Structural studies by H-1 NMR and molecular modeling of peptide substratesof thermolysin in relation with its proteasic activity in water and glycerol

In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cl eavages, five peptides of various length were studied in two solvents H2O a nd glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficiently rough constraints to ensure a convergence of a refinement process for such short and flexible peptides, the conformatio nal space was first searched using the MCMM method. The generated structure s were then clustered in families using a 0.3 Angstrom rmsd criterion and t he derived structural characteristics were compared to the experimental NMR parameters. In a first approach, the NMR consistent conformations were com pared with the structure of a thermolysin bound peptidic inhibitor ZG(P)LL to characterize the free-ligand predisposition to be cleaved. Further molec ular dynamic calculations were performed at 300 K on the conformations corr esponding to families in agreement with the ZG(P)LL structure in order to o btain information on their stability and on the trajectories of the torsion angles involved in the active site recognition. In conclusion, for four st udied peptides, some conformations were found to be in agreement with 5 of the 8 cleavages experimentally observed.