| Co-Investigator(Kenkyū-buntansha) |
WINSLOW S.CA コロラド州立大学, 生物化学科, 教授
ROBERT O.POY コロラド大学, 分子生物学科, 教授
TSUKIHARA Tomitake Department of Biotechnology, Tokushima University, 工学部, 教授 (00032277)
CAUGHEY Winslow S. Department of Biochemistry, Colorado State University
POYTON Robert O. Department of Molecular Biology, University of Colorado
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| Research Abstract |
Among many factors affecting crystallization conditions of cytochrome c oxidase, structure of detergent for stabilizing the solubilized enzyme protein has turned out to be the most critical. We have discovered seven species of synthetic non ionic detergents which provide crystallizable enzyme preparations. An alkyloxyethylene, BL8SY(CH_3(CH_2)_<11>O(CH_2CH_2)_<11>OH), most extensively examined so far, gave tetragonal crystals which diffract x-ray with the correlation coefficient larger than 0.5 at 6A resolution and 0.8 at 7A resolution. Occasionally X-ray diffraction was found at higher than 5A. Heavy atom derivatives of the hexagonal crystal were obtained by Hg, W and Au compounds. For molecular replacement analysis, 2D crystals in various space groups were prepared and the electron diffractions obtained indicates a three dimensional structure significantly different from that previously given by Henderson et al. Furthermore, formation of dimer affects clearly the 3D structure of the
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monomer.
Preparation methods of yeast and bacterial(Paracoccas)enzymes have improved to establish much better methods than previously published ones in terms of the yeald, purity and reproducibility. The bacterial enzyme gave two dimensional crystals. The yeast preparation we obtained is soluble to any neutral buffer without any detergent. This property is quite promising for 3D crystallization.
Method for infrared anisotropy of the single crystal of carbonyl cytochrome c oxidase has been essentially established. The most critical point was how to make small and polished tubes of CaF_2 for both infrared and optical measurements for a single crystal at various direction. During this investigation, crystallization conditions for fully reduced and fully reduced CO forms were examined to obtain X-ray diffraction. The space groups and lattice constants of these derivatives obtained coincide with those of the resting oxidized form within the experimental accuracy, suggesting that the conformational changes during the electron transfer and ligand binding are not so large as affecting quaternary structure of the enzyme molecule. Thus, once a crystal at an oxidation state are obtained at a certain level of resolution, crystals with the same quality may be easily obtained at other oxidation and binding states probably by treatment of the crystal with appropriate oxidant, reductant or ligand. Less
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