Crane J C, Koepf E K, Kelly J W, Gruebele M
School of Chemical Sciences and Beckman Institute for Advanced Science and Technology, Urbana, IL 61801, USA.
J Mol Biol. 2000 Apr 28;298(2):283-92. doi: 10.1006/jmbi.2000.3665.
The folding kinetics of a three-stranded antiparallel beta-sheet (WW domain) have been measured by temperature jump relaxation. Folding and activation free energies were determined as a function of temperature for both the wild-type and the mutant domain, W39F, which modifies the beta(2)-beta(3) hydrophobic interface. The folding rate decreases at higher temperatures as a result of the increase in the activation free energy for folding. Phi-Values were obtained for thermal perturbations allowing the primary features of the folding free energy surface to be determined. The results of this analysis indicate a significant shift from an "early" (Phi(T)=0. 4) to a "late" (Phi(T)=0.8) transition state with increasing temperature. The temperature-dependent Phi-value analysis of the wild-type WW domain and of its more stable W39F hydrophobic cluster mutant reveals little participation of residue 39 in the transition state at lower temperature. As the temperature is raised, hydrophobic interactions at the beta(2)-beta(3) interface gain importance in the transition state and the barrier height of the wild-type, which contains the larger tryptophan residue, increases more slowly than the barrier height of the mutant.
通过温度跳跃弛豫测量了三链反平行β折叠（WW结构域）的折叠动力学。测定了野生型和突变结构域W39F（其修饰了β(2)-β(3)疏水界面）的折叠自由能和活化自由能随温度的变化。由于折叠活化自由能的增加，折叠速率在较高温度下降低。通过对热扰动获得的Phi值，可以确定折叠自由能表面的主要特征。该分析结果表明，随着温度升高，从“早期”（Phi(T)=0.4）到“晚期”（Phi(T)=0.8）过渡态发生了显著转变。对野生型WW结构域及其更稳定的W39F疏水簇突变体进行的温度依赖性Phi值分析表明，在较低温度下，39位残基在过渡态中的参与度较低。随着温度升高，β(2)-β(3)界面处的疏水相互作用在过渡态中变得更加重要，并且含有较大色氨酸残基的野生型的势垒高度比突变体的势垒高度增加得更慢。
