Kinetic description of structural changes linked to membrane import of the colicin E1 channel protein.

Abstract

Upon binding to membranes, the 178-residue colicin E1 C-terminal channel protein forms a steady-state closed-channel intermediate that is a flexible extended two-dimensional helical array [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. Analysis of the kinetics of binding-insertion to liposome membranes of the channel protein, P178, and of changes of spectral parameters associated with structure transitions allowed a correlation of the sequence of tertiary and secondary structure changes with binding-insertion. Binding and insertion were distinguished by use of lipids modified with quenchers of Trp fluorescence attached to lipid headgroups or acyl chains. Secondary and tertiary structure changes were inferred, respectively, from changes in far-UV circular dichroism and relative changes of interresidue distances by fluorescence resonance energy transfer (FRET). "Single Trp" mutants were used in FRET analysis, with the background Tyr contribution determined through use of a "zero Trp" mutant. The sequence of distinguishable events and the pseudo-first-order rate constants under "standard" conditions (large unilamellar vesicles, pH 4.0, I = 0.1 M) was binding (30 +/- 5 s(-)(1)) --> unfolding (12.6 +/- 0.5 s(-)(1)) --> helix elongation (9.0 +/- 1.0 s(-)(1)) --> insertion (6. 6 +/- 0.5 s(-)(1)). Thus, helix elongation on the surface of the membrane can occur after unfolding and does not require insertion. Binding-insertion and structural transitions of P178 occur significantly faster with small unilamellar vesicles. The relevance to general mechanisms of protein import of the structural changes associated with import of the colicin channel is discussed.