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

The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral me mbrane protein localized in the Escherichia coli cytoplasmic membrane, prot ects the cell from the lethal, channel forming activity of the bacteriocin, colicin El. Utilizing its solubility in organic solvents, ImmE1 was purifi ed by I-butanol extraction of isolated membranes, followed by gel filtratio n and ion-exchange chromatography in a chloroform/methanol/H2O (4:4:1) solv ent system. Circular dichroism analysis indicated that the alpha-helical co ntent of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydr ophobic transmembrane helical domains, H1-H3, Each of these extended hydrop hobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure o f purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H2O (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 si ngle and double Cys-to-X mutants of the Imm protein and their in vivo activ ity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- H SQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broa dening and disappearance of the indole H-1 proton resonance from Trp94 in H 3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near -UV circular dichroism spectra with a prominent ellipticity band centered a t 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded confo rmation in chloroform/methanol/H2O (4:4:1) that is stabilized by helix-heli x interactions. Analysis of the probable membrane folding topology indicate d that several Tyr residues in the bilayer region of the three transmembran e helices could contribute to the near-UV CD spectrum through helix-helix i nteractions.