Interaction of colicin E1 with membranes and its immunity protein

Yan-Liang Zhang, Purdue University

Abstract

Three important aspects of Colicin E1 are: (1) the transformation of the water-soluble to membrane-bound state; (2) structure of surface-bound intermediate; mechanism of voltage-gating; (3) interaction with its immunity protein. 1. Electrostatic interactions in the initial binding of the colicin E1 channel fragment have a major role, as inferred from the dependence of the binding capacity (lipid molecules/channel domain) and affinity (dissociation constant of the fragment) on (a) ionic strength of the reaction medium, (b) net positive charge of the channel fragment, and (c) negative surface charge density of the membranes. Neutralization by mutagenesis of some of the positively charged lysines, suggested by analogy with colicin A to be important in channel-formation, can change the binding parameters. The fragment has a lipid affinity that is on the order of nanomolar. This high affinity might be partly attributed to the involvement of hydrophobic interactions subsequent to the initial electrostatic interactions, because the net binding was not reversible upon shifting the pH from 4 to 8 in the presence of high ionic strength. 2. An unfolded surface-bound state is indicated by increased protease susceptibility of the bound fragment relative to that of the fragment in aqueous solution. Mutations in the voltage-gating segment of K420-K461 that either eliminate a positive (e.g., 440H-$>$C) or negative charge (e.g., 446D-$>$C), or alter the sign of the positive charge (440H-$>$E), do not change the ion selectivity of the channel or voltage dependence of the rate of "turn-off". A four helix channel model is proposed for colicin E1, in which the channel lumen consists of a trans-membrane hydrophobic hairpin and an amphiphilic voltage-gating hairpin that are almost protease-inaccessible (the C-terminal 140 residues). The remaining N-terminal segments that modulate the channel activity remain outside the membrane, as indicated by extensive protease accessibility. 3. By constructing a hybrid colicin molecule, IaE1, containing channel domain of colicin E1, and the translocation and receptor domains of colicin Ia, and altering charged residues in all peripheral regions of the immunity protein to neutral residues, the involvement of translocation tol proteins in the immunity mechanism of colicin E1 was ruled out. It is concluded that the immunity protein exerts its specific effect through rapid lateral diffusion in the cytoplasmic membrane, and helix-helix recognition and interaction with at least one hydrophobic and one amphiphilic trans-membrane helix of the colicin channel. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biochemistry

COinS