Thrust Area 1: Purification and Biophysical Characterization of MR-1 Redox Proteins (LDRD 1: Specific Aims 5&6)
EMSL Project ID
14091
Abstract
A facultative anaerobic bacterium, Shewanella oneidensis MR-1 is able to use metal oxides, such as Mn(IV) and Fe(III), as terminal electron acceptors during anaerobic respiration. Since Mn(IV) and Fe(III) are highly insoluble under neutral pH, S. oneidensis MR-1cells have developed a sophisticated electron transport network that transports electrons across cell walls, which include cytoplasmic membrane or inner membrane (IM), periplasmic space and outer membrane (OM), to the cell surfaces to reduce these insoluble metal oxides. C-type cytochromes are the major components of the electron transport network of S. oneidensis MR-1 that contains 39 putative c-type cytochromes (Heidelberg et. al., 2002). Located at OM, MtrC is required for efficient reduction of Mn (IV) and Fe (III) by S. oneidensis MR-1. Because of its OM location, it is speculated that MtrC transfers electrons directly to metal oxides. Despite the essential role played by MtrC in metal oxide reduction, the molecular mechanism deployed by MtrC to reduce metal oxides has not been determined. The goal of this project will be to produce, isolate and characterize MtrC and associated proteins such as MtrA using biochemical and biophysical methods.Summary of Expected Results
We expect to generate milligram quantities of purified MtrC, MtrA and associated or related proteins for biophysical characterization. Polyclonal antibodies of MtrC, MtrA and associated proteins will be generated as needed for biochemical and cellular studies. Several novel expression constructs and expression strategies MtrC and MtrA will be developed over the course of the project. We expect to generate three-dimensional crystals of MtrA and MtrC that will used for subsequent structural studies using x-ray crystallography. We will attempt to identify MtrC-associated proteins by characterizing intact MtrC complexes isolated from the outer membrane of S. oneidensis MR-1 cells. Model membrane and membrane-mineral systems will be developed and tested for their ability to enable spectroscopic interrogation of heme centers and their orientation in environments representative of the gram-negative bacterial membrane surface.
Specific Aim #5: EPR Studies of Heme Centers, Heme Ligand Orientations and Redox Properties of MtrC in Intact Membranes.
Specific Aim #6: EPR Studies of Heme Centers, Heme Ligand Orientations and Redox Properties for Membrane-Associated MtrC in on mineral surfaces.
Project Details
Project type
Grand Challenge
Start Date
2005-03-17
End Date
2006-12-28
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members