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.