The pH-trigger mechanism of colicin E1 channel peptide, previously proposed by our laboratory (Merrill 'et al.', 1997), was confirmed through the design of mutant proteins with altered pH profiles for membrane binding, insertion and activity. These mutant proteins featured cumulative substitutions of serine for the acidic residues, D-408, D-410, and D-423, that were earlier described as the principal determinant of the low pH-induced helix-to-coil conformational transition of the helix-turn-helix motif containing helices 4 and 5a. The activating helix-to-coil transition of this motif was previously probed by the low pH sensitive fluorescence decay and quantum yield parameters of the two single tryptophan mutant channel peptides, F413W and W424. In this study, the time-resolved and steady-state fluorescence behaviour of F413W single tryptophan mutants, featuring the double aspartateto-serine substitutions, D408S/D423S, indicated a random-coil-like conformational state for helix 4 of this mutant channel peptide at near neutral pH. These observations provide structural and functional credence to the proposed pH-trigger mechanism and highlight a possible role for the site-specific protonation of D-408, D-410 and D-423 in the 'in vivo' mechanism of action of pore-forming colicins.