Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 430, Issue 12, Pages 1799-1813Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2018.04.025
Keywords
multi-domain protein; beta-sheet; on-pathway intermediate
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Funding
- JSPS KAKENHI [JP20870043, JP16K07314]
- MEXT KAKENHI [JP20107009]
- Astellas Foundation for Research on Metabolic Disorders
- Sumitomo Electric Industries CSR Foundation
- Kato Memorial Bioscience Foundation
- Intelligent Cosmos Research Institute Foundation
- Naito Foundation
- Waksman Foundation of Japan
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Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a beta-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer beta-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer beta-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer beta-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. (C) 2018 Elsevier Ltd. All rights reserved.
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