Mizuguchi M, Arai M, Ke Y, Nitta K, Kuwajima K
Graduate School of Science, Hokkaido University, Sapporo, 060, Japan.
J Mol Biol. 1998;283(1):265-77. doi: 10.1006/jmbi.1998.2100.
The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of alpha-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.
通过远紫外和近紫外区域的圆二色光谱，研究了钙离子结合蛋白马肝溶菌酶的平衡去折叠以及去折叠和重折叠的动力学。在230nm和292.5nm处测量的盐酸胍诱导去折叠的转变曲线，以及该蛋白的脱辅基和全酶形式表明，去折叠过程可以很好地用三态机制来描述，其中熔融球状体状态作为一个稳定的中间体出现。该蛋白的熔融球状体状态比马肝溶菌酶的同源蛋白α-乳白蛋白的熔融球状体状态更稳定且更类似天然状态。通过变性剂浓度跃变诱导该蛋白的动力学去折叠和重折叠，并通过停流圆二色性进行测量。观察到的去折叠和重折叠曲线都与单指数函数吻合良好。然而，在低于3M盐酸胍的动力学重折叠反应中，圆二色性存在一个突发相变化，并且动力学重折叠中的突发相中间体被证明与平衡去折叠中观察到的熔融球状体状态相同。在强天然条件下，几乎所有马肝溶菌酶分子将结构从去折叠状态转变为熔融球状体，随后的重折叠从熔融球状体状态发生。通过研究盐酸胍浓度对重折叠和去折叠速率常数的依赖性，表征了可能存在于熔融球状体和天然状态之间的折叠过渡态。该蛋白超过80%的疏水表面在过渡态被掩埋，因此它比熔融球状体更接近天然状态，在熔融球状体中只有36%的表面被埋在分子内部。得出的结论是，目前所有的结果最好用蛋白质折叠的顺序模型来解释，其中熔融球状体状态是折叠途径上一个必不可少的折叠中间体。
