Kim Yun Ho, Stites Wesley E
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701-1021, USA.
Biochemistry. 2008 Aug 19;47(33):8804-14. doi: 10.1021/bi800297j. Epub 2008 Jul 26.
To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.
为了探究分子拥挤和排阻体积对蛋白质稳定性的影响，我们使用了一系列交联剂与葡萄球菌核酸酶的九个不同单半胱氨酸突变体来制备共价连接的二聚体。这些交联剂的长度从10.5埃到21.3埃不等，这种间距通常只会在最浓缩的蛋白质溶液中出现。通过盐酸胍和热变性来测定二聚体蛋白质和单体对照的稳定性。与单体对照的两态行为不同，具有短连接子的二聚体倾向于表现出明显的三态变性行为。连接子长度增加会导致三态行为不那么明显。三态行为在一个三态模型中得到解释，其中交联的天然蛋白质二聚体N-N在一个两态转变中与一个其中一个蛋白质亚基变性的二聚体N-D相互转化。剩余的天然蛋白质进而可以在另一个两态转变中变性为两个相连蛋白质都变性的状态D-D。三态行为最好用变性状态下的排阻体积效应来解释。对于许多二聚体来说，连接子长度超过17埃会消除大部分三态特征。这为变性状态的灵活性和大小设定了一个限制。值得注意的是，与理论预测相反，这些交联二聚体并没有得到稳定。这些预测的失败可能是由于忽略了伴随变性状态构象空间任何显著减少而发生的疏水暴露变化。
