The overall goal is to test the hypothesis that the method of interaction of a particular LPS-binding protein with LPS is the insertion of the LPS within the interior of the beta-helical structure of that protein. As a first step towards this goal, studies are proposed to define the amino acids on the tailspike protein (TSP) of Salmonella typhimurium phage P22 which bind and hydrolyze the bacterial LPS. These studies may lead to the development of LPS binding protein vehicles for diagnosis and therapy. Because of the rapid rise in bacteria resistance to all known antibiotics, it becomes critical to explore non-antibiotic- based diagnosis and treatment regimes as soon as possible. These studies involve the selection of phage host range mutants and the mutagenesis of a plasmid DNA containing the P22 tailspike gene. Extracts fromcells containing these mutant tailspike genes will be screened for mutants which are representative of LPS interaction mutants which contain a native structure as indicated by the reaction to monoclonal antibodies to the native trimeric TSP structure and by production of SDS-PAGE profile indicative of a trimeric structure. Physcial binding to LPS and enzymatic activity will be measured and those with limited activity or binding will be tentatively identified as LPS recognition mutants. Having shown the close structural similarity between the P22 and epsilon34 TSPs, efforts will be continued at the purification of the epsilon34 TSP. The TSPs will be instructive in identifying the LPS binding site.
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