A molecular level understanding of how key signaling proteins distinguish between nitric oxide (NO) and oxygen is the central focus of this proposal. NO is a key signaling molecule that plays a central role in blood vessel dilation, penile erections and other smooth muscle related responses. NO also is a signaling agent in the central nervous system and is used by the immune system to kill infectious organisms and tumor cells. NO is toxic and chemically reactive and major questions remain concerning how this molecule is used with specificty in humans and other animals. Recent observations clearly show that bacteria contain a family of proteins that are closely related to the enzyme guanylate cyclase, the NO receptor (sensor) in humans. However, while in some cases our hypothesis is that these prokaryotic proteins are involved in NO sensing, in others is appears that they are O2 sensors. How are these proteins able to distinguish NO from oxygen is the broad overall goal of this proposal as well as understanding function. The results obtained are likely to explain not only how NO and oxygen are sensed in bacteria but will also shed light on how these molecules are sensed and used in humans and other animals. Further, the work will provide novel data on why some bacterial pathogens are not killed by NO. Experimentally this project will involve general tools of molecular biology and protein characterization plus advanced spectroscopic techniques such as resonance Raman spectroscopy. 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 oxygen. 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 to be contained in a number of facultative aerobic bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070671-03
Application #
7279906
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Jones, Warren
Project Start
2005-09-22
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
3
Fiscal Year
2007
Total Cost
$324,064
Indirect Cost
Name
University of California Berkeley
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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

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