Experimental goals outlined in this proposal are to characterize the structural changes in the colicin E1 (ColE1) channel domain that accompany membrane binding, insertion and channel formation and develop a model for interaction of colicin with immunity protein. Long-term objectives of this research are to gain a better understanding of the mechanism of action of pore-forming toxins, protein unfolding and interaction with membranes, structure and dynamics of membrane channels and inhibition of channel formation by secondary proteins. (i) Using the structure of the soluble ColE1 channel domain to guide their placement, cysteine residues will be introduced into pairs of alpha-helices and linked by disulfide bonds, thereby restricting the movement of cross- linked helices. Effects on membrane binding, insertion and channel formation will be evaluated by measuring changes in binding forces, fluorescence quenching and channel activity. Residues lining the channel lumen will be identified by individually replacing consecutive residues in helix 7 with cysteine and measuring channel blockage by methane- sulfonate derivatives in a planar bilayer system. (ii) A model for interaction with immunity protein will be developed using the Col10 system, which is similar in sequence to the ColE1 system yet differs in the recognition pattern of its immunity protein. Guided by information on structure, sequence and phenotype, ColE1 and its immunity protein will be selectively mutagenized tested for changes in recognition and their regions of interaction identified.
