Song Jianhui, Gomes Gregory-Neal, Shi Tongfei, Gradinaru Claudiu C, Chan Hue Sun
School of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao, Shandong, China; Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada; Department of Physics, University of Toronto, Toronto, Ontario, Canada.
Biophys J. 2017 Sep 5;113(5):1012-1024. doi: 10.1016/j.bpj.2017.07.023.
A mathematico-physically valid formulation is required to infer properties of disordered protein conformations from single-molecule Förster resonance energy transfer (smFRET). Conformational dimensions inferred by conventional approaches that presume a homogeneous conformational ensemble can be unphysical. When all possible-heterogeneous as well as homogeneous-conformational distributions are taken into account without prejudgment, a single value of average transfer efficiency 〈E〉 between dyes at two chain ends is generally consistent with highly diverse, multiple values of the average radius of gyration 〈R〉. Here we utilize unbiased conformational statistics from a coarse-grained explicit-chain model to establish a general logical framework to quantify this fundamental ambiguity in smFRET inference. As an application, we address the long-standing controversy regarding the denaturant dependence of 〈R〉 of unfolded proteins, focusing on Protein L as an example. Conventional smFRET inference concluded that 〈R〉 of unfolded Protein L is highly sensitive to [GuHCl], but data from SAXS suggested a near-constant 〈R〉 irrespective of [GuHCl]. Strikingly, our analysis indicates that although the reported 〈E〉 values for Protein L at [GuHCl] = 1 and 7 M are very different at 0.75 and 0.45, respectively, the Bayesian R distributions consistent with these two 〈E〉 values overlap by as much as 75%. Our findings suggest, in general, that the smFRET-SAXS discrepancy regarding unfolded protein dimensions likely arise from highly heterogeneous conformational ensembles at low or zero denaturant, and that additional experimental probes are needed to ascertain the nature of this heterogeneity.
需要一个数学物理上有效的公式来从单分子Förster共振能量转移（smFRET）推断无序蛋白质构象的性质。通过假定均匀构象集合的传统方法推断出的构象维度可能不符合实际情况。在不预先判断的情况下考虑所有可能的异构以及均匀构象分布时，两个链端染料之间的平均转移效率〈E〉的单个值通常与高度多样的多个回转半径平均值〈R〉一致。在这里，我们利用来自粗粒度显式链模型的无偏构象统计来建立一个通用逻辑框架，以量化smFRET推断中的这种基本模糊性。作为一个应用，我们以蛋白L为例，解决了关于未折叠蛋白〈R〉的变性剂依赖性的长期争议。传统的smFRET推断得出未折叠的蛋白L的〈R〉对[盐酸胍（GuHCl）]高度敏感，但小角X射线散射（SAXS）的数据表明，无论[GuHCl]如何，〈R〉几乎恒定。令人惊讶的是，我们的分析表明，尽管报道的在[GuHCl] = 1和7 M时蛋白L的〈E〉值分别为0.75和0.45，差异很大，但与这两个〈E〉值一致的贝叶斯R分布重叠高达75%。我们的研究结果总体表明，关于未折叠蛋白尺寸的smFRET-SAXS差异可能源于低或零变性剂下高度异构的构象集合，并且需要额外的实验探针来确定这种异质性的性质。
