Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.
Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.
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DOI：
10.1021/jacs.6b05917
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发表时间：
2016-09-14
影响因子：
15
通讯作者：
Schuler B
中科院分区：
文献类型：
--
作者：
Borgia A;Zheng W;Buholzer K;Borgia MB;Schüler A;Hofmann H;Soranno A;Nettels D;Gast K;Grishaev A;Best RB;Schuler B
关于化学变性剂对未折叠和内在无序蛋白质尺寸的影响一直存在争议：广泛的实验技术表明，多肽链随着变性剂浓度的增加而扩展，但使用小角X射线散射（SAXS）的几项研究报告没有这样的回转半径（Rg）增加。这种不一致性挑战了我们目前对化学变性剂机制的理解，化学变性剂被广泛用于研究蛋白质折叠和稳定性。在这里，我们使用单分子Förster共振能量转移（FRET），SAXS，动态光散射（DLS）和双焦点荧光相关光谱（2f-FCS）的组合来表征在两种不同的变性剂中的spectrin结构域R17和内在无序蛋白ACTR的未折叠状态的变性剂依赖性。对原始数据的标准分析清楚地表明，无论使用何种蛋白质、变性剂或实验方法，随着变性剂浓度的增加，未折叠状态都会扩大。这是SAXS和FRET在应用于相同蛋白质时产生关于变性剂扩增的甚至定性一致的结果的第一种情况。为了更直接地说明这种自洽性，我们已经使用了SAXS和FRET数据在贝叶斯过程中，以改善所观察到的未折叠状态的结构合奏代表。该分析表明，这两个实验探针与每个变性剂浓度的蛋白质构型的共同集合是相容的。此外，由此产生的合奏再现的趋势，增加流体动力学半径与变性剂浓度获得2f-FCS和DLS。因此，我们能够调和的结果，从所有四个实验技术定量，获得一个全面的结构图片变性剂诱导的展开状态扩展，并确定最可能的来源早期的差异。
There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we have used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.
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影响因子：
16.6
作者：
通讯作者：
--
影响因子：
64.8
作者：
Demarest, SJ;Martinez-Yamout, M;Wright, PE
通讯作者：
Wright, PE
影响因子：
5.6
作者：
Foerster, Friedrich;Webb, Benjamin;Krukenberg, Kristin A.;Tsuruta, Hiro;Agard, David A.;Sali, Andrej
通讯作者：
Sali, Andrej
DOI：
10.1021/j150451a002
发表时间：
1947-01-01
期刊：
JOURNAL OF PHYSICAL AND COLLOID CHEMISTRY
影响因子：
--
作者：
DEBYE, P
通讯作者：
DEBYE, P
影响因子：
3.3
作者：
Antonov, L. D.;Olsson, S.;Hamelryck, T.
通讯作者：
Hamelryck, T.