Preparation and Characterization of Novel Lipid and Proteolipid Membranes from Polymerizable Lipids
AdvisorSaavedra, S. Scott
Committee ChairSaavedra, S. Scott
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe work described here has focused on two types of supramolecular assemblies, supported lipid bilayers (SLBs) and giant vesicles (GVs) from polymerizable lipids. SLBs are explored extensively as structural models in biophysical studies of cell membranes and biosensor coatings. With regard to implementation as biocompatible scaffoldings for receptor-based molecular devices, fluid SLBs lack chemical, thermal and mechanical stability as lipids are self-organized by weak, noncovalent forces. One possible solution is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how polymerization affects transmembrane protein structure and activity. Specifically it is unclear if lipid cross-linking can be achieved without adversely affecting the activity of incorporated proteins. In this work the effect of lipid polymerization on transmembrane protein activity was studied with rhodopsin. The protein was reconstituted into SLBs composed of polymerizable lipids, bis-SorbPC, bis-SorbPC:mono-SorbPC, bis-DenPC and bis-SorbPC:mono-SorbPE. Rhodopsin photoactivity was monitored using plasmon waveguide spectroscopy. The results show that reconstitution of rhodopsin into SLBs composed of phosphatidylcholine with the polymerizable moiety in the acyl chain terminus, followed by photoinduced cross-linking of the lipids, does not significantly perturb protein function. A possible explanation is that a bilayer with relatively low Xn retains sufficient elasticity to accommodate the membrane deformation that accompanies the conformational change associated with rhodopsin photoactivation when polymerized in the acyl chain terminus. GVs have diameters ranging from several to few hundred micrometers and thus can be observed by optical microscopic methods. This allows manipulation of individual vesicles and observation of their transformations in real time. GVs have attracted attention as microcontainers for enzymes and drugs, and as biosensors. With the aim of increasing stability for these types of applications, GVs were prepared from synthetic dienoyl lipids that can be polymerized to form robust vesicles. The stability of these vesicles after polymerization was investigated by surfactant treatment, drying and rehydration, and temperature variations. The structure of poly(GVs) was largely retained under these conditions which destroy unpolymerized vesicles. Permeability studies on poly(GVs) suggests that they could be potentially used in a variety of technological applications, including sensors, macromolecular carriers, and microreactors.