A. Anthrax Protective Antigen: Protective antigen (PA) undergoes a pH induced conformational change to form a pore through which edema factor and lethal factor are transported into the cell, resulting in cell death. This conformation change is characterized using oxidative protein footprinting. The prepore (pH 7.5) and pore (pH 5.5) structures are probed using OPF using laser photolysis flow cell system, the differentially modified residues are identified and mapped to the structure to identify solvent accessible residues of each conformation. Under physiological conditions PA is bound to a transmembrane anthrax toxin receptor (capillary morphogenesis gene 2, CMG2) associated with the cell membrane. In the previous experiments the lack of receptor and cell membrane may have serious implications in the structure of the PA complex. To overcome this limitation a lipoparticle system which incorporates a large number of the anthrax toxin receptors (CMG2) onto the surface of a stable nanoscale membrane particle was implemented. These non-infectious lipoparticles are derived from cells using retroviral structural proteins (GAG) and closely mimics in-vivo conditions for pH induced conformational change of PA. Although we were able to see oxidation, it was only on one methionine in the PA sequence. The remaining oxidized species were below our limits of detection. We have also investigated changes in digestion conditions to obtain better coverage of the oxidized PA. Using appropriate detergents and enzymes, we have been able to obtain better coverage of the pore peptides. B. Characterization of anti-Streptococcus mutans P1 protein neutralizing antibody (collaboration with Prof. Jeanne Brody/Paula Crowley, UF. The long-term goal of the project is to understand the molecular basis of bacterial adhesion and biofilm formation, particularly with regard to the oral pathogen Streptococcus mutans and to probe the mechanisms of protective immunity. The S. mutans P1 work is directed at elucidating the molecular mechanisms of antibody-mediated immunomodulation and to use monoclonal antibodies as tools to probe structural features of the antigen with the goal of guided re-engineering and improvement in vaccine design. Our role in this project is to characterize the epitope on S. mutans P1 that is recognized by the antibody MAb 4-10A. P1 is characterized by a number of highly homologous sequences in the region of the protein recognized by MAb 4-10A. Our goal is to identify the specific sequence/region that the antibody recognizes. Our initial experiments involve differential oxidation of P1 constructs with and without the antibody being bound. Data have been acquired and are being analyzed. C. Aprataxin. Aprataxin (APTX) is a novel DNA repair protein whose dysfunction causes the neurodegenerative disease, called Ataxia Oculomotor Apraxia 1 (AOA1). As a means of correcting failed DNA ligation events, APTX hydrolyzes 5'-adenylated damaged DNA ends left by abortive ligase reactions, to regenerate catalytically ligatable DNA 5'-phosphates. Using photolytic oxidation, chemical modification, and H/D exchange coupled to mass spectrometric analyses, the solvent-accessibility of APTX with and without nicked DNA binding has been probed.
