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We have identified the steady-state and time-resolved fluorescence of the three tryptophan residues (Trp-86, Trp-130, and Trp-140) of the pore-forming domain of colicin A using site-directed mutagenesis in order to construct two- and one-tryptophan-containing mutant proteins. Fluorescence lifetimes were measured via multifrequency phase fluorometry. The fluorescence of the pore-forming domain of colicin A is dominated by Trp-140 which contributes almost 53% to the fluorescence intensity. Mutation of Trp-140 results in a decrease in fluorescence quantum yield and average lifetime. Colicin A wild-type and all mutant proteins display multiple lifetimes which belong to three different lifetime classes: 0.38-0.57 ns for tau 1, 1.6-1.87 ns for tau 2, and 3.6-4.41 ns for tau 3 at pH 5. At pH 7, the three classes are 0.64-0.89 ns for tau 1, 2.01-2.19 ns for tau 2, and 4.23-4.94 ns for tau 3. This pH effect influences all the lifetimes and must be attributed to a general conformational change. In wild-type colicin A, tau 3 originates mainly from Trp-140 while Trp-86 and Trp-130 both provide a major contribution to tau 2. The pH dependence of the fluorescence intensity gives rise to a pKa of 5.2. The different lifetime components of two of the three single-tryptophan-containing mutants show different quenching properties toward acrylamide, indicating that each lifetime is coupled to a different microenvironment. The linear combination of the lifetimes of the single tryptophans into pairs simulates very well the behavior of the two-tryptophan-containing mutants except for one, the mutant containing Trp-86 and Trp-130. The lifetimes of the wild-type protein can only be obtained by the linear combination of the lifetimes from the mutant containing the tryptophan pair Trp-86/Trp-130 and the mutant containing Trp-140. Mutual energy transfer between Trp-86 and Trp-130 is assumed to be the explanation of this deviation since the mutant proteins display no structural or dynamic aberrances. The calculated energy transfer efficiency amounts to 65% for energy transfer from Trp-86 to Trp-130 and 21% for the reverse transfer and is in agreement with our measurements.

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).