Stevens J M, Hornby J A, Armstrong R N, Dirr H W
Protein Structure-Function Research Programme, Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa.
Biochemistry. 1998 Nov 3;37(44):15534-41. doi: 10.1021/bi981044b.
Solvent-induced equilibrium unfolding of a homodimeric class sigma glutathione transferase (GSTS1-1, EC 2.5.1.18) was characterized by tryptophan fluorescence, anisotropy, enzyme activity, 8-anilino-1-naphthalenesulfonate (ANS) binding, and circular dichroism. Urea induces a triphasic unfolding transition with evidence for two well-populated thermodynamically stable intermediate states of GSTS1-1. The first unfolding transition is protein concentration independent and involves a change in the subunit tertiary structure yielding a partially active dimeric intermediate (i.e., N2 left and right arrow I2). This is followed by a protein concentration dependent step in which I2 dissociates into compact inactive monomers (M) displaying enhanced hydrophobicity. The third unfolding transition, which is protein concentration independent, involves the complete unfolding of the monomeric state. Increasing NaCl concentrations destabilize N2 and appear to shift the equilibrium toward I2 whereas the stability of the monomeric intermediate M is enhanced. The binding of substrate or product analogue (i.e., glutathione or S-hexylglutathione) to the protein's active site stabilizes the native dimeric state (N2), causing the first two unfolding transitions to shift toward higher urea concentrations. The stability of M was not affected. The data implicate a region at/near the active site in domain I (most likely alpha-helix 2) as being highly unstable/flexible which undergoes local unfolding, resulting initially in I2 formation followed by a disruption in quaternary structure to a monomeric intermediate. The unfolding/refolding pathway is compared with those observed for other cytosolic GSTs and discussed in light of the different structural features at the subunit interfaces, as well as the evolutionary selection of this GST as a lens crystallin.
通过色氨酸荧光、各向异性、酶活性、8-苯胺基-1-萘磺酸盐（ANS）结合以及圆二色性对溶剂诱导的同二聚体σ类谷胱甘肽转移酶（GSTS1-1，EC 2.5.1.18）的平衡去折叠进行了表征。尿素诱导了一个三相去折叠转变，有证据表明GSTS1-1存在两个丰度较高的热力学稳定中间态。第一个去折叠转变与蛋白质浓度无关，涉及亚基三级结构的变化，产生一个部分活性的二聚体中间体（即，N2⇄I2）。接着是一个与蛋白质浓度相关的步骤，其中I2解离成紧密的无活性单体（M），显示出增强的疏水性。第三个去折叠转变与蛋白质浓度无关，涉及单体状态的完全去折叠。增加NaCl浓度会使N2不稳定，并似乎使平衡向I2移动，而单体中间体M的稳定性增强。底物或产物类似物（即谷胱甘肽或S-己基谷胱甘肽）与蛋白质活性位点的结合稳定了天然二聚体状态（N2），导致前两个去折叠转变向更高的尿素浓度移动。M的稳定性不受影响。数据表明结构域I中活性位点处/附近的一个区域（很可能是α-螺旋2）高度不稳定/灵活，会发生局部去折叠，最初导致I2形成，随后四级结构破坏为单体中间体。将该去折叠/重折叠途径与其他胞质谷胱甘肽转移酶观察到的途径进行了比较，并根据亚基界面处的不同结构特征以及该谷胱甘肽转移酶作为晶状体蛋白的进化选择进行了讨论。
