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Proc Natl Acad Sci U S A. 2017 Aug 1;114(31):E6342-E6351. doi: 10.1073/pnas.1704692114. Epub 2017 Jul 17.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Author information

1
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
2
European Molecular Biology Laboratory, 22607 Hamburg, Germany.
3
Center for Biological Systems Engineering, Department of Biomedical Engineering, School of Engineering & Applied Science, Washington University in St. Louis, St. Louis, MO 63130.
4
Heidelberg Institut für Theoretische Studien, 69118 Heidelberg, Germany.
5
Interdisciplinary Center for Scientific Computing, 69120 Heidelberg, Germany.
6
Micromechanics Section, Zernike Institute for Advanced Materials, University of Groningen, 9747AG Groningen, The Netherlands.
7
Barcelona Supercomputing Center, 08034 Barcelona, Spain.
8
IDP Discovery Pharma SL, 08028 Barcelona, Spain.
9
Center for Biological Systems Engineering, Department of Biomedical Engineering, School of Engineering & Applied Science, Washington University in St. Louis, St. Louis, MO 63130; lemke@embl.de pappu@wustl.edu svergun@embl-hamburg.de.
10
European Molecular Biology Laboratory, 22607 Hamburg, Germany; lemke@embl.de pappu@wustl.edu svergun@embl-hamburg.de.
11
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; lemke@embl.de pappu@wustl.edu svergun@embl-hamburg.de.
12
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

Abstract

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

KEYWORDS:

denatured-state ensemble; intrinsically disordered proteins; polymer theory; protein folding; single-molecule FRET

PMID:
28716919
PMCID:
PMC5547626
DOI:
10.1073/pnas.1704692114
[Indexed for MEDLINE]
Free PMC Article

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