Research is focused towards understanding the mechanism of action and molecular recognition features of proteins involved in bacterial protection. Energetic and biochemical methods, combined with NMR spectroscopy, are used to investigate the actions and interactions of two classes of proteins: (i) transition state regulators and (ii) response regulators. Transition state regulators are able to control bacterial """"""""decision making"""""""" by their ability to recognize and bind to a diverse array of DNA sequences. Response regulators constitute one-half of a ubiquitous communication module known as a two-component switch. Two-component switches are found in all bacteria and have been shown to be vital components in all signal transduction pathways leading to the development of virulence. As a model system, the signal transduction pathway that initiates sporulation in B. subitlis has been chosen. In response to nutrient deprivation and/or high cell density, B. subtilis passes from its vegetative-state, through a transition-state, to a self-protective stationary phase that involves the development of a spore. This system contains the transition-state regulator AbrB and a pair of two-component switches, including the response regulators Spo0F and Spo0A. It is regarded as a paradigm for bacterial signal transduction systems required for virulence, environmental sensing, cell-cycle control and cell-cell communication. The studies are designed to: (a) determine the DNA binding nature of the transition state regulator AbrB and to explain its mechanism of widespread DNA recognition. Initial investigations have revealed a putative novel DNA-binding motif which may have the capacity to adopt different conformations due to the flexible nature of the protein backbone; (b) address critical issues of molecular recognition and mechanisms involving response regulator proteins. Both the molecular recognition properties and many of the mechanistic features of the response regulators Spo0F and Spo0A have recently been shown to depend, not only on their structure, but also equally on their inherent dynamic characteristics. Moreover, the investigations have allowed Dr. Cavanagh to propose practical models for response regulator action. The research described in the proposal is designed to test the veracity of these models, subsequently extend them, and provide general insight into the two-component mode of action.
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