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.
Author: | György Pintér, Harald SchwalbeORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-638447 |
DOI: | https://doi.org/10.1002/anie.202006945 |
ISSN: | 1521-3773 |
Parent Title (English): | Angewandte Chemie |
Publisher: | Wiley-VCH |
Place of publication: | Weinheim |
Document Type: | Article |
Language: | English |
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 |
Volume: | 59 |
Issue: | 49 |
Page Number: | 6 |
First Page: | 22086 |
Last Page: | 22091 |
Note: | 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. |
HeBIS-PPN: | 493368418 |
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 | |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - Namensnennung 4.0 |