Heme Protein Sensors in Prokaryotes

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DESCRIPTION (provided by applicant): We seek a molecular level understanding of how key signaling proteins distinguish between nitric oxide and oxygen. Recent observations clearly show that prokaryotes contain a family of proteins that are closely related to the heme NO sensor in the enzyme guanylate cyclase. However, while in some cases our hypothesis is that these prokaryotic proteins are involved in NO sensing, in others is appears that they are 02 sensors. How are these proteins able to distinguish NO from 02 is the broad overall goal of this proposal as well as understanding function. The specific aims are: (1) clone, express and purify heme protein sensors (HPS) from Nostoc punctiliforme, Caulobacter crescentus, Vibrio cholerae, and Legionaella pneumophilia, (2) characterize the ligand binding properties of the HPSs from specific aim 1, (3) clone express and purify the histidine kinases associated with each HPS from Nostoc punctiliforme, Caulobacter crescentus, Vibrio cholerae, and Legionaella pneumophilia, (4) characterize the HPS:Histidine Kinase signaling events, (5) clone, express and purify the relevant response regulators to enable the characterization of the their potential interaction with the appropriate histidine kinase, (6) characterization of a Caulobacter crescentus HPS knockout, (7) clone, express and purify the HPS from Thermoanaerobacter tengcogensis and Clostridium botulinum, (8) probe the function of the Thermoanaerobacter tengcogensis and Clostridium botulinum HPS-MCP (methyl-accepting chemotaxis domain) fusion protein, (9) structure determination of representative HPSs, (10) clone and characterize the putative HPS from Rhodobacter sphaeroides. Experimentally this project will involve general tools of molecular biology and protein characterization plus advanced spectroscopic techniques such as resonance Raman. In addition, protein crystallization structure determination and functional studies by gene transfection into E. coli will be used. The structural determinants that allow for this difficult discrimination are not obvious and remain at the heart of biological recognition and specificity of signaling. What appeared for the last 10 years to be a characterization of proteins that bind NO has now been expanded to include 02. CO signaling is an active area of research lacking among other things a specific receptor. Proteins in this family are likely possibilities for this key, missing component. Furthermore, when pathogens respond to a NO challenge from the immune system, they could use a receptor system similar to the apparent NO signaling system hypothesized