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 b y Bacillus lentus, have been studied using N-15 relaxation measurement s derived from proton-detected two-dimensional H-1-N-15-NMR spectrosco py. 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 f ormalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Sec. 104, 4546-45 59] was used to derive values for a generalized order parameter, S-2, interpretable as a measure of the amplitude of motion on the picosecon d-nanosecond timescale, for each N-H bond vector. Additional terms use d to fit the data include an effective correlation time for internal m otions (tau(e)) and an exchange term (R(ex)) to account for exchange c ontributions 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 de gree of secondary structure and compact tertiary fold. Residues S125-S 128, located in the substrate-binding region, represent the longest st retch of protein which exhibits disorder on the picosecond- nanosecond timescale. These residues also exhibit significant exchange terms, po ssibly 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 a nd G219 in the substrate-binding region also show flexibility on the p icosecond-nanosecond timescale. There is also significant motion in th e turn, G258-T260, of a small solvent-exposed loop region which may ma ke the protein vulnerable to autolysis at that point. Some residues in both calcium-binding sites and nearby also show mobility.