N-15 NMR RELAXATION STUDIES OF THE FK506 BINDING-PROTEIN - BACKBONE DYNAMICS OF THE UNCOMPLEXED RECEPTOR

Backbone dynamics of the major tacrolimus (FK506) binding protein (FKBP-12, 107 amino acids) have been studied using N-15 relaxation data derived from proton-detected two-dimensional H-1-N-15 NMR spectroscopy. N-15 spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and heteronuclear NOEs were determined for over 85% of the backbone amide N-15 nuclei. A model free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4559-45701 was used to derive values for the generalized order parameter (S2), the effective correlation time for internal motions (tau(e)), and the chemical exchange line width (R(ex)) for each N-H bond vector. The final optimized overall correlation time (tau(m)) was 9.2 ns. The average order parameter (S2) describing the amplitude of motions on the picosecond time scale was found to be 0.88 +/- 0.06. Motions on the picosecond time scale are restricted at the N and C termini, consistent with previous NMR structural studies indicating well-defined beta-strands in these regions. With the exception of the flap region from residues 82 to 87, no regions appear to be significantly disordered on the picosecond time scale. Residues in several regions of the protein exhibit high R(ex) terms, indicating possible motions on the millisecond to microsecond time scale due to chemical exchange and/or conformational averaging effects. Possible effects of tacrolimus (FK506) binding on FKBP-12 dynamics are discussed in the context of previously determined solution structures for FKBP-12 in the uncomplexed [Michnick et al. (1991) Science 252, 836-839; Moore et al. (1991) Nature 351, 248-250] and complexed [Meadows et al. (1993) Biochemistry 32, 754-765] states.