Protein function is largely dependent on conformational fluctuations. Outer surface protein A (OspA) is composed of N- and C-terminal globular domains linked by a central β-sheet. OspA is expressed on the surface of the Lyme disease-causing bacterium. Solution NMR studies have revealed that the central β-sheet and C-terminal domain containing receptor recognition sites are less stable than the N-terminal domain, indicating a locally-disordered intermediate conformation between the native folded and completely unfolded conformation. We previously proposed that the exposure of receptor-binding sites following denaturation of the C-terminal domain is advantageous for OspA binding to the receptor. Here, pressure perturbation and single-point mutagenesis were found to amplify a specific protein fluctuation. Based on previous NMR protein structure and dynamics studies, we designed the salt-bridge-destabilized mutant E160D and cavity-enlarged mutant I243A of OspA. As expected, the E160D and I243A mutations significantly reduced the stability of the C-terminal domain and stabilized the intermediate. Using high-pressure NMR studies, we found that the proportion of the intermediate accounted for almost 100% in E160D at 250 MPa and 313 K. The peak intensities of the N-terminal domain (β1-β7) remained constant or increased, indicating that the N-terminal domain is not denatured. In contrast, pressurization reduced the peak intensities of most residues in the center and C-terminal domains (β9-β21). Additionally, new unassigned peaks were observed. These results indicate that the center and C-terminal domains are disordered. Interestingly, both behaviors were observed for β8, indicating that the transition to the disordered conformation began in β9, and the locally-disordered intermediate has a structured N-terminal domain and highly disordered central and C-terminal domains. These structural characteristics of the locally disordered intermediate were similar to those of the WT. Strategies employing a suitably chosen point mutation in conjunction with high pressure are generally applicable for the amplification of specific conformational fluctuations and may enhance our understanding of the intermediate conformations of proteins.