INTEGRATED LIGHT-SCATTERING SPECTROSCOPY, A SENSITIVE PROBE FOR PEPTIDE-VESICLE BINDING - APPLICATION TO THE MEMBRANE-BOUND COLICIN E1 CHANNEL PEPTIDE

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamel lar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditi ons were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The in crease in vesicle size upon the insertion of the channel peptide was m easured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were deter mined for five different systems having peptide/vesicle ratios of appr oximately 0, 77, 154, 206, and 257. The experiment was repeated four t imes (twice at two different vesicle concentrations) to determine repr oducibility. The relative changes in vesicle radius upon peptide bindi ng to the membrane vesicles was remarkably reproducible even though th ese changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made be tween ILS and dynamic light scattering (DLS) data and showed similar r esults. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was similar to 177 +/- 22 nm(3). These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane result ing in a significant displacement of the lipid bilayer as evidenced fr om the dose-dependent increase in the vesicle radius. These results in dicate that ILS spectroscopy is a sensitive sizing technique that is c apable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.