Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding.

摘要: Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with toxin (CT) and zonula occludens (Zot), that causes endemic disease cholera. Structural characterization of Ace has been restricted because limited production this toxic protein by V. cholerae. We have cloned, overexpressed, purified from strain O395 in Escherichia coli to homogeneity determined its biological activity. The unfolding was investigated using circular dichroism intrinsic tryptophan fluorescence. Because predominantly a hydrophobic protein, degree exposure regions identified spectral changes environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) quenches fluorescence residues concentration-dependent manner. Results showed bis-ANS binds one monomeric unit 1:1 stoichiometry K' 0.72 μM. exists as dimer, higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports binding virstatin, small molecule inhibits virulence regulation cholerae, Ace. constant (K=9×10(4) M(-1)) standard free energy change (ΔG°=-12 kcal mol(-1)) Ace-virstatin interaction evaluated quenching method. does not affect status A cell viability assay antibacterial activity performed various microbial strains. homology model Ace, consistent experimental results, constructed.