19F-NMR studies on anthrax toxin protective antigen

Abstract

The anthrax protective antigen (PA) is a 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin (two-component protein complexes secreted by a number of pathogenic bacteria) secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the pre-pore, and at acidic pH undergoes a major conformational change to form a membrane spanning pore. In order to follow these structural changes at a residue-specific level, we have carried out initial studies where we have biosynthetically incorporated 5- fluorotryptophan (5-FTrp) into PA, and have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using 19F-NMR. Our studies highlight the positive impact of receptor binding on protein stability, and the use of 19F-NMR in gaining insight into structural changes in a large molecular weight protein. During conversion of the prepore to the pore, the seven phenylalanine-427 (Phe427) residues form a structure called the "phi-clamp". In order to follow the structural changes the phi-clamp at a residue-specific level we have utilized the Fuerter method to site-specifically incorporate p-fluorophenylalanine at Phe427. We have shown by 19F-NMR that the Phe427 resonance is visible in both the monomeric and prepore forms. We present our recent achievement on this work determines the effect of pore formation on 19FPhe and 19FTrp resonances. Our results show the utility of 19F-NMR in the ability to follow changes in a single residue in a large (440 kDa) protein.