Qin W, Sanishvili R, Plotkin B, Schejter A, Margoliash E
Department of Biological Sciences, University of Illinois at Chicago 60607, USA.
Biochim Biophys Acta. 1995 Sep 27;1252(1):87-94. doi: 10.1016/0167-4838(95)00124-d.
Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild-type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur-iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about.
通过定点诱变对大鼠细胞色素c中两个并非血红素铁配体的组氨酸对蛋白质稳定性的重要性进行了比较研究。将组氨酸26替换为缬氨酸，并测量了由此对Met-80硫与血红素铁键稳定性随pH和温度变化的影响，以及蛋白质在尿素溶液中展开时整体稳定性的影响。结果表明，His-26咪唑与Asn-3l主链酰胺之间氢键的丧失导致了观察到的局部稳定性下降；此外，该咪唑与Pro-44羰基之间氢键消除导致蛋白质右侧下部环（残基41-47）的流动性增加及其与中间环（残基26-31）距离增大，这可能导致分子内部右侧水化程度更高。这些变化导致蛋白质整体稳定性下降。将Asn-52进一步突变为Ile导致硫铁键野生型稳定性完全恢复，蛋白质整体稳定性部分恢复。用苯丙氨酸取代His-33不会改变硫铁键，但会导致蛋白质整体稳定性显著增加。据推测，这种效应是由于Phe-33侧链与蛋白质右侧下部环的疏水相互作用所致。这种相互作用也解释了以下观察结果：相同的突变逆转了由Val取代His-26导致的整体稳定性丧失，但没有恢复该突变所导致的硫铁键强度。
