Huang F, Lerner E, Sato S, Amir D, Haas E, Fersht A R
MRC Center for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
Biochemistry. 2009 Apr 21;48(15):3468-76. doi: 10.1021/bi801890w.
The B domain of protein A (BDPA), a three-helix bundle of 60 residues, folds via a nucleation-condensation mechanism in apparent two-state kinetics. We have applied a time-resolved FRET (tr-FRET) approach to characterize the ensembles of BDPA during chemical denaturation. The distribution of the distance between residues 22 and 55, which are close and separated by helices 2 and 3 in the native state, was determined by global analysis of the time-resolved fluorescence decay curves of the probes. Narrow distributions were observed when the protein was equilibrated in guanidinium chloride (GdmCl) concentrations below 1.5 M (native state, N) and above the transition zone at 2.6-3.0 M GdmCl (denatured state, D). Considerably broader distributions were found around the transition point (2.0 M GdmCl) or much higher GdmCl concentrations (>3.0 M). Comparative global analysis of the tr-FRET data showed a compact denatured state of the protein, characterized by narrow distribution and relatively small mean distance between residues 22 and 55 that was observed at mild denaturing conditions (<3 M GdmCl). This experiment supports the two-state folding mechanism of BDPA and indicates the existence of effective nonlocal, probably hydrophobic, intramolecular interactions that stabilize a pretty uniform ensemble of compact denatured molecules at intermediate denaturing conditions.
蛋白A的B结构域（BDPA）由60个残基组成，呈三螺旋束状，通过成核凝聚机制折叠，呈现出明显的两态动力学。我们应用时间分辨荧光共振能量转移（tr-FRET）方法来表征化学变性过程中BDPA的集合体。通过对探针的时间分辨荧光衰减曲线进行全局分析，确定了天然状态下由螺旋2和螺旋3靠近并隔开的残基22和55之间的距离分布。当蛋白质在氯化胍（GdmCl）浓度低于1.5 M（天然状态，N）以及高于2.6 - 3.0 M GdmCl的转变区（变性状态，D）中达到平衡时，观察到窄分布。在转变点（2.0 M GdmCl）或更高的GdmCl浓度（>3.0 M）附近发现分布明显更宽。对tr-FRET数据的比较全局分析显示，在温和变性条件（<3 M GdmCl）下观察到蛋白质的紧密变性状态，其特征是分布窄且残基22和55之间的平均距离相对较小。该实验支持BDPA的两态折叠机制，并表明存在有效的非局部（可能是疏水的）分子内相互作用，这些相互作用在中等变性条件下稳定了紧密变性分子的相当均匀的集合体。
