Taisho Pharmaceutical Co., Ltd., Yoshino-cho, Kita-ku, Saitama 331-9530, Japan.
J Chem Phys. 2011 Jan 14;134(2):025101. doi: 10.1063/1.3519814.
We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25 °C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c(551), PH c(552), HT c(552), and AA c(555)) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c(555). However, AA c(555) has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c(555) is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c(555) has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25 °C the α-helix content of the apo state of AA c(555) is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c(551), PH c(552), and HT c(552)).
在 25°C 下折叠时的水熵增益除以残基数，该方法使用角度相关积分方程理论与多极水分子模型和形态测量方法的混合体计算。具有较大测量值的蛋白质在热稳定性方面更优越。在这里，我们将研究扩展到分析血红素对四种细胞色素 c（PA c(551)、PH c(552)、HT c(552)和 AA c(555)）的热稳定性的影响，尽管它们具有显著的高序列同源性和相似的三维折叠，但它们的变性温度却有很大的差异。主要结论如下。对于所有四种细胞色素 c，血红素结合在很大程度上增强了热稳定性，就水熵而言。对于全酶状态，AA c(555)的测量值最大。然而，AA c(555)的血红素结合效率最低，具有全酶样结构的脱辅基多肽，这不利于水熵。AA c(555)的最高稳定性主要归因于脱辅基多肽本身侧链包装效率最高。对于所有四种细胞色素 c，我们认为由于共价连接的血红素，变性状态的可及构象数量由于血红素的空间位阻而减少，折叠时的构象熵损失变小，导致热稳定性增强。对于模拟去除血红素的天然结构的脱辅基状态，AA c(555)的测量值比其他三种都大得多。总体而言，理论结果与实验观察结果非常一致（例如，在 25°C 时，AA c(555)的脱辅基状态的α-螺旋含量几乎等于全酶状态，而 PA c(551)、PH c(552)和 HT c(552)的脱辅基状态中几乎所有的螺旋都塌陷）。
