We have obtained diffracting crystals of two membrane proteins involved in the glycerol metabolic pathway in bacteria, glycerol facilitator and glycerol-3-phosphate dehydrogenase. This is a significant step towards crystal structure determination and these structures will yield valuable new insight, linking protein folds to function and protein-protein interactions as well as mechanisms of catalysis, regulation, and transmembrane uptake of solute molecules. Underlying effective mechanisms of bacterial proliferation are means of mediating oxidative and carbohydrate metabolism. This structural study focuses on elucidating structure-function relationships of key bacterial membrane proteins that mediate fundamental oxidative and carbohydrate metabolism. We have been engaged in structural studies of these metabolic proteins for several years and are at pivotal point in integrating all of our results into a comprehensive and coherent picture of metabolism in these pathogenic bacteria. We have determined the structures of three other members of the oxidative and glycerol metabolism pathways, including NADH peroxidase, NADH oxidase, and glycerol kinase. This proposal addresses key questions correlating structure to function and regulation. Gly-3-phosphate dehydrogenase is of particular medical importance as it is a key player in providing triose phosphate intermediates for biosynthesis of polysaccharides that form biofilm and protects the bacterium from dehydration as well as antibiotic therapy. This membrane-protein structural study has the potential to yield new and novel results, which are not provided by high-throughput structural genomics. We have overcome some of the most difficult and rate-limiting hurdles in membrane protein structural studies by obtained diffracting crystals of the glycerol facilitator (2.4 A) from Gram-positive Streptococcus pneumonia, a membrane protein involved in glycerol uptake and likely to be regulated via protein-protein interactions with glycerol kinase, whose structure we've recently determined. Subtle but significant differences exist between this and the Gram-negative E. coli facilitator. We have diffracting crystals of the glycerol-3-phosphate dehydrogenase (3.3 A) from Pseudomonas aeruginosa and have promising indications for better diffracting crystals.
Our specific aims are to obtain atomic resolution structures of both membrane proteins, both of which are likely to be novel targets for antibiotic design and therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066466-06
Application #
7092203
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Chin, Jean
Project Start
2002-08-01
Project End
2010-07-31
Budget Start
2006-08-01
Budget End
2010-07-31
Support Year
6
Fiscal Year
2006
Total Cost
$212,272
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Yeh, Joanne I; Levine, Arthur S; Du, Shoucheng et al. (2012) Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair. Proc Natl Acad Sci U S A 109:E2737-46
Brillet, Thomas; Marden, Michael C; Yeh, Joanne I et al. (2012) Interaction of haptoglobin with hemoglobin octamers based on the mutation ?Asn78Cys or ?Gly83Cys. Am J Mol Biol 2:1-10
An, Jihyun; Farha, Omar K; Hupp, Joseph T et al. (2012) Metal-adeninate vertices for the construction of an exceptionally porous metal-organic framework. Nat Commun 3:604
Du, Shoucheng; Betts, Laurie; Yang, Ruifeng et al. (2011) Structure of the HIV-1 full-length capsid protein in a conformationally trapped unassembled state induced by small-molecule binding. J Mol Biol 406:371-86
Triroj, Napat; Jaroenapibal, Papot; Shi, Haibin et al. (2011) Microfluidic chip-based nanoelectrode array as miniaturized biochemical sensing platform for prostate-specific antigen detection. Biosens Bioelectron 26:2927-33
Yeh, Joanne I; Pohl, Ehmke; Truan, Daphne et al. (2010) The crystal structure of non-modified and bipyridine-modified PNA duplexes. Chemistry 16:11867-75
Yeh, Joanne I; Shivachev, Boris; Rapireddy, Srinivas et al. (2010) Crystal structure of chiral gammaPNA with complementary DNA strand: insights into the stability and specificity of recognition and conformational preorganization. J Am Chem Soc 132:10717-27
Yeh, Joanne I; Shi, Haibin (2010) Nanoelectrodes for biological measurements. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2:176-88
Yeh, Joanne I; Kettering, Regina; Saxl, Ruth et al. (2009) Structural characterizations of glycerol kinase: unraveling phosphorylation-induced long-range activation. Biochemistry 48:346-56
Yeh, Joanne I; Chinte, Unmesh; Du, Shoucheng (2008) Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism. Proc Natl Acad Sci U S A 105:3280-5

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