Key Residues in Vibrio Cholerae Cytolysin Involved in Membrane Binding

Abstract

Recent studies using pore forming membrane proteins in single-molecule detection have concentrated exclusively on analyte interaction with the socalled ''canonic pore'', displaying a certain conductance under given ionic conditions and presumably corresponding to a pore structure assembled from a particular number of monomers. For instance, for the pore-forming toxin alpha-hemolysin, a conductance of approximately 1 nS in 1 M KCl at pH 7.5 is thought to correspond to a heptameric assembly, while a smaller conductance level is likely to be due to pores formed by a smaller number of monomers [1-4] Using a recently developed microarray device for parallel and high-resolution recording from suspended micro-bilayres (Microelectrode cavity array, MECA [5-7] we have begun to study the properties of the lower con-ductance form of aHL-mediated pores. The adavntage of the parallel recording system for these studies is that single canonical pores can be recorded simultaneously with low conductance forms under identical conditions. We found that low-conductance variants tend to appear after prolonged incubation of mono-mers in recording solution (3 M KCl). Interestingly, these smaller pores are blocked by poly-ethyleneglycol (PEG) oligomers in a fashion very similar to the larger canonical pores [8], showing a similar resolution of PEG mass [6,9-11]. However, surprisingly, the relative position of the maxima in the histo-gram of relative residual conductances are shifted to larger values for the smaller pores. This finding has potential implications for the mechanism of the block by PEG, in that it suggests that PEG entry into the pore adds a resistance in series with the resistance of the internal constriction site.