Conformational fluctuation, namely,
protein interconversion between
different conformations, is crucial to protein function. Outer surface
protein A (OspA), comprising N- and C-terminal globular domains linked
by a central β-sheet, is expressed on the surface of Borrelia burgdorferi, the causative agent of Lyme disease,
and recognizes the TROSPA receptor in the tick gut. Solution nuclear
magnetic resonance studies have shown that the central β-sheet
and C-terminal domain containing TROSPA recognition sites are less
stable than the N-terminal domain, revealing an intermediate conformation
between the basic folded and completely unfolded proteins. We previously
suggested that exposure of receptor-binding sites following denaturation
of the C-terminal domain is advantageous for OspA binding to the receptor.
Here, we observed amplification of a specific protein fluctuation
by pressure perturbation and site-specific mutagenesis. The salt-bridge-destabilized
mutant E160D and the cavity-enlarged mutant I243A favored the intermediate.
The proportion of the intermediate accounted for almost 100% in E160D
at 250 MPa. Strategies using a suitably chosen point mutation with
high pressure are generally applicable for amplification of specific
conformational fluctuation and potentially improve our understanding
of the intermediate conformations of proteins. Knowledge of various
conformations, including OspA intermediates, may be useful for designing
a vaccine for Lyme disease.