The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways.

Autor:	Tiwari VP; Tata Institute of Fundamental Research, Hyderabad 500107, India., Toyama Y; Department of Biochemistry, University of Toronto, ON M5S 1A8, Canada.; Department of Molecular Genetics, University of Toronto, ON M5S 1A8, Canada.; Department of Chemistry, University of Toronto, ON M5S 3H6, Canada., De D; Tata Institute of Fundamental Research, Hyderabad 500107, India., Kay LE; Department of Biochemistry, University of Toronto, ON M5S 1A8, Canada; kay@pound.med.utoronto.ca pramodh@tifrh.res.in.; Department of Molecular Genetics, University of Toronto, ON M5S 1A8, Canada.; Department of Chemistry, University of Toronto, ON M5S 3H6, Canada.; Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada., Vallurupalli P; Tata Institute of Fundamental Research, Hyderabad 500107, India; kay@pound.med.utoronto.ca pramodh@tifrh.res.in.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Nov 16; Vol. 118 (46).
DOI:	10.1073/pnas.2115113118
Abstrakt:	Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s -1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES. Competing Interests: The authors declare no competing interest.
Databáze:	MEDLINE
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