Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
J Biochem. 2012 Dec;152(6):521-9. doi: 10.1093/jb/mvs098. Epub 2012 Aug 25.
We have previously shown that polymerization of cytochrome c (cyt c) occurs by successively domain swapping its C-terminal α-helix in the presence of ethanol. However, the factors that govern the conversion process of monomers to domain-swapped oligomers remain unknown. We found that oligomeric cyt c is produced in the presence of ethanol and the oligomers precipitate due to low solubility. The optical absorption spectra revealed that in the presence of 30-40% ethanol, the Met-heme coordination in cyt c is dissociated. However, according to circular dichroism and small-angle X-ray scattering measurements, the α-helical structure of cyt c is maintained in solution with a little perturbation and the radius of gyration increases slightly but without dissociation of the C-terminal α-helix from the rest of the protein by the addition of ethanol. Solid-state nuclear magnetic resonance spectra showed that oligomeric cyt c in the precipitate also retains most of its α-helical structure. In the transmission electron microscopic image of the precipitate obtained by the addition of ethanol to cyt c, spherical particles with diameters of about 3 nm were detected. These results indicate that oligomeric cyt c forms through a state with the Met80 region locally unfolded, while maintaining the secondary structure, possibly an open monomer.
我们之前已经表明，在乙醇存在的情况下，细胞色素 c（cyt c）通过依次交换其 C 末端α-螺旋来聚合。然而，控制单体转化为域-swapped 寡聚物的因素尚不清楚。我们发现，在乙醇存在的情况下会产生寡聚细胞色素 c，并且由于低溶解度，寡聚体会沉淀。光吸收光谱表明，在存在 30-40%乙醇的情况下，细胞色素 c 中的 Met-血红素配位被解离。然而，根据圆二色性和小角 X 射线散射测量，细胞色素 c 的α-螺旋结构在溶液中保持不变，只有轻微的扰动，并且随着乙醇的加入，回转半径略有增加，但 C 末端α-螺旋并未从蛋白质的其余部分解离。固态核磁共振光谱表明，沉淀中的寡聚细胞色素 c 也保留了其大部分α-螺旋结构。在向细胞色素 c 中添加乙醇后获得的沉淀的透射电子显微镜图像中，检测到直径约为 3nm 的球形颗粒。这些结果表明，寡聚细胞色素 c 通过局部展开 Met80 区域的状态形成，同时保持二级结构，可能是开放的单体。
