Ferguson Neil, Day Ryan, Johnson Christopher M, Allen Mark D, Daggett Valerie, Fersht Alan R
MRC Centre for Protein Engineering and MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
J Mol Biol. 2005 Apr 8;347(4):855-70. doi: 10.1016/j.jmb.2004.12.061.
We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.
我们使用Phi值分析来表征嗜热蛋白在325 K相对较高温度下折叠的过渡态。通过在无化学变性剂的情况下进行温度跳跃实验，确定了嗜热脂肪芽孢杆菌三螺旋束外周亚基结合结构域（E3BD）折叠的PhiF值。E3BD在微秒内通过高度弥散的过渡态折叠。实验结果与在373 K下进行的八次（独立）分子动力学展开模拟结果之间观察到了极好的一致性。我们结合异核核磁共振实验和分子动力学模拟来表征变性集合体，发现其中几乎没有持久的残余结构。然而，通过模拟发现，那些在天然状态下形成螺旋结构的区域在变性状态下易于形成螺旋。这些区域在折叠的过渡态中也具有显著结构。整体折叠途径似乎是成核-凝聚。
