Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Biophys J. 2018 Feb 6;114(3):592-601. doi: 10.1016/j.bpj.2017.12.016.
Nitroxide- and Cu-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important α-helix of the human glutathione S-transferase A1. Distance measurements on various spin-labeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu-based ESR distance measurements. Cu-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.
氮氧自由基和铜基电子自旋共振（ESR）相结合，深入了解了人谷胱甘肽 S-转移酶 A1 中功能重要的α-螺旋的构象状态。对各种标记的二聚体人谷胱甘肽 S-转移酶 A1-1 的距离测量均导致双峰距离分布，表明 C 末端在溶液中存在两种不同的构象，其中一种与配体结合酶的晶体结构中发现的构象非常匹配。这些测量允许生成未配体构象的模型。室温 ESR 表明，第二种构象具有高迁移率，可能使酶具有高度的底物混杂性。然后使用计算建模和进一步的 Cu 基 ESR 距离测量来验证该模型。Cu 基 ESR 还提供了证据表明，第二种构象的二级结构具有螺旋性质。添加 S-己基谷胱甘肽会导致相对丰度的变化，有利于类似于先前已知的配体结合酶结构的状态。
