Toshitaka Nakada

Effects of a magnetic field on the nucleation and growth of protein crystals

Crystallization of hen egg-white lysozyme and horse-spleen ferritin was carried out under a steady and uniform magnetic field of 10 T and compared with the crystals grown under 0 T. It is clearly demonstrated that a magnetic field reduced the number of nuclei and not only oriented the crystals but also modified the habit of protein crystals. The present experimental result indicates that application of a magnetic field is an efficient method for growing a small number of large crystals.

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.

Observation of the concentration distribution around a growing lysozyme crystal

The optical path length distribution of the solution around a growing lysozyme crystal was observed in situ using a Michelson interferometer of the reflection type. From the interferograms, a concentration profile around a growing crystal and the growth rate were obtained and thus, we could evaluate the diffusion coefficient of the lysozyme molecules to be 1.59 × 10-10 m2/s. The measurement of the viscosity of the solution rendered it possible to estimate the Stokes radius of the diffusing entities which coincided with the crystallographic size of a lysozyme molecule.

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.

Substrate Recognition Mechanism of α-1,6-Glucosidic Linkage Hydrolyzing Enzyme, Dextran Glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the alpha-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce alpha-glucose. By using the mutant of catalytic acid Glu236-->Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 A resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (beta/alpha)(8)-barrel in domain A, which is common to the alpha-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites -1 to +2. The environment of the glucose residue at subsite -1 is similar to the environment of this residue in the alpha-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite -1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile alpha-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile alpha-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the alpha-amylase structure from Bacillus subtilis. The comparison with the alpha-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of alpha-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.

Effect of a magnetic field on the orientation of hen egg-white lysozyme crystals

Recent experiments have shown that lysozyme crystallization in a magnetic field of the order of 1 T can result in a significant degree of orientation of the crystals. We present more extensive experimental results and propose a model to account for this phenomenon. Because of the small susceptibility anisotropy of most protein molecules, the orienting effect is unimportant for smaller aggregates, even those much larger than a critical nucleus. However, during sedimentation crystals grow larger and are more likely to become aligned. The degree of orientation thus depends on crystal growth rate and container geometry, in addition to magnetic field strength, as we have confirmed experimentally.

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.

Characterization of indium tin oxide film and practical ITO film by electron microscopy

In order to clarify the structure of indium oxide film containing tin and tin oxides, various In2O3 based films prepared by vacuum evaporation were studied using high-resolution electron microscope (HREM). Indium tin oxide (ITO) film was composed of In2O3 and SnO. SnO crystal also contained (110) or (101) crystallographic shear (CS) structures that indicate excess amounts of tin. The CS structure was also found in a commercial ITO film having the resistivity of 2 £ 10 24 V cm. q 2000 Elsevier Science S.A. All rights reserved.

An interferometric study of the solubility of lysozyme crystals under high pressure

The effect of hydrostatic pressure on the solubility of tetragonal hen egg-white lysozyme crystals was measured in situ by a two-beam interferometer combined with a high-pressure optical cell which was newly designed by our group. The change in the concentration with time during equilibration was measured accurately and continuously starting from a supersaturated state (growth relaxation) and an undersaturated state (dissolution relaxation). The solubility value thus measured at 100 MPa was 28% larger than that at 0.1 MPa, and this increase was much smaller than those of previous reports.

Atomic Force Microscopic Study of Subsurface Ordering and Structural Transforms in n-Alcohol on Mica and Graphite.

Structure forces in n-alcohols (Cn H2n +1OH (8≤ n≤12)) on the mica or graphite surfaces were investigated using an atomic force microscope (AFM). The structure force on graphite has a periodicity of ~0.5 nm, which is independent of temperature. The structure force on mica has a periodicity of 0.86 nm when T 40° C. This phase transition corresponds to a change in the alignment of molecules on the surface from parallel (lying, at T 40° C).