This proposal is to investigate the putative role of the ubiquitous lipidic polymer, polyhydroxybutyrate, in the sorting and membrane assembly to OmpA, the major outer membrane protein of Escherichia coli. The localization of proteins to different cellular compartments is a central process in the construction of a cell. Outer membrane proteins of Gram- negative bacteria must avoid entrapment in the cytoplasmic membrane, and yet achieve recognition by the outer membrane and proper assembly within it. These processes, known as protein sorting and assembly, are not understood. Studies have indicated that, in addition to the information within the signal sequence, information required for the localization and assembly of an outer membrane protein exists in a short amino acid sequence within the mature polypeptide. Such a sequence has been identified in OmpA. Our preliminary studies have shown that short-chain poly-3-hydroxybutyrate is covalently bound to an amino acid residue within this sorting sequence. OmpA protein will be fragmented by chemical and enzymatic digestion, and peptides containing polyhydroxybutyrate will be identified with anti- polyhydroxybutyrate IgG. The selected peptides will be purified by reverse-phase liquid chromatography, and sequenced by matrix-assisted laser desorption ionization mass spectroscopy to identify the amino acid covalently bonded to polyhydroxybutyrate. Site-directed mutagenesis, followed by in vitro translation, will be employed to study the effects of substitutions or deletions of this and neighboring amino acids on the incorporation of polyhydroxybutyrate into OmpA. Mutants which produced polyhydroxybutyrate-deficient OmpA protein will be developed by transformation of selected constructs into ompA-strains. The cellular location and proper assembly of the in vivo produced mutant OmpA proteins will be investigated by cell fractionation and immuno-electron-microscopy, and by examination of characteristic properties of properly assembled OmpA-heat modifiability and sensitivity to proteases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM054090-01A2
Application #
2614295
Study Section
Special Emphasis Panel (ZRG5-MBC-1 (03))
Project Start
1998-05-01
Project End
2000-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Michigan State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Reusch, Rosetta N (2015) Poly-(R)-3-hydroxybutyrates (PHB) are Atherogenic Components of Lipoprotein Lp(a). Med Hypotheses 85:1041-3
Reusch, Rosetta N (2012) Biogenesis and functions of model integral outer membrane proteins: Escherichia coli OmpA and Pseudomonas aeruginosa OprF. FEBS J 279:893
Reusch, Rosetta N (2012) Insights into the structure and assembly of Escherichia coli outer membrane protein A. FEBS J 279:894-909
Negoda, A; Negoda, E; Reusch, R N (2010) Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA. Biochim Biophys Acta 1798:1480-4
Negoda, Alexander; Negoda, Elena; Reusch, Rosetta N (2010) Resolving the native conformation of Escherichia coli OmpA. FEBS J 277:4427-37
Dai, Dongsheng; Reusch, Rosetta N (2008) Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli. Biochem Biophys Res Commun 374:485-9
Xian, Mo; Fuerst, Michelle M; Shabalin, Yuri et al. (2007) Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate. Biochim Biophys Acta 1768:2660-6
Negoda, Alexander; Xian, Mo; Reusch, Rosetta N (2007) Insight into the selectivity and gating functions of Streptomyces lividans KcsA. Proc Natl Acad Sci U S A 104:4342-6
Zakharian, E; Reusch, R N (2005) Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer. Biochemistry 44:6701-7
Zakharian, E; Reusch, R N (2004) Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA. Biochem Biophys Res Commun 322:1059-65

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