Raman Microscopy Investigation of GLP-1 Peptide Association with Supported Phospholipid Bilayers

A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into the bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. These results were compared with previous measurements of the association of fluorescently labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid bilayer on the porous silica support, suggesting that the fluorescent label alters the GLP-1 lipid bilayer association.