Rodgers K K, Sligar S G
Department of Chemistry, University of Illinois, Urbana.
J Mol Biol. 1991 Oct 20;221(4):1453-60. doi: 10.1016/0022-2836(91)90945-3.
In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed. An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers. Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive. We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation. The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces. Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains. We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex. Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins. Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein. Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal. This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.
在电子传递蛋白的结合中，有人提出静电作用在维持所形成的生物分子复合物的稳定性和特异性方面发挥作用。一个出色的模型系统是哺乳动物细胞色素b5和细胞色素c之间的相互作用，其中单个组分的X射线结构揭示了围绕其各自氧化还原中心的电荷互补不对称性。然而，确定静电相互作用的确切程度以及识别参与电子传递复合物形成的特定残基却更具挑战性。我们在此报告利用高压技术结合定点诱变来绘制生物分子复合物形成过程中的相互作用域图谱。高压的应用会破坏大分子结合，因为复合物的解离会导致系统体积减小，这是由于先前隔离在界面区域的电荷发生溶剂化以及疏水表面被迫溶剂化。通过对大鼠肝细胞色素b5的全合成基因进行定点诱变，该基因在大肠杆菌中表达这种哺乳动物蛋白作为含血红素的可溶性成分，用于将细胞色素b5带负电荷的残基选择性地改变为中性酰胺侧链。我们已经证明，通过比较这些修饰的细胞色素b5 - 细胞色素c复合物与天然复合物的解离体积，可以绘制出细胞色素b5与细胞色素c的相互作用域。利用这些技术，我们可以具体研究特定残基在这两种电子传递蛋白平衡结合中的作用。相互作用域中的单点突变对测量的解离体积产生几乎相同的影响，但识别表面之外酸性残基的去除产生的体积与野生型蛋白相似。在所提出的蛋白质 - 蛋白质相互作用位点的多个突变被发现允许界面具有更大的溶剂可及性，这反映在后续电荷去除时体积变化的减小。这表明该复合物中的蛋白质间盐桥提供了一种机制，可使溶剂从复合物的界面区域被更大程度地排除，从而导致更稳定的结合。
