Structure of a cyclic peptide with a catalytic triad, determined by computer simulation and NMR spectroscopy

Summary We report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the -turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 . At a later stage in the simulation, a transition at Ser³ produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.     

Selection of a cyclic nonapeptide inhibitor to α-chymotrypsin using a phage display peptide library

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against α-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with α-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of α-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for α-chymotrypsin was estimated to be 10^-6 M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified α-chymotrypsin than for natural α-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to α-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of α-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature. This revised version was published online in August 2006 with corrections to the Cover Date.