Abstract
AbstractStructural 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.
Highlights
In the past decades, an enormous variety of protein structures has been accumulated experimentally by employing sophisticated high-resolution techniques such as X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR).[1]
We examine the direct impact of Förster resonance energy transfer (FRET) dyes on small-angle X-ray scattering (SAXS) measurements and derived quantities
We find that FRET dyes attached to a protein significantly affect SAXS measurements on that system, as the dyes change both its size and shape
Summary
An enormous variety of protein structures has been accumulated experimentally by employing sophisticated high-resolution techniques such as X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR).[1] With cellular function, being dictated by the interplay between static structures and dynamic conformational changes, alternative methods have been catching up so as to elucidate the dynamic nature of the structure-function paradigm. SAXS can be used to study average structures of various systems and enables even time-resolved analyses of conformational transitions in direct response to altered external conditions.[2] A solution of biomolecules is exposed to X-rays and the integrated scattered intensity is recorded in the small-angle regime, which contains information on structural features of the solute molecules. After labeling specific molecular sites with fluorescent dyes, the distance-dependent energy transfer efficiency between them is measured
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