Combining Footprinting Mass Spectrometry and Molecular Dynamics Simulation for Structural Studies in Membrane Proteins

• DOI: 10.7936/52k5-gf85
• Publication Date: 2018-01-01
• Authors: Fengbo Zhou
• Keywords: Footprinting; Dynamics

Membrane proteins are essential in many cellular processes and represent ~60% of all current drug targets. Due to technical limits, membrane proteins of various types were not studied extensively in the past and the biochemistry and functionality of them remain unclear. The structural biology methodologies require pure isolated protein samples for us to resolve their structure and study their biochemical functions. For such in vitro studies, however, membrane proteins often become unstable when isolated from their native lipid bilayer environment. To overcome the challenge, I employed a novel methodology of solubilizing membrane proteins in solution without detergent. I reconstituted ferroportin, an iron exporter, into saposin A lipoprotein discs, which provide a phospholipid bilayer environment for stabilizing the ferroportin. Those ferroportin picodiscs samples were further used for mass spectrometry footprinting experiments. Based on the analysis, different footprinting methods are an efficient and extensive probe of specific residues over the entire sequence of ferroportin. Footprinting results by fast photochemical oxidation of proteins (FPOP) show that the extramembrane regions of ferroportin in picodiscs are extensively oxidized by free hydroxyl radicals, whereas transmembrane regions are more protected, suggesting the native structure of ferroportin is retained throughout the labeling process. In contrast, footprinting by NEM, a membrane-permeable reagent, showed extensive labeling of cysteines in both the transmembrane (hydrophobic) regions and extramembrane (hydrophilic) regions. HDX digestion of ferroportin was done in ferroportin picodiscs and a ~92% sequence coverage was achieved for footprinting. Those results demonstrate saposin picodiscs to be a feasible tool for membrane protein studies with footprinting mass spectrometry methods. Because membrane proteins remain a challenge for experimental studies, I performed molecular dynamics (MD) simulation for studying membrane proteins and started a ...