Identification of a chameleon-like pH-sensitive segment within the colicin E1 channel domain that may serve as the pH-activated trigger for membrane bilayer association.

In vitro, the channel-forming domain of colicin E1 requires activation by acidic pH (<4.5) or detergents. The activation of this domain to its insertion-competent state results in an increased ability of the protein to dock onto and to form channels in artificial membranes. Fluorescence methods were used to characterize the conformational changes occurring in a channel-forming peptide of colicin E1 in solution with pH. The 178-residue thermolytic fragment of colicin E1 contains three Trp residues, W-424, W-460, and W-495. In order to study the structural and dynamic requirements for activation of the C-terminal domain of colicin E1, single-Trp-containing peptides were prepared by site-directed mutagenesis. All of the mutant peptides displayed in vitro channel activity and cellular cytotoxicity similar to the those of wild-type peptide. Two Trp residues, W-413 and W-424, exhibited pH-sensitive fluorescence parameters. Upon acidification (pH 6.0 --> 3.5), the fluorescence quantum yield of W-413 and W-424 increased 50% and 80%, respectively, indicating a significant change in the local environment of the peptide segment containing these two Trp residues. The fluorescence decay of W-413 and W-424 was best fit by three fluorescence decay components, two of which were sensitive to pH. However, only small changes in spectral shape and position were observed for W-424 fluorescence, whereas there were larger changes in these fluorescence parameters for W-413. The quantum yields for the Trp residues in the seven other single-Trp mutant peptides and the wild-type peptide were distinct but only slightly affected by changes in pH. Time-resolved fluorescence measurements showed that W-460, -484, and -495 each had two fluorescence decay components with similar decay times, with one component dominating the fluorescence decay behavior. Furthermore, the individual fluorescence decay times for all the single-Trp peptides, except for W-413 and W-424, were insensitive to pH changes. At pH 3.5, the fluorescence of the wild-type peptide was fit by three decay time components, with the two longer decay times being quite different from the fluorescence decay times of the single-Trp mutant proteins (W-424, -460, and -495, the naturally occurring Trp residues). In contrast, at pH 6.0, the wild-type peptide showed double-exponential decay kinetics. Time-resolved fluorescence anisotropy decay measurements of the three single-Trp mutant proteins, containing a naturally occurring Trp residue, suggest that local segmental motion of the peptide as reported by each of the three tryptophans is highly restricted and largely insensitive to changes in pH. On the other hand, the anisotropy decay profiles of the wild-type protein were consistent with energy transfer occurring between Trp residues, likely between W-460 and W-495. These steady-state and time-resolved fluorescence results show that W-413 and W-424 report conformational changes which may be associated with the insertion-competent state and reside on the protein segment(s) which form the pH-activated trigger of the channel peptide.