Our current specific aims are to understand the molecular mechanisms underlying the function of the protein complexes that drive bacterial chemotaxis, and the complex proteins that are central to histidine kinase mediated signal transduction in the fungi. 1. We have described a family of homologous chemotaxis proteins and genes in mesophilic bacteria and in thermophilic bacteria. We will study the atomic structure of specific functional domains of these proteins in an effort to understand how they interact with each other and how they act to transduce and transmit information. The thermophilic and hyperthermophilic organisms provide versions of these proteins that lend themselves more readily to structural studies at atomic resolution and to studies of the stability of protein-protein interaction. The homologous mesophilic systems allow us to use mutagenesis, genetic manipulation and physiology to test ideas generated by the structural studies. 2. We will further elucidate the pathways involved in hybrid-kinase function in Neurospora and in some of the pathogenic fungi. We have found two hybrid histidine kinases Nik-1 and Nik-2 that regulate cell growth and morphology. Homologs of these kinases are found in a variety of organisms including Candida. We will use genetic and molecular techniques to isolate the other components of these pathways in Neurospora to determine their localization and to reconstitute their biochemical interactions. We will characterize the domain structure of these systems and develop ideas about how specific protein-protein interactions are controlled and how they in turn regulate information transmission and transduction. We plan to develop assay systems for specific small molecule inhibitors of these pathways and to study the biochemical mechanisms underlying the inhibition. Ultimately we will be able to trace the information transduction process from ligand binding events, through a series of specific stabilized conformational changes to a change in the mechanism or rate of a catalytic or regulatory function. These insights will allow us to understand the parameters, that are critical to the evolution of these systems and, that control their versatility and ubiquity. Finally, comparable histidine kinases may not be present in mammalian systems and thus may present an excellent target for the development of therapeutic agents, e.g. antibiotics, fungicides, and herbicides. We will explore this possibility.
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