Takuro Matsui

A unique dye-decolorizing peroxidase, DyP, from Thanatephorus cucumeris Dec 1: heterologous expression, crystallization and preliminary X-ray analysis

The dye-decolorizing peroxidase DyP is a key enzyme in the decolorizing fungus Thanatephorus cucumeris Dec 1 that degrades azo and antraquinone dyes. The gene dyp from T. cucumeris Dec 1, which has low homology to other peroxidase genes, was cloned and transformed into Aspergillus oryzae and glycosylated DyP was expressed at high levels. Purified DyP was deglycosylated using GST Endo F1 and then crystallized in a strong magnetic field (10 T) at 283 K using ammonium sulfate as precipitant. X-ray diffraction data to 2.96 A resolution collected from a native crystal at the Photon Factory (Tsukuba, Japan) showed that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 136.15, c = 363.46 A. The asymmetric unit of the crystal contained four DyP molecules, with a corresponding Matthews coefficient (V(M)) of 2.50 A(3) Da(-1) and a solvent content of 51%. Heavy-atom derivatives of DyP have been obtained and electron-density maps have been calculated. The haem is visible and continuous electron density between the haem and protein clearly indicates the location of the proximal histidine ligand.

Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric alpha-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast alpha-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A.

Structural consequences of hen egg-white lysozyme orthorhombic crystal growth in a high magnetic field: validation of X-ray diffraction intensity, conformational energy searching and quantitative analysis of B factors and mosaicity.

A novel method has been developed to improve protein-crystal perfection during crystallization in a high magnetic field and structural studies have been undertaken. The three-dimensional structure of orthorhombic hen egg-white (HEW) lysozyme crystals grown in a homogeneous and static magnetic field of 10 T has been determined and refined to a resolution of 1.13 angstroms and an R factor of 17.0%. The 10 T crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.54 (3), b = 73.86 (6), c = 30.50 (2) angstroms and one molecule per asymmetric unit. A comparison of the structures of the 0 T and 10 T crystals has been carried out. The magnitude of the structural changes, with a root-mean-square deviation value of 0.75 angstroms for the positions of all protein atoms, is similar to that observed when an identical protein structure is resolved in two different crystalline lattices. The structures remain similar, with the exception of a few residues e.g. Arg68, Arg73, Arg128 and Gln121. The shifts of the arginine residues result in very significant structural fluctuations, which can have large effects on a proteins crystallization properties. The high magnetic field contributed to an improvement in diffraction intensity by (i) the displacement of the charged side chains of Arg68 and Arg73 in the flexible loop and of Arg128 at the C-terminus and (ii) the removal of the alternate conformations of the charged side chains of Arg21, Lys97 or Arg114. The improvement in crystal perfection might arise from the magnetic effect on molecular orientation without structural change and differences in molecular interactions. X-ray diffraction and molecular-modelling studies of lysozyme crystals grown in a 10 T field have indicated that the field contributes to the stability of the dihedral angle. The average difference in conformational energy has a value of -578 kJ mol(-1) per charged residue in favour of the crystal grown in the magnetic field. For most protein atoms, the average B factor in the 10 T crystal shows an improvement of 1.8 angstroms(2) over that for the 0 T control; subsequently, the difference in diffraction intensity between the 10 T and 0 T crystals corresponds to an increase of 22.6% at the resolution limit. The mosaicity of the 10 T crystal was better than that of the 0 T crystal. More highly isotropic values of 0.0065, 0.0049 and 0.0048 degrees were recorded along the a, b and c axes, respectively. Anisotropic mosaicity analysis indicated that crystal growth is most perfect in the direction that corresponds to the favoured growth direction of the crystal, and that the crystal grown in the magnetic field had domains that were three times the volume of those of the control crystal. Overall, the magnetic field has improved the quality of these crystals and the diffracted intensity has increased significantly with the magnetic field, leading to a higher resolution.

In Situ Observation of Elementary Growth Processes of Protein Crystals by Advanced Optical Microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

In-situobservation of elementary growth steps on a protein crystal, surface diffusion of protein molecules and dislocations inside a protein crystal

C62 MS46.27.3 Acta Cryst. (2005). A61, C62 In-situ Observation of Elementary Growth Steps on a Protein Crystal, Surface Diffusion of Protein Molecules and Dislocations inside a Protein Crystal Gen Sazaki , Masashi Okada , Takuro Matsui, Hideo Higuchi, Tomonobu Watanabe, Katsuo Tsukamoto, Kazuo Nakajima, IMR, Tohoku Univ., Japan. CIR, Tohoku Univ., Japan. ATRL, Matsushita Electric Ind. Co., Ltd., Japan. TUBERO, Tohoku Univ. , Japan. Grad. School Sci, Tohoku Univ., Japan. E-mail: sazaki@imr.tohoku.ac.jp