The purpose of this proposal is to elucidate the mechanism of action of the bacterial chemotaxis receptor, consisting of the methylation enzyme and the cytoplasmic H-protein. The current knowledge regarding the molecular mechanism of bacterial chemotaxis is very incomplete. During the past year, we have succeeded in developing in vitro a system which, unlike other experimental approaches, provides the advantage of allowing us to follow the reaction directly from substrate to product and reaction intermediates by several different types of physical measurements. To this end we have purified in sufficient quantities, both the modification enzyme (methylase) and the associated transducer H-protein from cells of the escherichia coli strain CB15. Using these preparations we have studied the reaction of methylase using a methylene labeled derivative of aspartate-gamma-carboxylate as the substrate. We have observed formation of a product which was identified by NMR to be succinate-alpha-amino-beta-carboxylate which is a precursor for several amino acids that are related to methionine. We have found that the product formation is dependent upon the presence of an H-protein. Furthermore, both unlabeled aspartate-gamma-carboxylate and methylene-labeled succinate-alpha-amino-beta-carboxylate inhibit the methylase activity. These results are consistent with our earlier finding that the product has been identified as the inhibitor of the methylase activity. We are currently investigating whether there is a direct interaction between the methylase and the H-protein in the methylase: H-protein complex. We are also attempting to identify possible reaction intermediates by using NMR. From these results we should be able to clarify the mechanism of action of the chemotaxis receptor. During the next year, we shall use these preparations to investigate the interaction of the methylase with the H-protein in further detail. Another aspect of the proposed study is to investigate the relation between the observed affinity of H-protein for the receptor in vivo and the affinity of the purified H-protein for methylase as measured in vitro. It has been shown that the methylase protein has to be associated with the H-protein in order for the chemotaxis system to function. To identify regions on the methylase and H-protein which are required for their proper association, we shall use systematic site directed mutagenesis on the methylase and H-protein genes. Preliminary experiments on systematic mutagenesis have already yielded some positive results. In order to map regions on the H-protein, we shall employ the recently developed method of random fragmentation of DNA followed by PCR (random fragmentation-PCR). By examining a collection of mutants on the H-protein, we should be able to identify regions on the H-protein which are required for the proper association of methylase with the receptor. This would help us in indentifying which amino acid residues on the H-protein are essential for the chemotaxis function. We have also started a collaborative effort with Dr. J.P. Schramm on this problem. They are carrying out the chemical synthesis of the H-protein. Our preliminary results suggest that they are making a peptide having a sequence which is 99% homologous to the H-protein of Salmonella typhimurium. Thus the structure of the synthetic peptide is the one expected from the gene sequence of the original H-protein which has been cloned. Using this peptide, we hope to learn about the structure-function relationship of the H-protein. A preliminary test of this hypothesis shows that the protein synthesis of E. coli is greatly decreased when such a peptide is added in sufficient amount to the cell suspension. Using this approach, we should be able to identify the region of the H-protein which is required for the proper association of methylase with the receptor. We shall then be able to modify this protein in order to obtain a soluble protein which will have no methylase and no ability to bind the receptor, and thus be able to identify the region on the H-protein which is responsible for its binding to the receptor. These results will also help us to identify the receptor recognition site on the H-protein.