Watkins Herschel M, Simon Anna J, Sosnick Tobin R, Lipman Everett A, Hjelm Rex P, Plaxco Kevin W
Interdepartmental Program in Biomolecular Science and Engineering and.
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; and.
Proc Natl Acad Sci U S A. 2015 May 26;112(21):6631-6. doi: 10.1073/pnas.1418673112. Epub 2015 May 11.
Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.
小角散射研究通常表明，未折叠的单结构域蛋白的尺寸与变性剂浓度无关（在百分之几的实验不确定性范围内）。相比之下，单分子荧光共振能量转移（smFRET）研究总是表明，当变性剂浓度从高（约6 M）降至低（约1 M）时，蛋白质的未折叠状态会显著收缩。在这里，我们通过使用聚乙二醇（PEG）进行一个此前未有的阴性对照来探究这种差异。这种不带电荷、高度亲水的聚合物已通过多种独立技术证明在水中表现为无规卷曲，这表明在添加变性剂时它不太可能进一步膨胀。与此观察结果一致，小角中子散射表明，盐酸胍或尿素的存在不会显著改变PEG的尺寸。然而，对用最常用的荧光共振能量转移染料对修饰的PEG构建体进行的smFRET测量，产生了与许多未折叠蛋白类似的、依赖于变性剂的转移效率变化。鉴于PEG和未折叠蛋白的化学性质差异巨大，且有大量证据表明不含染料的PEG可以很好地描述为与变性剂无关的无规卷曲，这种相似性引发了关于在低变性剂条件下根据氢键或疏水驱动的未折叠状态收缩来解释smFRET数据的问题。
