Solution NMR spin-relaxation experiments were used to compare mus-ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 +/- 1.0 kcal/mol and 4.5 +/- 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data suggests that the magnitude of motion is also similar for these two forms and that it is likely that apo enzyme interconverts to a structure that resembles a ligand-bound form. Likewise, it appears that the bound conformation samples the apo enzyme form even when ligand is present. Taken together the data imply that RNase A is in a preexisting dynamic equilibrium between two conformations that represent the open and closed enzyme forms. These data suggest that ligand binding stabilizes the bound conformer but does not induce it.

The dynamic properties of the enzyme ribonuclease A (RNase A) were investigated through the use of solution NMR spin relaxation experiments. As determined by "model-free" analysis, RNase A is conformationally rigid on time scales faster than overall rotational tumbling (picoseconds to nanoseconds). The average order parameter, S(2), for RNase A is 0.910 +/- 0.051. However, 28 of the amino acid residues in RNase A were identified as undergoing chemical exchange on the microsecond to millisecond time scale. For 16 of these residues the microscopic chemical exchange rates, k(ex), were quantitated through the use of the relaxation-compensated CPMG (rcCPMG) experiment. The value of k(ex) was identical for all residues with an average of 1640 s(-1) and is similar to the RNase A k(cat) value of 1900 s(-1). Many of these mobile residues localize to the active site in RNase A and include the catalytically crucial amino acids His119 and Asp121. Additional motion is found in the B1, B2, and P0 subsites, suggesting a coupling of motion between the binding and catalytic sites. The activation energy of the observed millisecond motion was measured by applying the rcCPMG experiment at temperatures of 283, 293, and 298 K and was determined to vary between 3.6 and 7.4 kcal/mol. The measured barrier to conformational motion is similar to the activation barrier for the RNase A catalyzed reaction and thus would not be thermodynamically limiting to catalysis. These studies suggest a correlation of conformational exchange kinetics and thermodynamics derived from NMR measurements with those determined by biochemical means and are suggestive of an important role for flexibility in enzyme function.

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Herein we describe the program FAST-Modelfree for the fully automated, high throughput analysis of NMR spin-relaxation data. This program interfaces with the program Modelfree 4.1 and provides an intuitive graphical user interface for configuration as well as complete standalone operation during the model selection and rotational diffusion parameter optimization processes. FAST-Modelfree is also capable of iteratively assigning models to each spin and optimizing the parameters that describe the diffusion tensor. Tests with the protein Ribonuclease A indicate that using this iterative approach even poor initial estimates of the diffusion tensor parameters will converge to the optimal value within a few iterations. In addition to improving the quality of the final fit, this represents a substantial timesaving compared to manual data analysis and minimizes the chance of human error. It is anticipated that this program will be particularly useful for the analysis and comparison of data collected under different conditions such as multiple temperatures and the presence and absence of ligands. Further, this program is intended to establish a more uniform protocol for NMR spin-relaxation data analysis, facilitating the comparison of results both between and within research laboratories. Results obtained with FAST-Modelfree are compared with previous literature results for the proteins Ribonuclease H, E. coli glutaredoxin-1 and the Ca(2+)-binding protein S100B. These proteins represent data sets collected at both single and multiple static magnetic fields and which required analysis with both isotropic and axially symmetric rotational diffusion tensors. In all cases results obtained with FAST-Modelfree compared favorably with the original literature results.