Characterizing The Structure Of Styrene Maleic Acid Copolymer Lipid Nanoparticles (Smalps) Using Raft Polymerization For Membrane Protein Spectroscopic Studies

Membrane proteins are necessary for the function of a variety of biological systems. They assist in transporting ions across membranes, acting as receptors affecting down-stream signaling pathways and providing structure and assembly to cells. The function of a majority of proteins are known, but little is known about their structure. Successful analysis of membrane proteins using biophysical techniques is difficult to accomplish due to protein denaturization. Membrane proteins must be in a native environment once extracted, which poses a challenge to extract the protein to analyze using spectroscopic techniques. Membrane mimetic systems such as detergent micelles, bicelles, nanodiscs, and lipodisqs have been shown to be successful for studying membrane proteins, but each system has its limitations. Polymers have recently become a promising mechanism for stabilizing membrane proteins and form a homogeneous membrane mimetic system. Recent studies have shown that a stabilizing polymer, styrene-maleic acid (StMA), can be synthesized using reversible-addition fragmentation chain transfer polymerization (RAFT) styrene-maleic acid copolymer-lipid nanoparticles (SMALPs). This homogeneous SMALP system was characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Electron Paramagnetic Resonance (EPR) spectroscopy. Three different lipid environments were titrated with styrene-maleic acid at a ratio of 3:1 (St:MA). This promising membrane mimetic system forms homogeneous SMALPs at a weight ratio of 1:1.5 (lipid:StMA) with nanoparticles ∼10 nm. V156C, a residue that lies in the hydrophobic interior of KCNQ1-Voltage Sensor Domain (Q1-VSD), was successfully incorporated into SMALPs. Continuous Wave- EPR line shape analysis shows two components of the spin label; one shows line shape broadening, indicating a lower rigid motional component and the second indicates a faster component. This study will open paths for researchers studying membrane proteins in more native membrane mimetic using biophysical techniques.