Preparation of crystals of cytochrome c oxidase and cytochrome bcl complex

细胞色素c氧化酶和细胞色素bcl复合物晶体的制备

• 批准号: 06453220
• 负责人: YOSHIKAWA Shinya
• 金额: $ 4.54万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06453220/
• 关键词: 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

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

洗涤剂结构对牛心细胞色素c氧化酶结晶的影响已被广泛研究。癸基麦芽糖苷提供的晶体可使X射线的分辨率达到2.2埃或更高，尽管重现性相当低。洗涤剂结构的轻微改变会显著影响结晶条件。例如，壬基和十一烷基麦芽糖苷都不能提供稳定的晶体。癸基麦芽糖苷中糖部分的结构对于结晶是关键的。实际上，除了麦芽糖以外，没有糖能提供牛心细胞色素氧化酶的微晶。已经建立了用癸基麦芽糖苷稳定的这种酶的各种衍生物的结晶条件。通过将完全氧化形式的晶体浸泡在含有每种配体的介质中来制备叠氮化物结合形式以及氰化物结合形式的晶体。完全还原形式和完全还原CO结合形式的晶体不能被刺穿。 ...更多信息 由于对O2的敏感性较强，在氮气气氛下进行了硅烷化处理。然而，在存在母液的情况下，不可能将晶体固定在毛细管中。因此，完全还原形式的晶体和完全还原的CO结合形式的晶体通过用含有抗坏血酸盐和细胞色素c作为还原系统的母液平衡并分别用N2和CO饱和的葡聚糖凝胶来稳定。通过放置在没有葡聚糖凝胶的母液中的晶体的吸收光谱检查氧化和配体结合状态。完全还原形式的晶体衍射X射线的分辨率高达2.2埃^^，而其他衍生晶体衍射X射线的分辨率优于2.9埃^^。细胞色素bcl复合物用蔗糖单壳层稳定后得到的晶体分辨率可达2.8埃。然而，这种结晶条件不像牛心细胞色素c氧化酶那样对洗涤剂的结构敏感。从线粒体内膜溶解细胞色素bcl复合物的脱氧胆酸盐的纯度似乎是结晶的关键。少

项目成果

T.Tsukihara: "The whole structure of the 13 subunit oxidized bovine heart cytochrome c oxidase at 2.8Å." Science. 272. 1136-1144 (1996)

T.Tsukihara：“氧化牛心脏细胞色素 c 氧化酶的 13 个亚基的整体结构，2.8Å。《科学》272. 1136-1144 (1996)。

S.Hirota, T.Ogura, K.Shinzawa-Itoh, S.Yoshikawa, and T.Kitagawa: "Observation of multiple CN-isotope-sensitive Raman bands for CN-adducts of hemoglobin, myoglobin, and cytochrome c oxidase : evidence for vibrational coupling berween the Fe-C-N bending and

S.Hirota、T.Ogura、K.Shinzawa-Itoh、S.Yoshikawa 和 T.Kitakawa：“血红蛋白、肌红蛋白和细胞色素 c 氧化酶的 CN 加合物的多个 CN 同位素敏感拉曼谱带的观察：证据

M.Kawamoto: "New crystal forms and preliminary X-ray deffraction studies of mitochondrial cytochrome bc_1 complex from bovine heart" J.Mol.Biol.244. 238-241 (1994)

M.Kawamoto：“牛心脏线粒体细胞色素 bc_1 复合物的新晶体形式和初步 X 射线衍射研究”J.Mol.Biol.244。

T.Tsukihara, H.Aoyama, E.Yamashita, T.Tomizaki, H.Yamaguchi, K.Shinzawa-Itoh, R.Nakashima, R.Yaono and S.Yoshikawa: "Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8A^^ﾟ" Science. 269. 1069-1074 (1995)

T.Tsukihara、H.Aoyama、E.Yamashita、T.Tomizaki、H.Yamaguchi、K.Shinzawa-Itoh、R.Nakashima、R.Yaono 和 S.Yoshikawa：“氧化牛心细胞色素 c 氧化酶的金属位点结构2.8A^^ﾟ" 科学。269. 1069-1074 (1995)

T.Tsukihara, K.Aoyama, E.Yamashita, T.Tomizaki, H.Yamaguchi, K.Shinzawa-Itoh, R.Nakashima, R.Yaono and S.Yoshikawa: "The whole structure of the 13 subunit oxidized bovine heart cytochrome c oxidase at 2.8A^^ﾟ" Science. 272. 1136-1144 (1966)

T. Tsukihara、K. Aoyama、E. Yamashita、T. Tomizaki、H. Yamaguchi、K. Shinzawa-Itoh、R. Nakashima、R. Yaono 和 S. Yoshikawa：“13 个亚基氧化牛心细胞色素的整体结构c 氧化酶在 2.8A^^ﾟ" Science. 272. 1136-1144 (1966)