Acrylamide quenching of the intrinsic fluorescence of tryptophan residues genetically engineered into the soluble colicin E1 channel peptide. Structural characterization of the insertion-competent state

Colicin E1 or any of its COOH-terminal channel peptides can be activated in vitro by acidic (< 4.5) pH or detergents. In its activated or insertion-competent state, the colicin E1 thermolytic (178 residue) channel peptide demonstrated an increased ability to bind and form channels in artificial membranes. An earlier report [Merrill et al. (1990) Biochemistry 29, 5829-5836] indicated that the structural change occurring in the channel peptide upon activation was not a large unfolding but seemingly involves a more subtle conformational change. To probe the solution structure of the colicin channel peptide and the structural changes occurring upon activation, 12 single-tryptophan-containing mutant peptides have been prepared. All of the peptides displayed cellular cytotoxicity comparable to the wild-type peptide. Fluorescence quenching by acrylamide of each Trp residue genetically engineered into the channel peptide indicated that tryptophyls located at positions 355, 367, 393, 413, and 443 report significant conformational changes which are associated with the insertion-competent state. Calculation of the bimolecular quenching constants for each single-Trp peptide showed that there are three classes of Trp residues found in the native colicin E1 channel peptide. None of the Trp residues were found to be completely inaccessible to acrylamide (buried). The NH2-terminal region near Trp-355 and -367 along with the COOH-terminal hydrophobic domain, including Trp-484, -495, and -507, was largely buried in the channel peptide soluble structure. Two peptide segments, one containing Trp-393, -404, and -413 and a second encompassing Trp-431 and -443, were moderately to very exposed regions in the soluble channel peptide.