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

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>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. <I>Borrelia</I> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Elucidation of mechanisms of multi-domain protein folding is an important issue. </LI> <LI> A two-domain protein OspA is a useful model for studying the multi-domain folding. </LI> <LI> We show evidence for the presence of an on-pathway folding intermediate in OspA. </LI> <LI> A rigid and stable intervening region between the domains played a critical role. </LI> <LI> The presence of such a region can lead to coupled folding behavior of the domains. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>