? 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 ? ?
| Plate, Lars; Marletta, Michael A (2013) Phosphorylation-dependent derepression by the response regulator HnoC in the Shewanella oneidensis nitric oxide signaling network. Proc Natl Acad Sci U S A 110:E4648-57 |
| Weinert, Emily E; Phillips-Piro, Christine M; Marletta, Michael A (2013) Porphyrin ?-stacking in a heme protein scaffold tunes gas ligand affinity. J Inorg Biochem 127:7-12 |
| Winter, Michael B; Woodward, Joshua J; Marletta, Michael A (2013) An Escherichia coli expression-based approach for porphyrin substitution in heme proteins. Methods Mol Biol 987:95-106 |
| Plate, Lars; Marletta, Michael A (2013) Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem Sci 38:566-75 |
| Winter, Michael B; Klemm, Piper J; Phillips-Piro, Christine M et al. (2013) Porphyrin-substituted H-NOX proteins as high-relaxivity MRI contrast agents. Inorg Chem 52:2277-9 |
| Plate, Lars; Marletta, Michael A (2012) Nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-GMP signaling network. Mol Cell 46:449-60 |
| Derbyshire, Emily R; Marletta, Michael A (2012) Structure and regulation of soluble guanylate cyclase. Annu Rev Biochem 81:533-59 |
| Tran, Rosalie; Weinert, Emily E; Boon, Elizabeth M et al. (2011) Determinants of the heme-CO vibrational modes in the H-NOX family. Biochemistry 50:6519-30 |
| Winter, Michael B; Herzik Jr, Mark A; Kuriyan, John et al. (2011) Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain. Proc Natl Acad Sci U S A 108:E881-9 |
| Weinert, Emily E; Phillips-Piro, Christine M; Tran, Rosalie et al. (2011) Controlling conformational flexibility of an O?-binding H-NOX domain. Biochemistry 50:6832-40 |
Showing the most recent 10 out of 25 publications
