Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
J Phys Chem B. 2012 Sep 6;116(35):10631-8. doi: 10.1021/jp211296e. Epub 2012 Aug 27.
The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor-acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (Phe(CN)) would exhibit different donor fluorescence lifetimes. Our results provide strong evidence suggesting that the native free energy basin of HP35 is populated with conformations that differ mostly in the position and mean helicity of the C-terminal helix. This finding is consistent with several previous experimental and computational studies. Moreover, this result holds strong implications for computational investigation of the folding mechanism of HP35.
头部结构域（HP35）由于其体积小和超快的折叠动力学，已成为蛋白质折叠研究中应用最广泛的模型系统之一。在这里，我们使用 HP35 作为测试平台，表明通过荧光共振能量转移（FRET）机制，附近猝灭剂（如色氨酸或 7-氮杂色氨酸）对非天然氨基酸 p-氰基苯丙氨酸（Phe(CN)）的荧光衰减动力学的调制，可以用于检测蛋白质构象异质性。该方法基于这样一种观点，即与供体（Phe(CN)）的荧光寿命相比，具有不同供体-受体距离且缓慢相互转化的蛋白质构象将表现出不同的供体荧光寿命。我们的结果提供了有力的证据表明，HP35 的天然自由能盆地中存在的构象主要在 C 末端螺旋的位置和平均螺旋性上有所不同。这一发现与之前的一些实验和计算研究一致。此外，这一结果对 HP35 折叠机制的计算研究具有重要意义。
