Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity.

Abstract

The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions.