An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

J Mol Biol. 2018 Jun 8;430(12):1799-1813. doi: 10.1016/j.jmb.2018.04.025. Epub 2018 Apr 27.


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 β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-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 β-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 β-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.

Keywords: beta-sheet; multi-domain protein; on-pathway intermediate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Surface / chemistry*
  • Antigens, Surface / genetics
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Vaccines / chemistry*
  • Bacterial Vaccines / genetics
  • Borrelia burgdorferi / chemistry
  • Borrelia burgdorferi / genetics
  • Borrelia burgdorferi / metabolism*
  • Circular Dichroism
  • Kinetics
  • Lipoproteins / chemistry*
  • Lipoproteins / genetics
  • Models, Molecular
  • Protein Domains
  • Protein Folding / drug effects
  • Protein Structure, Secondary
  • Urea / pharmacology*


  • Antigens, Surface
  • Bacterial Outer Membrane Proteins
  • Bacterial Vaccines
  • Lipoproteins
  • OspA protein
  • Urea