Detergent Solubilization of Mixed-Lipid Liposomes and Mixed Liposomes for Incorporation of Photosynthetic Membrane Proteins

This dissertation examines two phenomena related to the use of photosystem proteins and hydrogenase enzymes in liposomes to form light-catalyzed fusion complexes for producing molecular hydrogen. First, the method to best form fusion proteins of photosystem I (PSI) using sortase-mediated ligation was examined. In this work, a surrogate for hydrogenase, green fluorescent protein, was used and the concentrations of the ligation reaction compounds were varied to most effectively achieve fusion complexes. Second, the process of detergent solubilization of liposomes needed for PSI incorporation was examined. In this work, mixtures of lipids were used to more closely imitate natural thylakoid membranes than single lipid systems. I looked at factors that affect the ability to drive insertion of amphiphilic detergent molecules into lipid bilayers by observing changes in lipid vesicle morphology as the concentration of detergent is increased. Detergent molecules interact with liposomes by inserting their hydrophobic portion into the bilayer, thereby partitioning the lipids and causing swelling of the vesicle. As the vesicles are saturated with detergent, mixed lipid- detergent micelles bud off until there is complete dissociation of liposomes. By studying the detergent concentration these stages occur, I gained understanding about lipid-lipid interactions and factors that affect the incorporation of PSI into liposomes. In this case, I found that lipid shape, conical vs. cylindrical respectively, is a major influence in the incorporation of detergent, Triton X-100 (TX). The conical nature of ePA leads to increased mismatching within the bilayer and promotes increased uptake of TX before dissociation. Additionally, the interaction of detergent with multiple membrane types present was examined with two populations of liposomes made from egg phosphatidic acid (ePA) and egg phosphatidylcholine (ePC). An equilibrium model was derived that predicts the point of saturation for mixtures of two liposomes types and aligns with experimental observations. Furthermore, the model estimates the number of detergent molecules incorporated into each of the liposome populations.