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Refolding of Cold‐Denatured Barstar Induced by Radio‐Frequency Heating: A New Method to Study Protein Folding by Real‐Time NMR Spectroscopy
Author(s) -
Pintér György,
Schwalbe Harald
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202006945
Subject(s) - chemistry , nuclear magnetic resonance spectroscopy , folding (dsp implementation) , denaturation (fissile materials) , protein folding , isomerization , downhill folding , spectroscopy , phi value analysis , crystallography , stereochemistry , biochemistry , physics , quantum mechanics , electrical engineering , engineering , nuclear chemistry , catalysis
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.