Refolding of cold-denatured barstar induced by radio-frequency heating: a new method to study protein folding by real-time NMR spectroscopy

  • The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio-frequency power to lossy aqueous samples, refolding of barstar from its cold-denatured state can be followed by real-time NMR spectroscopy. Since temperature-induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real-time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low-temperature-unfolded state, which can potentially induce multiple folding pathways. The high time resolution real-time 2D-NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state-correlated spectroscopy, an alternative folding pathway circumventing the rate-limiting cis-trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

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Author:György Pintér, Harald SchwalbeORCiDGND
Parent Title (English):Angewandte Chemie
Place of publication:Weinheim
Document Type:Article
Date of Publication (online):2020/08/03
Date of first Publication:2020/08/03
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2022/03/09
Tag:NMR spectroscopy; barstar; proline isomerization; protein folding; temperature jump
Page Number:6
First Page:22086
Last Page:22091
This work was supported by Deutsche Forschungsgemeinschaft (GRK 1986 “CLiC”) and European Union Horizon 2020 Program (Marie Sklodowska-Curie Grant 642773). Open access funding enabled and organized by Projekt DEAL.
Institutes:Biochemie, Chemie und Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - Namensnennung 4.0