Conformational ensembles of an intrinsically disordered protein consistent with NMR, SAXS and single-molecule FRET.
 

Conformational ensembles of an intrinsically disordered protein consistent with NMR, SAXS and single-molecule FRET.

Related Articles Conformational ensembles of an intrinsically disordered protein consistent with NMR, SAXS and single-molecule FRET.

J Am Chem Soc. 2020 Aug 25;:

Authors: Gomes GW, Krzeminski M, Namini A, Martin EW, Mittag T, Head-Gordon T, Forman-Kay JD, Gradinaru CC

Abstract
Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging, but of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it mutually supports a consistent structural picture. We used integrative modelling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET) reach concordance on structural ensembles for Sic1 and phosphorylated Sic1 (pSic1). To resolve apparent discrepancies between smFRET and SAXS, we integrated SAXS data with non-smFRET (NMR) data and reserved the new smFRET data for Sic1 and pSic1, as an independent validation. Given the consistency of the SAXS/PRE restrained ensembles with smFRET, which was not guaranteed a priori, indicates that the perturbative effects of NMR or smFRET labels on the Sic1 and pSic1 ensemble are minimal. Furthermore , the mutual agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence, such as overall compactness and end-to-end distance fluctuations, to reveal distinguishing features of Sic1 vs. pSic1. From the experimentally well supported ensembles, we find they are consistent with independent biophysical models of Sic1's ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling and validation in calculating and drawing biological conclusions from IDP conformational ensembles.


PMID: 32840111 [PubMed - as supplied by publisher]



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