Gokhale R S, Ray S S, Balaram H, Balaram P
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Biochemistry. 1999 Jan 5;38(1):423-31. doi: 10.1021/bi981087s.
The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.
利用圆二色性、荧光和尺寸排阻色谱法，研究了恶性疟原虫磷酸丙糖异构酶（TIMWT）在尿素和氯化胍（GdmCl）溶液中的构象稳定性。该二聚体酶在尿素溶液中非常稳定。即使在8M尿素中，它仍保留相当多的二级、三级和四级结构。相比之下，在2.4M GdmCl时解折叠转变完成。虽然二级和三级相互作用在四级结构受到扰动之前就已消失，但这些研究表明，二聚体解离成单体最终会导致结构坍塌，这表明界面相互作用在决定多聚体蛋白质稳定性方面起主要作用。与其他单体和二聚体蛋白质相比，磷酸丙糖异构酶的Cm(尿素)/Cm(GdmCl)比值（其中Cm是转变中点所需变性剂的浓度）异常高。一种经工程改造在界面处形成两个二硫键交联（13-74'）和（13'-74）的二硫键交联突变蛋白（Y74C）在尿素中显著不稳定。在6M尿素时解折叠转变完成，涉及通过分子内硫醇-二硫键交换实现二聚体解离的新机制。
