Our preliminary studies, which include immuno-, chemical, and enzymatic assays, Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and proton nuclear magnetic resonance spectroscopy ('H-NMR) indicate that E. coli protein OmpA is modified by cPHB, an amphiphilic, salt-solvating molecule. OmpA is a major outer membrane protein that has served as a paradigm for studies of sorting and assembly of proteins of Gram-negative bacteria into the outer membrane, but despite extensive investigations these mechanisms are still not understood. Previous studies by other investigators have determined that the sorting signal is contained within the eighth B membrane strand. Our preliminary studies indicate that the binding site for cPHB is a tyrosine residue in the eighth f3-strand. Site-directed mutagenesis of this residue produced cPIIB-minus OmpA. OmpA is also widely regarded as a porin, but the question of whether it is capable of forming open pores remains controversial. Liposome-swelling assays and single-channel planar bilayer studies indicate a small percentage of molecules have open pores, whereas high-resolution X-ray structures show no evidence of a continuous water channel through the protein. Our hypothesis is that cPHB-modification is implicated in the sorting and proper membrane assembly and/or pore formation by OmpA. Here we propose to investigate the effects of cPHB on OmpA structure and function. The ability of wild type and cPHB-minus OmpA proteins to incorporate and assembly properly in the outer membrane will be examined in studies of heat-modifiability, sensitivity to proteases, and sensitivity to OmpA-specific phages. The capacity of wild type and cPFIBminus OmpA molecules to form open pores in the membrane will be investigated in single-channel planar bilayer studies. Finally, enzyme(s) responsible for cPHB synthesis in E. coli will be identified.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054090-03A2
Application #
6429958
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Chin, Jean
Project Start
1998-05-01
Project End
2006-02-28
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
3
Fiscal Year
2002
Total Cost
$203,976
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

Showing the most recent 10 out of 12 publications