Lo T P, Guillemette J G, Louie G V, Smith M, Brayer G D
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Biochemistry. 1995 Jan 10;34(1):163-71. doi: 10.1021/bi00001a020.
A combination of structural, functional, and mutagenic experiments has been used to study the roles of the invariant Phe82 and highly conserved Leu85 residues in cytochrome c, especially with respect to the complexation interface with electron transfer partners and maintenance of the hydrophobic heme pocket. Structural analyses show that the F82Y, L85A, and F82Y/L85A mutant proteins all retain the characteristic cytochrome c fold, but that conformational alterations are introduced in the direct vicinity of the mutation sites. In particular, the additional hydroxyl group of Tyr82 is in direct spatial conflict with the side chain of Leu85 in the F82Y mutant protein, leading to rotation of the side chain of Tyr82 out toward the protein surface. This strain is relieved in the F82Y/L85A mutant protein where the phenyl ring of Tyr82 is accommodated in a conformation comparable to that of the phenylalanine normally present at this location. In addition, the available space vacated by the replacement of Leu85 with an alanine allows for the inclusion of two new internal water molecules, one of which is bound to Tyr82 and the other to Arg13. In contrast, in the L85A mutant protein, no internal water molecules are observed in this exclusively hydrophobic pocket, which is partially filled by shifts in nearby side chains. Overall, the conformational changes observed result from the optimization of side chain packing to reflect the spatial requirements of new side chains, the minimization of both vacant internal space and the solvent exposure of hydrophobic groups, and the attainment of maximal hydrogen bonding between available polar groups.(ABSTRACT TRUNCATED AT 250 WORDS)
结构、功能和诱变实验相结合，用于研究细胞色素c中不变的苯丙氨酸82（Phe82）和高度保守的亮氨酸85（Leu85）残基的作用，特别是关于与电子传递伙伴的络合界面以及疏水血红素口袋的维持。结构分析表明，F82Y、L85A和F82Y/L85A突变蛋白均保留了细胞色素c的特征折叠，但在突变位点的直接附近引入了构象改变。特别是，F82Y突变蛋白中酪氨酸82（Tyr82）的额外羟基与亮氨酸85的侧链存在直接空间冲突，导致Tyr82的侧链向蛋白质表面旋转。在F82Y/L85A突变蛋白中这种张力得到缓解，其中Tyr82的苯环以与通常位于该位置的苯丙氨酸相当的构象容纳。此外，用丙氨酸取代Leu85腾出的可用空间允许包含两个新的内部水分子，其中一个与Tyr82结合，另一个与精氨酸13（Arg13）结合。相比之下，在L85A突变蛋白中，在这个完全疏水的口袋中未观察到内部水分子，该口袋部分被附近侧链的移动填充。总体而言，观察到的构象变化源于侧链堆积的优化，以反映新侧链的空间需求，使内部空置空间和疏水基团的溶剂暴露最小化，并在可用极性基团之间实现最大程度的氢键结合。（摘要截断于250字）
