Gating of Protein Channels in Lipid Bilayer Membranes

DESCRIPTION (provided by applicant): The long term objectives of this project are two-fold: (1) to develop a general understanding of metabolite and protein translocation through ? -barrel channels, along with a detailed description and analysis of transport through one of them - the channel formed by PA63, the channel-forming part of anthrax toxin; (2) to develop detailed molecular models of the ion-conducting pathways in channels formed by several bacterial toxins, that account for their ion permeability and protein translocation characteristics. The methodology for achieving these goals is the reconstitution of these channels in planar phospholipid bilayer membranes and studying both protein and metabolite translocation through them, and also their ion permeability characteristics. The channels focused on are those formed by the outer bacterial membrane ?- barrel channels FhuA, Cir and BtuB, the ? -barrel channel formed by PA63, and the channels formed by colicin Ia, colicin A, and the T-domain of diphtheria toxin. With respect to the first objective: we shall determine if, and under what conditions, colicin Ia can be translocated through Cir (the outer membrane transporter it has parasitized) and if ferrichrome can be transported through FhuA. We already know that the PA63 channel of anthrax toxin is the conduit for protein translocation, and we shall determine whether the secondary structure of the protein is preserved as it traverses the channel, what is the intrinsic rate of peptide movement through the channel, how does a pH gradient drive translocation, and where in the channel does the exclusion of negative charges on the translocated protein occur. With respect to the second objective: by mutating one at a time residues to cysteine and then determining whether sulfhydryl-specific reagents react with them, we shall identify the residues lining the ion-conducting pathways of the colicin Ia four-transmembrane and three- membrane segments channel and the channel formed by diphtheria toxin's T-domain. By forming chimeric channels by helix swapping between colicins Ia and A, and between diphtheria toxin's T-domain and colicin A, we shall identify which parts of colicin A are responsible for its anomalously high H+ selectivity, and in particular which residues within those helices. The insights we gain of the mechanism by which essential metabolites are transported through the outer membrane channels may lead to the development of drugs that can block this transport and thereby kill (starve) infectious pathogens. Elucidation of the details of protein translocation through the anthrax toxin channel may lead to strategies that can block this process and prove useful as a defense against this terrorist weapon. PUBLIC HEALTH RELEVANCE: The long term objectives of this project are two-fold: (1) to develop a general understanding of metabolite and protein translocation through ?-barrel channels, along with a detailed description and analysis of transport through one of them - the channel formed by PA63, the channel-forming part of the protective antigen (PA) component of anthrax toxin; (2) to develop detailed molecular models of the ion-conducting pathways in channels formed in membranes by several bacterial toxins, that account for their ion permeability and protein translocation characteristics. The methodology for achieving these goals is the reconstitution of these channels in planar phospholipid bilayer membranes and studying both protein and metabolite translocation through them, and also their ion selectivity and single channel conductance. The channels focused on are those formed by the outer bacterial membrane 2- barrel channels FhuA, Cir and BtuB, the 2-barrel channel formed by PA63, and the channels formed by colicin Ia, colicin A and the T-domain of diphtheria toxin.