Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain
Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain
复制标题
DOI：
10.1073/pnas.0505432102
复制
发表时间：
2005-11-15
影响因子：
11.1
通讯作者：
Dyer, RB
中科院分区：
文献类型：
--
作者：
Brewer, SH;Vu, DM;Dyer, RB
采用平衡傅里叶变换红外光谱（FTIR）和温度跃变红外光谱（T-jump IR）技术研究了小分子三螺旋蛋白质--绒毛头部螺旋亚结构域（HP 36）的去折叠热力学和动力学。还研究了使该蛋白的疏水核心不稳定的双苯丙氨酸突变体（HP 36 F47 L，F51 L）。双突变体比野生型（WT）稳定性差，并且已显示在其未折叠状态下含有较少的残留二级结构和三级接触。研究了HP 36和HP 36 F47 L、F51 L在T跃扰动后的弛豫动力学。这两种蛋白质表现出双相松弛动力学响应T-跳跃。发现WT（60.2 ℃时为3.23 μ s）和双苯丙氨酸突变体（49.9 ℃时为3.01 μ s）在其热去折叠转变的近似中点处的折叠时间是相似的。WT的折叠时间在49.9 ℃下测定为3.34 μ s，与双苯丙氨酸突变体在该温度下的折叠时间相似。然而，双苯丙氨酸突变体在49.9 ℃下展开更快，展开时间为3.01 μ s，而WT为6.97 μ s。
Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 mu s at 60.2 degrees C) and double phenylalanine mutant (3.01 mu s at 49.9 degrees C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 mu s at 49.9 degrees C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 mu s compared with 6.97 mu s for the WT at 49.9 degrees C.