Kinetic description of structural changes linked to membrane import of thecolicin El channel protein

Upon binding to membranes, the 178-residue colicin E1 C-terminal channel pr otein forms a steady-state closed-channel intermediate that is a flexible e xtended two-dimensional helical array [Zakharov et al. (1998) Proc. Natl. A cad. Sci, U.S.A. 95, 4282-4287]. Analysis of the kinetics of binding-insert ion to liposome membranes of the channel protein, P178, and of changes of s pectral parameters associated with structure transitions allowed a correlat ion of the sequence of tertiary and secondary structure changes with bindin g-insertion, Binding and insertion were distinguished by use of lipids modi fied with quenchers of Trp fluorescence attached to lipid headgroups or acy l chains. Secondary and tertiary structure changes were inferred, respectiv ely, from changes in far-UV circular dichroism and relative changes of inte rresidue distances by fluorescence resonance energy transfer (FRET), "Singl e Trp" mutants were used in FRET analysis, with the background Tyr contribu tion determined through use of a "zero Trp" mutant. The sequence of disting uishable 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 signi ficantly faster with small unilamellar vesicles. The relevance to general m echanisms of protein import of the structural changes associated with impor t of the colicin channel is discussed.