Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, México DF, Mexico.
PLoS One. 2011 Apr 18;6(4):e18791. doi: 10.1371/journal.pone.0018791.
For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.
为了更好地理解蛋白质的结构-功能关系，有必要确定与可测量蛋白质功能相关的氨基酸。由于氨基酸在蛋白质内建立了众多的接触点，并且它们的相互作用具有协同性，因此很难实现这一目标。因此，蛋白质-配体相互作用的研究通常集中在局部环境结构差异上。在这里，我们使用来自两个不同生物体的一对具有极高同源性的磷酸丙糖异构酶酶，证明了界面半胱氨酸反应性的 70 倍差异的控制位于几个氨基酸上，这些氨基酸与对半胱氨酸敏感的巯基试剂甲硫氨酸磺酸甲酯不接触，也不与紧邻的残基接触。反应性的变化是由于突变残基导致界面半胱氨酸的表观 pKa 增加所致。我们的工作涉及系统地将一个蛋白质的部分片段嫁接到另一个蛋白质上，揭示了意想不到的和多部位的远程相互作用，这些相互作用调节了界面半胱氨酸的性质，并对未来的蛋白质结构-功能关系研究具有普遍意义。
