The use of peptides to reconstruct conformational determinants; a brief review.

In many biological processes, defined regions of proteins are involved in selective recognition. These regions can often be mimicked with peptides and are the main targets for vaccine and drug development. The authors review the use of peptides, to define and ultimately mimic defined protein regions of interest. Especially the role of the Pepscan method is emphasized. This method has been proven to be a useful and fast tool in defining protein regions of interest. It is based on the simultaneously synthesis of multiple peptides coupled to solid supports. Hundreds of peptides can be produced and tested in a relatively short period of time. With the construction of random peptide libraries in recombinant DNA systems, it is now even possible to screen for peptidic determinants without the requirement of preliminary knowledge of primary structure. Having this information, the affinity of peptides can be further enhanced with the Pepscan approach. The power of this approach will be illustrated with results from studies on the development of synthetic vaccines and hormone analogues.

Antigenicity and immunogenicity of synthetic peptides of foot-and-mouth disease virus.

Peptides reactive with two neutralizing monoclonal antibodies raised against intact foot-and-mouth disease virus A10 were identified with the aid of all overlapping (hexa)peptides of the outer structural viral protein VP1 and located on the viral surface. Using this procedure, it was possible to define those amino acids within a peptide which were critical in the binding of antibody to that peptide. One eight amino acid long peptide, containing six such amino acids, was virtually indistinguishable from viral antigen in its ability to bind monoclonal antibody as determined by competition tests. Another peptide, which was able to induce neutralizing activity as well, showed no competition and possessed fewer amino acids contributing to binding. This peptide appeared to be an incomplete epitope. Comparison of our data with those of others suggests that this may apply commonly to the reactive peptides described.

Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.

It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.