Synthesis and Characterization of Biomimetic Compartments

This dissertation will first explore methods for the structural characterization of artificial phospholipid membrane assemblies. We employed confocal microscopy, negative stain transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and small angle x-ray scattering (SAXS) to analyze the structure of two artificial lipid systems from the millimeter to atomic scale. These biophysical techniques reveal their necessity as a general toolkit for the structural characterization of artificial lipid systems in a range of spatial resolutions.Next, this dissertation will discuss the biochemical and biophysical characterization of lipid sponge droplets that have potential application as an artificial organelle. The spatial organization afforded by organelles can possibly expand the functions of synthetic reaction systems, especially within artificial cells. Here, we describe highly stable nonlamellar, galactolipid-based sponge phase droplets as programmable synthetic organelles. We use various biochemical and biophysical techniques to structurally characterize the dense network of lipid bilayers and nanometric aqueous channels of the sponge phase. In the third section of this dissertation, a novel approach for the semisynthesis and reconstitution of transmembrane (TM) proteins in giant unilamellar vesicles (GUVs) will be presented. Herein, we test an alternative solution to detergent-based TM protein reconstitution strategies involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic GUV membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. Finally, this dissertation will discuss the total synthesis of protein-based compartments as drug delivery vehicles. Common vehicles are empty viral capsids and bacterial microcompartments comprised of protein subunits. Usually, these compartments are expressed in E. coli, purified, disassembled, and reassembled with a cargo (e.g. a therapeutic) of interest. The total synthesis of protein-based compartments would increase the versatility of the protein sequence and enable the incorporation of noncanonical amino acids, fluorescent or radioactive labels, and chemical tags. Herein, we describe the total synthesis and characterization of the 129-mer bacteriophage MS2 coat protein for its future application as a versatile and robust drug delivery vehicle