Unfolding pathway of the colicin E1 channel protein on a membrane surface

The channel-forming domain of colicin E1 is composed of a soluble helical b undle which, upon membrane binding, unfolds to form an extended, two-dimens ional helical net in the membrane interfacial layer. To characterize the pa thway of unfolding of the protein and the structure of the surface-bound in termediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes i n the efficiency of fluorescence resonance energy transfer, and of the dono r-acceptor overlap integral, between each of six individual tryptophan resi dues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Compariso n of the rate constants revealed the following order of events associated w ith unfolding of the protein at the membrane surface: (A) movement of the h ydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys5 09 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V ; (C) a slow (time-scale, seconds) condensation of the surface-bound helice s. The rate of protein unfolding events increased with increasing anionic l ipid content. Unfolding did not occur below the lipid thermal phase transit ion, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the ste ady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydr ophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, i s the first described for a membrane-active protein. It is proposed that th e pathway and principles described for the colicin protein are relevant to membrane protein import. (C) 2000 Academic Press.