Membrane topography of Escherichia coli ColE1 gene products: The channel-forming domain and the immunity protein of colicin E1
Abstract
The ColE1 plasmid of Escherichia coli encodes colicin E1, a bactericidal protein and an immunity protein. The COOH-terminal channel-forming domain of colicin E1 and the immunity protein have been studied to determine their topography in the membrane. It was important to determine whether the COOH-terminal hydrophobic anchor of colicin E1 could span the membrane once or twice to test (i) the $\alpha$-helical "hairpin" insertion model, and (ii) the possibility that helices shorter than 20 residues can also span the membrane bilayer. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic domain caused a large decrease in cytotoxicity of the purified mutant colicin E1 protein. Another 17 mutants were made in the upstream highly amphipathic segment to identify two additional trans-membrane segments. Based on the results of the mutagenesis in the channel-forming domain of colicin E1, four trans-membrane segments, two hydrophobic and two amphipathic, were predicted. The colicin E1 channel is probably monomeric and composed of four or six trans-membrane helices. It was proposed that the E1 immunity protein caused an inoperative channel protein. The topography of the colicin E1 immunity (imm) protein was determined from the pattern of phoA and complementary lacZ fusions, and site-directed substitution of charged for non-polar residues. These data imply that the 113 residue E1 immunity protein folds in the membrane as three trans-membrane $\alpha$-helices, with the NH$\sb2$- and COOH-termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The specificity of the E1 immunity protein for interaction with the translocation apparatus and the colicin E1 ion channel is proposed to reside in the peripheral segments of the immunity protein, exposed on the surface of the inner membrane. A new plasmid construct was made, with which the colicin E1 channel-forming peptide could be directly expressed. The channel peptide was readily crystallized and diffracted to about 2.6 A. Preliminary characterization of the crystals showed that the crystal has a tetragonal unit cell with a space group of I4, and the unit cell parameters, a = b = 85.6A, c = 59.2A. (Abstract shortened with permission of author.)
Degree
Ph.D.
Advisors
Cramer, Purdue University.
Subject Area
Microbiology
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