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.
Verfasserangaben: | 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 |
Titel des übergeordneten Werkes (Englisch): | Angewandte Chemie |
Verlag: | Wiley-VCH |
Verlagsort: | Weinheim |
Dokumentart: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Veröffentlichung (online): | 03.08.2020 |
Datum der Erstveröffentlichung: | 03.08.2020 |
Veröffentlichende Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Datum der Freischaltung: | 09.03.2022 |
Freies Schlagwort / Tag: | NMR spectroscopy; barstar; proline isomerization; protein folding; temperature jump |
Jahrgang: | 59 |
Ausgabe / Heft: | 49 |
Seitenzahl: | 6 |
Erste Seite: | 22086 |
Letzte Seite: | 22091 |
Bemerkung: | 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 |
Institute: | Biochemie, Chemie und Pharmazie |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie | |
Sammlungen: | Universitätspublikationen |
Lizenz (Deutsch): | Creative Commons - Namensnennung 4.0 |