Probing Protein-Lipid Interactions at the Single Molecule Level

Abstract

Cell membranes consist mostly of lipids and embedded proteins, forming a shell around the cell that controls information and material fluxes into and out of the cell. Protein-lipid interactions thus play a crucial role in numerous cellular mechanisms. Understanding these interactions would provide basic biophysical knowledge in addition to advance pharmaceutical developments, many of which target membranes. Despite their significance, protein-lipid interactions are not fully understood and have not yet been fully investigated from a mechanistic standpoint. In this study we investigated synthesized peptide constructs that insert into lipid bilayers via force spectroscopy. The constructs used here are based on SecA, a membrane-associated ATPase of the general secretory system. We investigated the first 10 amino terminal residues of SecA (SecA2-11) in three different arrangements to provide control experiments and guide interpretation. In one arrangement, two copies of SecA2-11 were covalently linked in series to the atomic force microscope tip, in another, they were linked in parallel. In all cases the constructs were brought into close proximity of a supported POPC lipid bilayer and the mechanical interaction between the peptide and lipid was recorded using single-molecule dynamic force spectroscopy. Investigating chemically similar yet geometrically different constructs provides important benchmarks for the interpretation of future studies involving longer constructs such as full length SecA.