Colicins of the E1 family are bactericidal proteins which exert their lethal action by forming voltage-gated ion channels in the cytoplasmic bacterial membrane. The crystallographic structure in the water soluble state of one colicin is known. The formation and gating of colicin E1 channels are being studied in voltage-clamped, solvent-free, planar bilayer phospholipid membranes. These studies will lead to knowledge of the movements, on an atomic scale, of the protein during refolding from its water soluble to membrane-bound form and the conformational changes responsible for opening and closing of the channels. The properties of colicin E1 channels in planar bilayers thus provide a well-defined system to delineate the physico-chemical principles that underly the physiological processes of channel gating and protein translocation through bilayers. The enumeration of these principles would facilitate strategies for coupling protein toxins (e.g. ricin, abrin, diptheria) to carrier proteins so that the toxins retain their activity and cross targeted (e.g. transformed) cell membranes. This is, for example, the goal when designing immunotoxins -- toxins coupled to antibodies. This proposal is specifically directed toward determining the regions and residues of colicin that are translocated when the channel is gated by voltage and resolving the folding pattern of these regions in the bilayer. The voltage-dependence of deactivation of site-directed mutants that have charges added or deleted at defined residues will be measured. This dependence will give the fraction of the applied voltage sensed by each of the altered residues. Because this fraction sets the location of the mutated residues within the bilayer, the folding pattern of the channel will be obtained. Direct confirmation of proposed folding patterns and translocated regions will be sought by complexing membrane-impermeant avidin, added to the trans-aqueous compartment, with biotinylated colicin to lock biotinylated residues to the trans side. If the residues are translocated, deactivation will be inhibited. The deactivation kinetics are strongly dependent on the pH of the trans-aqueous compartment. Acidic residues, facing the trans compartment, that are neutralized by protonation at low pH (<4 - 5) are thought to be responsible for this pH dependence. Acidic residues that are candidates to face the trans compartment will be mutated to neutral ones to determine if the rates of deactivation are increased at high pH.
| Cramer, W A; Heymann, J B; Schendel, S L et al. (1995) Structure-function of the channel-forming colicins. Annu Rev Biophys Biomol Struct 24:611-41 |
