Structural analyses in biophysics aim at revealing a relationship between a molecule's dynamic structure and its physiological function. Förster resonance energy transfer (FRET) and small‐angle X‐ray scattering (SAXS) are complementary experimental approaches to this. Their concomitant application in combined studies has recently opened a lively debate on how to interpret FRET measurements in the light of SAXS data with the popular example of the radius of gyration, commonly derived from both FRET and SAXS. There still is a lack of understanding in how to mutually relate and interpret quantities equally obtained from FRET or SAXS, and to what extent FRET dyes affect SAXS intensities in combined applications. In the present work, we examine the interplay of FRET and SAXS from a computational simulation perspective. Molecular simulations are a valuable complement to experimental approaches and supply instructive information on dynamics. As FRET depends not only on the mutual separation but also on the relative orientations, the dynamics, and therefore also the shapes of the dyes, we utilize a novel method for simulating FRET‐dye‐labeled proteins to investigate these aspects in atomic detail. We perform structure‐based simulations of four different proteins with and without dyes in both folded and unfolded conformations. In‐silico derived radii of gyration are different with and without dyes and depend on the chosen dye pair. The dyes apparently influence the dynamics of unfolded systems. We find that FRET dyes attached to a protein have a significant impact on theoretical SAXS intensities calculated from simulated structures, especially for small proteins. Radii of gyration from FRET and SAXS deviate systematically, which points to further underlying mechanisms beyond prevalent explanation approaches.

中文翻译：

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FRET染料显着影响蛋白质的SAXS强度

生物物理学中的结构分析旨在揭示分子的动态结构与其生理功能之间的关系。福斯特共振能量转移（FRET）和小角度X射线散射（SAXS）是对此的补充实验方法。它们在组合研究中的伴随应用最近引起了关于如何根据SAXS数据以及通常从FRET和SAXS得出的普遍的回转半径示例来解释FRET测量的激烈辩论。在如何相互关联和解释从FRET或SAXS中平均获得的量以及FRET染料在组合应用中对SAXS强度的影响程度方面仍缺乏理解。在当前的工作中，我们从计算仿真的角度检查了FRET和SAXS的相互作用。分子模拟是实验方法的宝贵补充，可提供有关动力学的指导性信息。由于FRET不仅取决于相互分离，而且取决于相对方向，动力学以及染料的形状，因此我们采用了一种新颖的方法来模拟FRET染料标记的蛋白质，以原子方式详细研究这些方面。我们对四种不同蛋白质的折叠和展开构象进行基于结构的模拟，无论有无染料。硅内衍生的回转半径在有无染料的情况下是不同的，并且取决于所选的染料对。染料显然影响展开系统的动力学。我们发现，附着在蛋白质上的FRET染料对从模拟结构计算得出的理论SAXS强度有重大影响，特别是对于小蛋白质。