Linda M Columbus
University Of Virginia Charlottesville
Project start date: 2009-04-01
Project end date: 2014-01-31
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STRUCTURE AND DYNAMICS OF BACTERIAL MEMBRANE PROTEIN - RECEPTOR INTERACTIONS
Linda M Columbus, Assistant Professor
University Of Virginia Charlottesville, Box 400195, Charlottesville, Va 22904-4195
Grant 5R01GM087828-02 from National Institute Of General Medical Sciences
Abstract: Many obligate bacterial membrane proteins hijack human cellular pathways by mimicking or manipulating host machinery. The goal of this research is to investigate the structure and dynamics of bacterial outer membrane proteins and their interactions with host receptors. Specifically, research is focused on the outer membrane opacity-associated proteins (Opa) from Neisseria gonorrhoeae and Neisseria meningitides, which induce engulfment of the bacterium in non-phagocytic cells by binding to host receptors. Opa proteins bind to various host receptors and are classified into two families based on host receptor selectivity. The larger class, OpaCEA, bind to carcinoembryonic antigen-like cellular adhesion molecules (CEACAMs), and the smaller class, OpaHS, bind to two different receptors; the heparansulfate proteoglycan receptors (HSPGs) directly and indirectly to integrin receptors via a heparin- mediated interaction with fibronectin or vibironectin. Opa proteins are integral outer membrane proteins and predicted to have an eight-stranded 2-barrel fold. Two of the extracellular loops (HV1 and HV2) have the most sequence variation between Opa proteins and determine the host receptors specificity. Not only do the HV loops discriminate between HSPG and CEACAM receptors, but OpaCEA proteins can be further divided into subgroups based on the selective binding to four of the seven CEACAM receptors. Using nuclear magnetic resonance, electron paramagnetic resonance, isothermal titration calorimetry, and mutagenesis, the molecular determinants of these interactions will be determined. The results will provide insight into the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitides and, therefore, the potential for the rational design of novel antibiotics. In addition, the reconstituted Opa proteins may be useful for vaccine development. However, the most novel application of this research lies in the ability of Opa proteins to target host receptors specifically via three different mechanisms to induce endocytosis in non-phagocytic cells. This ability may be useful for liposome pharmaceutical carriers. The potential ability of liposome encapsulated therapeutics (e.g. enzymes, inhibitors, and peptides) to enter the cytoplasm of living cells and possibly tissue selectively is of crucial importance to the treatment of many diseases. Understanding the molecular determinants of the three Opa-mediated entry mechanisms may facilitate the development of liposome delivery mechanisms. This research aims to determine how bacteria interact with human cells. By gaining an understanding of these molecular interactions, insights into the rational design of novel antibiotics, vaccine development, and targeting of liposome pharmaceutical carriers will be obtained
Keywords: No Project Terms available
Relevance: This research aims to determine how bacteria interact with human cells. By gaining an understanding of these molecular interactions, insights into the rational design of novel antibiotics, vaccine development, and targeting of liposome pharmaceutical carriers will be obtained
Project start date: 2009-04-01
Project end date: 2014-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-253
5R01GM087828-02 (2010): $280936
Grants awarded to Linda M Columbus
NMR Structure Of Membrane Proteins Of T. Maritima
Linda M Columbus
Scripps Research Institute La Jolla, Ca 920371000
Grant 5F32GM068286-03 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Membrane protein structure determination is a difficult task for structural biologists. Membrane proteins comprise 30% of the proteome. However, only ~30 unique structures have been determined, making their contribution to the protein data bank less than 0.5%. The majority of membrane proteins are transmembrane helical bundles, but of the known structures roughly half are beta-barrel proteins, which are limited to the outer membrane of bacteria, mitochondria, and chloroplasts. This study targets helical membrane proteins with two, three, and four predicted transmembrane segments for NMR structure determination in order to significantly diversify the different types of membrane protein structures known. Defective membrane proteins have been identified in many diseases due to misfolding and loss of function making them targets for drug design. Structural information could advance the effectiveness of these drug pursuits. In addition, more helical transmembrane structures will aid in the understanding of membrane protein stability and increase the likelihood of predicting membrane protein structures.
Keywords: Archaea, bacterial protein, membrane protein, protein structure function, proteomics, conformation, functional /structural genomics, circular dichroism, nuclear magnetic resonance spectroscopy, polymerase chain reaction, postdoctoral investigator, protein purification, site directed mutagenesis
Project start date: 2003-06-16
Project end date: 2006-06-15
5F32GM068286-03 (2005): $49928
5F32GM068286-02 (2004): $47296
1F32GM068286-01 (2003): $41608
Structure And Dynamics Of Bacterial Membrane Protein - Receptor Interactions
Linda M Columbus
Chemistryuniversity Of Virginia Charlottesville
Grant 1R01GM087828-01 from National Institute Of General Medical Sciences IRG: BBM
Abstract: Many obligate bacterial membrane proteins hijack human cellular pathways by mimicking or manipulating host machinery. The goal of this research is to investigate the structure and dynamics of bacterial outer membrane proteins and their interactions with host receptors. Specifically, research is focused on the outer membrane opacity-associated proteins (Opa) from Neisseria gonorrhoeae and Neisseria meningitides, which induce engulfment of the bacterium in non-phagocytic cells by binding to host receptors. Opa proteins bind to various host receptors and are classified into two families based on host receptor selectivity. The larger class, OpaCEA, bind to carcinoembryonic antigen-like cellular adhesion molecules (CEACAMs), and the smaller class, OpaHS, bind to two different receptors; the heparansulfate proteoglycan receptors (HSPGs) directly and indirectly to integrin receptors via a heparin- mediated interaction with fibronectin or vibironectin. Opa proteins are integral outer membrane proteins and predicted to have an eight-stranded 2-barrel fold. Two of the extracellular loops (HV1 and HV2) have the most sequence variation between Opa proteins and determine the host receptors specificity. Not only do the HV loops discriminate between HSPG and CEACAM receptors, but OpaCEA proteins can be further divided into subgroups based on the selective binding to four of the seven CEACAM receptors. Using nuclear magnetic resonance, electron paramagnetic resonance, isothermal titration calorimetry, and mutagenesis, the molecular determinants of these interactions will be determined. The results will provide insight into the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitides and, therefore, the potential for the rational design of novel antibiotics. In addition, the reconstituted Opa proteins may be useful for vaccine development. However, the most novel application of this research lies in the ability of Opa proteins to target host receptors specifically via three different mechanisms to induce endocytosis in non-phagocytic cells. This ability may be useful for liposome pharmaceutical carriers. The potential ability of liposome encapsulated therapeutics (e.g. enzymes, inhibitors, and peptides) to enter the cytoplasm of living cells and possibly tissue selectively is of crucial importance to the treatment of many diseases. Understanding the molecular determinants of the three Opa-mediated entry mechanisms may facilitate the development of liposome delivery mechanisms. This research aims to determine how bacteria interact with human cells. By gaining an understanding of these molecular interactions, insights into the rational design of novel antibiotics, vaccine development, and targeting of liposome pharmaceutical carriers will be obtained
Project start date: 2009-04-01
Project end date: 2014-01-31