Commonly used FRET fluorophores promote collapse of an otherwise disordered protein
Commonly used FRET fluorophores promote collapse of an otherwise disordered protein
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DOI：
10.1073/pnas.1813038116
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发表时间：
2019-04-30
影响因子：
11.1
通讯作者：
Sosnick, Tobin R.
中科院分区：
文献类型：
--
作者：
Riback, Joshua A.;Bowman, Micayla A.;Sosnick, Tobin R.
未折叠蛋白质，包括内在无序蛋白质（IDP），在没有变性剂的情况下采用的尺寸仍然存在争议。我们开发了一个小角X射线散射（SAXS）配置文件的分析程序，并使用它来证明，即使相对疏水的IDP仍然几乎在水中扩展，因为它们是在高变性剂浓度。相反，如这里所示，大多数荧光共振能量转移（FRET）测量已经表明，相对疏水的IDP在不存在变性剂的情况下显著收缩。我们使用两个独立的方法来进一步探讨这一争议。首先，使用SAXS，我们表明，在FRET中使用的荧光团可以有助于观察到的差异。具体来说，我们发现，添加Alexa-488到正常扩展的IDP中会导致额外的15%收缩，该值与基于FRET的研究中报告的收缩值合理雅阁。其次，使用我们的模拟和分析程序从SAXS轮廓中准确提取回转半径（Rg）和端到端距离（Ree），我们测试了最近的建议，即如果Rg和Ree是“非偶联的”（即，不再简单地成比例），与无规行走均聚物的情况相反。然而，我们发现，即使是未折叠的蛋白质，这两个措施的未折叠状态尺寸保持成比例。总之，这些结果表明，改进的分析程序和校正显着的，荧光团驱动的相互作用是足以调和以前的SAXS和FRET研究，从而提供了一个统一的图片的性质展开的多肽链在变性剂的情况下。
The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.