Analysis of O^sub 2^-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A

Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O^sub 2^-binding sites by O^sub 2^-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O^sub 2^-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O^sub 2^-accessible sites in the protein based on the 1/r^sup 6^ distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O^sub 2^ generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O^sub 2^ within the largest cavity, egress of O^sub 2^ from the cavity, and ingress of O^sub 2^ in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O^sub 2^-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.