Research
Anthrax protective antigen
The anthrax protective antigen is a protein secreted by Bacillus anthracis, the causative agent of anthrax disease. Protective antigen binds to a host cellular receptor, and recruits two other toxins secreted by B anthracis: edema factor and lethal factor. The assembly of the complex of edema factor or lethal factor and protective antigen constitutes the anthrax toxin, and is the major cause of death in individuals that contract the disease. Our research is directed towards understanding how the protective antigen forms a membrane spanning pore at low pH. Formation of the pore is absolutely critical for the pathogenesis of the toxin, since the pore provides a conduit for entry of the edema or lethal factor into the cell cytosol.
The exact mechanism of pore formation is not understood, but can be prevented by mutating certain residues within protective antigen. We are currently investigating how these mutations prevent pore formation, using methods that include circular dichroism spectroscopy, fluorescence, and fluorine NMR.
Fluorinated amino acids
Our laboratory has developed methods to biosynthetically incorporate fluorinated amino acids into proteins, and a major focus of our laboratory is to expand the repertoire of fluorinated amino acids that have unique functionalities, and to incorporate these amino acids into proteins for structural studies. Fluorine (19F), is only slightly larger than hydrogen, is a spin ½ nucleus, is 100% naturally abundant and is almost as sensitive as 1H. The NMR spectrum of a fluorinated protein typically exhibits well resolved resonances which are sensitive to environment, allowing the researcher to follow structurally changes in select regions of the protein at a residue-specific level. Along with fluorinated aromatic amino acids, we have shown that both 2- and 4-fluorohistidine can be incorporated into proteins. 2-fluorohistidine has a side-chain pKa of 1, and uniform labeling of the protective antigen with this amino acid has been used to address the role of histidine protonation in governing the conformational change from a prepore to a membrane spanning pore, for instance. We have also synthesized 3-(R and S) fluoroproline, which exhibits properties similar to that of proline in terms of the equilibrium ratio of cis and trans isomers. This is important for studies aimed at understanding the influence of proline isomerization on protein folding. We are continuing to develop new fluorinated amino acids to address specific questions related to protein structure or function.