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

进化距离较远的生物的同源蛋白的氨基酸序列在很大程度上是不同的，这是蛋白质分子进化的结果。例如，马和酵母的细胞色素c （cyt c’s）序列差异约为0.45%。然而，同源蛋白的空间结构却惊人地相似。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，蛋白质中发生的每一次氨基酸替换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守恒，则种间杂交蛋白在结构上是不稳定的。本研究采用半合成法制备了马酵母杂交细胞c。马体细胞和杂交体细胞之间有12个氨基酸序列的差异。并与亲本分子进行了光谱和理化性质的比较。杂种cyt - c的光谱特征表明，杂种cyt - c具有与亲本分子相同的血红素配位结构。杂种分子与亲本cyt - c的盐酸胍变性曲线分析表明，杂种分子的稳定性介于亲本分子之间。此外，对这些cyt - c的咪唑结合常数的测量表明，杂交cyt - c的闭合血红素缝隙结构的稳定性也介于亲本分子之间。这些结果表明，在杂交cyt c中发现的12个氨基酸取代被安置在它们的空间位置上，只对马cyt c的结构产生了很小的扰动。此外，这些氨基酸取代加在一起改变了cyt c的整体和局部结构的稳定性。

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.