A Study of the Molecular Evolution and Structural Stability of Cytochrome c by using Its Interspecies Hybrids

由于蛋白质的分子进化，来自进化上遥远的生物体的同源蛋白质的氨基酸序列在很大程度上是不同的。例如，马和酵母细胞色素c（cyt c's）之间的序列差异约为45%。然而，同源蛋白质的空间结构惊人地相似。这种结构守恒可以通过两种替代机制产生。在第一种机制中，蛋白质中发生的每个氨基酸取代都不会引起结构变化。在第二种机制中，由氨基酸取代引起的结构变化被随后的取代所补偿。如果第二种机制实现了结构保守性，种间杂交蛋白可能是结构不稳定的。本研究采用半合成法制备了马-酵母杂交细胞色素c。在马和杂交细胞色素C之间观察到12个氨基酸序列差异。并将其光谱和理化性质与其母体分子进行了比较。杂化细胞色素c的光谱特征表明，杂化细胞色素c具有与母体分子相同的血红素配位结构。对杂交体及其亲本细胞色素c的盐酸胍变性曲线的分析表明，杂交体的稳定性介于亲本分子之间。此外，这些细胞色素c的咪唑结合常数的测量表明，封闭的血红素裂隙结构的混合细胞色素c的稳定性也是中间的父母分子。这些结果表明，在杂合细胞色素c中看到的12个氨基酸取代被容纳在它们的空间位置，而马细胞色素c的结构只有微小的扰动。此外，这些结果表明，这些氨基酸取代的累加改变的整体和局部结构的稳定性的细胞色素c。

The amino acid sequences of homologous proteins from evolutionarily distant organisms are different to large extents as a result of the molecular evolution of the proteins. For example, difference of ca.45% in sequence is seen between horse and yeast cytochromes c (cyt c's). The spatial structures of homologous proteins are, however, strikingly similar. This structural conservation can be produced by two alternative mechanisms. In the first mechanism, no structural change is caused by each of the amino acid substitutions that occur in the proteins. In the second mechanism, structural changes induced by amino acid substitutions are compensated by substitutions that follow them. Interspecies hybrid proteins can be structurally unstable if the second mechanism realizes the structural conservation. In the present study, horse-yeast hybrid cyt c was prepared by semisynthesis. 12 amino acid sequence differences are seen between horse and the hybrid cyt c's. The spectroscopic and physicochemical properties of the hybrid were compared with those of its parent molecules. The spectral features of the hybrid cyt c indicated that the hybrid cyt c has the same heme coordination structure as those of the parent molecules. Analysis of the guanidine hydrochloride denaturation curves of the hybrid and its parent cyt c's showed that the stability of the hybrid is intermediate between those of the parent molecules. In addition, the measurement of the imidazole binding constants of these cyt c's revealed that the stability of the closed heme crevice structure of the hybrid cyt c is also intermediate between those of the parent molecules. These results indicate that the 12 amino acid substitutions seen in the hybrid cyt c are accommodated at their spatial positions with only minor perturbation of the structure of horse cyt c. Furthermore, the results suggest that these amino acid substitutions change additively the stabilities of the global and local structures of cyt c.