SYNTHESIS AND CONFORMATIONAL-ANALYSIS BY H-1-NMR AND RESTRAINED MOLECULAR-DYNAMICS SIMULATIONS OF THE CYCLIC DECAPEPTIDE [SER-TYR-SER-MET-GLU-HIS-PHE-ARG-TRP-GLY]

The design of enzyme mimics with therapeutic and industrial applicatio ns has interested both experimental and computational chemists for sev eral decades. Recent advances in the computational methodology of rest rained molecular dynamics, used in conjunction with data obtained from two-dimensional H-1 NMR spectroscopy, make it a promising method to s tudy peptide and protein structure and function. Several issues, howev er, need to be addressed in order to assess the validity of this metho d for its explanatory and predictive value. Among the issues addressed in this study are: the accuracy and generizability of the GROMOS pept ide molecular mechanics force field; the effect of inclusion of solven t on the simulations; and the effect of different types of restraining algorithms on the computational results. The decapeptide Ser-Tyr-Ser- Met-Glu-His-Phe-Arg-Trp-Gly, which corresponds to the sequence of ACTH (1-10), has been synthesized, cyclized, and studied by two-dimensional H-1 NMR spectroscopy. Restrained molecular dynamics (RMD) and time-av eraged restrained molecular dynamics (TARMD) simulations were carried out on four different distance-geometry starting structures in order t o determine and contrast the behavior of cyclic ACTH(1-10) in vacuum a nd in solution. For the RMD simulations, the structures did not fit th e NOE data well, even at high values of the restraining potential. The TARMD simulation method, however, was able to give structures that fi t the NOE data at high values of the restraining potential. In both ca ses, inclusion of explicit solvent molecules in the simulation had lit tle effect on the quality of the fit, although it was found to dampen the motion of the cyclic peptide. For both simulation techniques, the number and size of the NOE violations increased as the restraining pot ential approached zero. This is due, presumably, to inadequacies in th e force field. Additional TARMD vacuum-phase simulations, run with a l arger memory length or with a larger sampling size (16 additional dist ance-geometry structures), yielded no significantly different results. The computed data were then analyzed to help explain the sparse NOE d ata and poor chymotryptic activity of the cyclic peptide. Cyclic ACTH( 1-10), which contains the functional moieties of the catalytic triad o f chymotrypsin, was evaluated as a potential mimic of chymotrypsin by measurement of the rate of hydrolysis of esters of L- and D-phenylalan ine. The poor rate of hydrolysis is attributed to the flexibility of t he decapeptide, the motion of the side chains, which result in the abs ence of long-range NOEs, the small size of the macrocycle relative to that of the substrate, and the inappropriate orientation of the Gly, H is, and Ser residues. The results demonstrate the utility of this meth od in computer-aided molecular design of cyclic peptides and suggest s tructural modifications for future work based on a larger and more rig id peptide framework.