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 bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 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 helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.
Lindeberg
M
M
Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392, USA.
Zakharov
S D
SD
Cramer
W A
WA
D003087:Colicins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D008051:Lipid Bilayers; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D055672:Static Electricity
D003087:Colicins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers