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Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS


Aznauryan, Mikayel; Delgado, Leonildo; Soranno, Andrea; Nettels, Daniel; Huang, Jie-Rong; Labhardt, Alexander M; Grzesiek, Stephan; Schuler, Benjamin (2016). Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS. Proceedings of the National Academy of Sciences of the United States of America, 113(37):E5389-E5398.

Abstract

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

Abstract

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:13 September 2016
Deposited On:17 Oct 2016 09:03
Last Modified:17 Oct 2016 09:03
Publisher:National Academy of Sciences
ISSN:0027-8424
Publisher DOI:https://doi.org/10.1073/pnas.1607193113
PubMed ID:27566405

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