Backbone dynamics of free barnase and its complex with barstar determined by N-15 NMR relaxation study

Backbone dynamics of uniformly N-15-labeled free barnase and its complex wi th unlabelled barstar have been studied at 40 degreesC, pH 6.6, using N-15 relaxation data obtained from proton-detected 2D {H-1}-N-15 NMR spectroscop y. N-15 spin-lattice relaxation rate constants (R-1), spin-spin relaxation rate constants (R-2), and steady-state heteronuclear {H-1}-N-15 NOEs have b een measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excl uding three prolines and the N-terminal residue) backbone amide N-15 sites of barnase. The primary relaxation data for both the cases have been analyz ed in the framework of the model-free formalism using both isotropic and ax ially symmetric models of the rotational diffusion tensor. As per the latte r, the overall rotational correlation times (tau (m)) are 5.0 and 9.5 ns fo r the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, th e changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the ac tual binding. For the residues where the order parameter increases, the mag nitudes vary significantly. It is observed that the complex has much less i nternal mobility, compared to free barnase. From the changes in the order p arameters, the entropic contribution of NH bond vector motion to the free e nergy of complex formation has been calculated. It is apparent that these m otions cause significant unfavorable contributions and therefore must be co mpensated by many other favorable contributions to effect tight complex for mation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for r emote effects and regulation of the enzyme action.