Kamatari Y O, Konno T, Kataoka M, Akasaka K
Graduate School of Science and Technology, Kobe University, Japan.
J Mol Biol. 1996 Jun 14;259(3):512-23. doi: 10.1006/jmbi.1996.0336.
Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering. At pH 3.0 and 25 degrees C, two methanol-induced non-native states were characterized. First, addition of methanol up to 25% (v/v) induced a compact denatured conformer (I(M)). Further addition of methanol transformed this I(M) state into the expanded and highly helical denatured state (H). The existence of the I(M) state was shown by the discrepancy in transition curves obtained from the ellipticity at 222 nm, the ellipticity at 282 nm, the tryptophan fluorescence monitored at 350 nm and the native peak intensity of the (1)H NMR spectrum. These CD, fluorescence and NMR results showed that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state. The radius of gyration of the I(M) state, 17.7 angstroms, obtained from the Guinier plot of the small-angle X-ray scattering data was significantly smaller than that of the acid-denatured state (30.1 angstroms) and was closer to that of the native state (14.6 angstroms), showing that the I(M) state is compact. The Kratky plot for the I(M) state exhibited a bell-shaped profile, indicating a globular conformation. These structural features indicate that the structure of the I(M) state is quite similar to that of the anion-induced molten globule state of this protein. Furthermore the alcohol-denatured state (H) of cyt C in 60% (v/v) methanol was structurally characterized. Though the H state had a helical content much higher than the native state monitored by far-UV CD spectroscopy, the radius of gyration, 31.7 angstroms, was similar to that of the acid-denatured state, showing that this H state is an expanded denatured state. The Kratky plot for the H state did not show a clear peak, indicating a chain-like conformation. Thus we conclude that the H state has an expanded and chain-like conformation with a high helical content. Finally, we constructed a phase diagram of cyt c involving the native, I(M), acid-denatured and H states against pH and the methanol concentration. The result indicates that the I(M) state is found in the pH range from 2.5 to at least 4.5 with a pH-dependent optimum methanol concentration of 10 to 40%.
通过结合使用远紫外和近紫外圆二色光谱、荧光光谱、核磁共振光谱以及小角X射线散射，研究了酸性pH值下甲醇诱导的细胞色素c（cyt c）构象转变。在pH 3.0和25℃条件下，鉴定出两种甲醇诱导的非天然状态。首先，加入高达25%（v/v）的甲醇会诱导形成一种紧密的变性构象（I(M)）。进一步添加甲醇会将这种I(M)状态转变为扩展的、高度螺旋化的变性状态（H）。从222 nm处的椭圆率、282 nm处的椭圆率、350 nm处监测的色氨酸荧光以及(1)H NMR谱的天然峰强度获得的转变曲线差异表明了I(M)状态的存在。这些圆二色光谱、荧光光谱和核磁共振结果表明，I(M)状态没有特定的三级结构，但具有与天然状态相似的二级结构含量和色氨酸环境。从小角X射线散射数据的吉尼埃图获得的I(M)状态的回转半径为17.7埃，明显小于酸变性状态的回转半径（30.1埃），且更接近天然状态的回转半径（14.6埃），表明I(M)状态是紧密的。I(M)状态的克拉特基图呈现钟形轮廓，表明其为球状构象。这些结构特征表明，I(M)状态的结构与该蛋白质的阴离子诱导熔融球状体状态的结构非常相似。此外，还对细胞色素C在60%（v/v）甲醇中的醇变性状态（H）进行了结构表征。尽管通过远紫外圆二色光谱监测发现H状态的螺旋含量远高于天然状态，但其回转半径为31.7埃，与酸变性状态的回转半径相似，表明这种H状态是一种扩展的变性状态。H状态的克拉特基图没有显示出明显的峰，表明其为链状构象。因此，我们得出结论，H状态具有扩展的链状构象且螺旋含量高。最后，我们构建了细胞色素c涉及天然状态、I(M)状态、酸变性状态和H状态与pH值和甲醇浓度的相图。结果表明，在pH值为2.5至至少4.5的范围内可发现I(M)状态，其最佳甲醇浓度与pH值相关，为10%至40%。
