Analyzing the visible conformational substates of the FK506-binding protein FKBP12.

The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43oC. The largest differences in chemical shift occur for the 80's loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the beta sheet, across the active site to the alpha helix and into the 50's loop. Each of the seven proline residues adopts a trans peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational linebroadening transition(s) that occur ~105-fold more rapidly, previously proposed to arise from a single global process. The 1.70 Å resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the linebroadening transition for residues near the 80's loop. Linebroadening was also decreased for the residues in the α-helix and 50's loop, while linebroadening in the 40's loop was unaffected. The K44V mutation selectively reduces the linebroadening in the 40's loop, verifying that at least three distinct conformational transitions underlie the linebroadening processes of FKBP12.