Channel formation of colicin E1 and purification and modeling of its immunity protein

Jacobus Bernardus Heymann, Purdue University

Abstract

I. Colicin E1. The E. coli cytotoxin, colicin E1, undergoes large transformations upon binding to the vitamin B$\sb{12}$ receptor at the cell surface, translocation across the periplasmic space aided by the Tol proteins, and insertion into the cytoplasmic membrane to form a voltage-gated channel. The open channel dissipates the cellular membrane potential and causes massive potassium efflux, resulting in cell death. Colicin E1 is expressed from a natural plasmid, pColE1, which also encodes an immunity protein, ImmE1, that protects the colicin-producing cells against colicin. In vitro channel-forming activity of colicin E1 is facilitated by a negatively charged membrane and acidic pH. Binding of colicin E1 to artificial membranes was assayed using lipophilic quenchers of tryptophan fluorescence. Initial binding of the colicin E1 channel-forming domain to a negatively charged membrane at low pH is predominantly electrostatic. It is followed by non-electrostatic binding of equivalent or larger magnitude indicating insertion into the membrane. Insufficient or excessive electrostatic binding decreased channel activity, suggesting that an optimal electrostatic interaction is required for subsequent insertion of the channel into the membrane. The open channel structure was probed in planar bilayer experiments using single-cysteine mutants of colicin E1 in residues 478-481. Only the V480C mutant channel could be blocked by a thiol-reactive compound, indicating that this residue faces the channel lumen. II. Colicin E1 immunity protein, ImmE1. ImmE1 was cloned behind a variety of promoters and a number of fusion proteins were constructed to facilitate purification. A glutathione S-transferase fusion was purified by affinity chromatography on a glutathione column, followed by cleavage with thrombin and reverse phase HPLC purification of the ImmE1 fragment. As an aid to mutagenesis, a three-dimensional model of ImmE1 was produced from finding the most probable orientations and interactions of the predicted three trans-membrane helices, consisting of a dimeric, six-helix bundle structure, consistent with the observation of a SDS-resistant dimer. In addition, a model for immunity was developed to indicate the likelihood of a mechanism for targeting the immunity protein to colicin entry sites in the cell envelope.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biophysics|Molecular biology|Biochemistry

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