Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Ttp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin El in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404W, F-413W, F-443W, F-484W, and W-495 peptides exhibited small and/or insignificant REES changes (DeltaREES) whereas W-424, F-431W, and Y-507W channel peptides possessed modest REES changes (3 mnless than or equal to5DeltaREESless than or equal to7 nm). In contrast, wild-type, Y-367W, W-460, Y-478W, and I-499W channel peptides showed large DeltaREES values upon membrane binding (7 nm