Backbone dynamics of the 269-residue protease Savinase determined from N-15-NMR relaxation measurements

Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using N-15 relaxation measurements derived from proton-detected two-dimensional H-1-N-15-NMR spectroscopy. N-15 spin-lattice rate constants (R(1)), spin-spin relaxation-rate constants (R(2)), and H-1-N-15 nuclear Overhauser effects (NOE) were determined for 84% of the backbone amide N-15 nuclei. The model-free formalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Sec. 104, 4546-4559] was used to derive values for a generalized order parameter, S-2, interpretable as a measure of the amplitude of motion on the picosecond-nanosecond timescale, for each N-H bond vector. Additional terms used to fit the data include an effective correlation time for internal motions (tau(e)) and an exchange term (R(ex)) to account for exchange contributions to R(2). The overall rotational correlation time (tau(m)) is 9.59 +/- 0.02 ns; the average order parameter (S-2) is 0.90 +/- 0.07, indicative of a rigid structure consistent with Savinase's high degree of secondary structure and compact tertiary fold. Residues S125-S128, located in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond- nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region also show flexibility on the picosecond-nanosecond timescale. There is also significant motion in the turn, G258-T260, of a small solvent-exposed loop region which may make the protein vulnerable to autolysis at that point. Some residues in both calcium-binding sites and nearby also show mobility.