Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies
Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies
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DOI：
10.1002/pro.5560071217
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发表时间：
1998-12-01
期刊：
影响因子：
8
通讯作者：
Tsou, CL
中科院分区：
文献类型：
--
作者：
Fan, YX;Zhou, JM;Tsou, CL
盐酸胍诱导的二聚体细胞质肌酸激酶的展开-重折叠的平衡和动力学研究已监测的内源性荧光，远紫外圆二色性，和1-苯胺基萘-8-磺酸结合。盐酸胍诱导的平衡展开曲线显示两个转变，表明在中等浓度的盐酸胍溶液中存在至少一个稳定的平衡中间体。该中间体是所有硫醇基团都暴露的无活性单体。通过使用三态模型分析获得的热力学参数表明，该中间体在能量上与完全未折叠状态相似。由于在停流装置中的混合死时间（15 ms）内形成中间体，在重折叠动力学中存在爆发相。在爆发相之后进一步重折叠至天然状态遵循双相动力学。研究并比较了猝发相和平衡中间体的性质。结果表明，这些中间体在某些方面是相似的，但在其他方面是不同的。两者的特征在于明显的二级结构，紧凑的球形，暴露的疏水表面积，和刚性侧链包装的情况下，类似于“熔融球”的状态。然而，爆发相中间体显示出更多的二级结构，更多的暴露的疏水表面积，和更灵活的侧链包装比平衡中间体。在爆发阶段之后，存在对应于单体折叠成紧凑构象的快速阶段。然后快速组装形成二聚体。这两种平衡解折叠转变都不依赖于蛋白质浓度。重折叠动力学也不依赖于浓度。这表明，协会的亚基是不限速的重折叠，并在平衡条件下，解离发生在该地区之间的两个展开过渡。基于上述结果，提出了肌酸激酶去折叠和复性的方案。
Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.