B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles

Abstract

Summary: Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches classically used to measure protein partitioning into lipid bilayers, such as fluorescence and circular dichroism, are hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe binding to lipid vesicles (B2LiVe), a simple, robust, and widely applicable nuclear magnetic resonance (NMR) method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. B2LiVe relies on previously described proton 1D-NMR fast-pulsing techniques. Membrane partitioning induces a large line broadening, leading to a loss of protein signals; therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The method uses low polypeptide concentrations and has been validated on several membrane-interacting polypeptides, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions. Motivation: Characterization of the interaction of peptides and proteins with lipid membranes is relevant for the study of various biological processes and is often challenging for polypeptides, which do not possess intrinsic fluorophores and do not exhibit significant structural content changes, as well as for those characterized by low membrane affinities. To meet these challenges, we have developed a label-free 1D-1H-NMR-based experimental approach, named B2LiVe, to measure the binding of polypeptides to lipid vesicles. B2LiVe complements the arsenal of label-free biophysical assays available to characterize protein-membrane interactions.

Keywords

• CP: Molecular biology
• CP: Cell biology