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 (FKB P-12, 107 amino acids) have been studied using N-15 relaxation data de rived 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 deriv e values for the generalized order parameter (S2), the effective corre lation time for internal motions (tau(e)), and the chemical exchange l ine width (R(ex)) for each N-H bond vector. The final optimized overal l correlation time (tau(m)) was 9.2 ns. The average order parameter (S 2) describing the amplitude of motions on the picosecond time scale wa s 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 struct ural studies indicating well-defined beta-strands in these regions. Wi th the exception of the flap region from residues 82 to 87, no regions appear to be significantly disordered on the picosecond time scale. R esidues in several regions of the protein exhibit high R(ex) terms, in dicating possible motions on the millisecond to microsecond time scale due to chemical exchange and/or conformational averaging effects. Pos sible effects of tacrolimus (FK506) binding on FKBP-12 dynamics are di scussed in the context of previously determined solution structures fo r FKBP-12 in the uncomplexed [Michnick et al. (1991) Science 252, 836- 839; Moore et al. (1991) Nature 351, 248-250] and complexed [Meadows e t al. (1993) Biochemistry 32, 754-765] states.