11 research outputs found
Antihydrophobic Evidence for the Single Electron Transfer Mechanism of Nucleophilic Substitution
Antihydrophobic Cosolvent Effects for Alkylation Reactions in Water Solution, Particularly Oxygen versus Carbon Alkylations of Phenoxide Ions â€
Structural Uncoupling between Opposing Domains of Oxidized Calmodulin Underlies the Enhanced Binding Affinity and Inhibition of the Plasma Membrane Ca-ATPase â€
Dynamic Motion of Helix A in the Amino-Terminal Domain of Calmodulin Is Stabilized upon Calcium Activation â€
Increased Catalytic Efficiency following Gene Fusion of Bifunctional Methionine Sulfoxide Reductase Enzymes from Shewanella oneidensis
Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of \u3ci\u3eShewanella oneidensis\u3c/i\u3e MR-1
Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., ∆omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the ∆omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4’,5’-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd \u3c 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1