| Project/Area Number |
06453220
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
YOSHIKAWA Shinya Himeji Institute of Technology, Department of Life Science, Professor, 理学部, 教授 (40068119)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1995: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1994: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Membrane proteins / Protein crystallization / X-ray crystallographic analysis / Cytochrome c oxidase / Cytochrome bcl complex / Detergent / チトクロムbc1複合体 / 結晶化 |
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| Research Abstract |
Effect of detergent structure on crystallization of bovine heart cytochrome c oxidase has been examined extensively. Decylmaltoside provides crystals which diffract x-rays up to 2.2 A^^ﾟ resolution or to better resolution, although at fairly low reproducibility A slight modification in the detergent structure affects the crystallization conditions significantly. For example, neither nonyl nor undecyl maltosides provides stable crystals. Structure of the sugar moiety in decylmaltoside is critical for the crystallization. Actually, no sugar other than maltose provides even microcrystals of bovine heart cytochrome oxidase.
Crystallization conditions for various derivalives of this enzyme stabilized with decylmaltoside has been established. Crystals of azide bound form as well as cyanide bound form are prepared from soaking the crystals of fully oxidized form in the medium containing each ligands. Crystals of the fully reduced form and the fully reduced CO bound form was not able to be stab
… More
ilized under nitrogen atomosphere, because of the strong sensitivity to O2. However, it is impossible to fix a crystal in a capillary in the presence of mother lizuer. Thus, the crystals of the fully reduced form and of the fully reduced CO bound form are stabilized by sephadex gel equilibrated with the mother liquer containing ascorbate and cytochrome c as the reducing system and saturated with N2 and CO respectively. The oxidation and ligand binding states are checked by the absorption spectra of the crystals placed in the mother liquer without sephadex gel. The crystals of fully reduced form diffracts x-rays up to 2.2 A^^ﾟ resolution and the other derivative crystals diffract to better than 2.9 A^^ﾟ resolution. Cytochrome bcl complex stabilized with sucrose monocaplate provides crystals which diffract to 2.8 A^^ﾟ resolution. However this crystallization conditions are not so sensitive to the structure of detergents as in the case of bovine heart cytochrome c oxidase. Purity of deoxycholate which is for solubilizing the cytochrome bcl complex from mitochondrial inner membrane seems critical for the crystallization. Less
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