materials and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC) that allows the collection of neutron diffraction data from crystals with unit cells of ≈250 A. Considering both the flux and pulse width necessary to realize data collection covering a minimum d-spacing of 2.0 A and with a unit cell constant of ≈250 A, we chose a decoupled moderator (DM) as the appropriate source for this high-resolution diffractometer. We considered a simple instrumentation model that includes a moderator, neutron guide, sample size, and neutron detector; we then investigated its spot separation performance and estimated the instrumental parameters for the design of a new diffractometer for protein crystals with large unit cells at J-PARC/MLF.