DEVELOPMENT OF NANODISC AFFINITY CAPILLARY ELECTROPHORESIS TO STUDY THE INTERACTION OF Giα PROTEIN WITH CELL MEMBRANES

Guanine nucleotide-binding proteins, or G-proteins, are crucial to cell signaling by acting as molecular switches through interactions with membrane-bound receptors. G-proteins have become of greater interest due to the possibility of novel drug targets. The G-protein alpha subunit, Gα, binds GTP before localizing to the membrane to interact with other G-protein subunits and G-protein-coupled receptors (GPCRs). Gi lacks a hydrophobic region to promote localization to the membrane, but a co-translational lipidation of the N-terminal residue stabilizes this interaction. Protein interactions with biological membranes are challenging to study due to the complex structure of membranes and the often-limited quantities of the sample protein. To simplify the system and structure of membranes, membrane mimics, such as nanodiscs, are often utilized. Capillary electrophoresis has been a proven technique for measuring protein interactions due to the small amounts of required sample. In this study, the interaction of Gα with styrene-maleic acid co-polymer belt nanodiscs was measured using nanodisc affinity capillary electrophoresis (NACE). The data obtained suggests a complicated kinetic system that might be dependent upon a third component or a conformation change of the protein. Here we show the potential for NACE to estimate dissociation constants for proteins with membranes and discuss the limitations of the technique