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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2020)

It is of great fundamental interest to develop experimental model systems to reconstruct design principles underlying formation of living cells and shed light on how life emerged on Earth and possibly elsewhere. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of basic building blocks with defined functionality to construct a life-like entity. This dissertation will explore the roles of miscellaneous lipid assemblies as building blocks of artificial cellular compartments.

The first part (Chapters 2 and 3) of this dissertation shows how lipid environments facilitate chemoselective reactions between amphiphilic partners through physical partitioning. Through examples of histidine ligation, and acyl phosphate chemistry we show functionalized micelle-forming amphiphiles react to form amidophospholipids which self-assemble into micron-sized vesicles.

The second part (Chapter 3) of the dissertation seeks to understand the origins of cellular phospholipid synthesis pathways by repurposing soluble fatty acid activating enzymes (fatty acyl adenylate ligase and fatty acyl CoA ligase) to synthesize phospholipids. Such simplified biochemical pathways may provide a hint at how lipids were likely synthesized in a minimal protocell without the necessity of transmembrane protein enzymes.

The third part (Chapters 4 and 5) of the dissertation discusses the vesicle formation from a novel class of single-chain amphiphiles derived from galactopyranosyl head groups and unsaturated fatty acid tails. The geometric parameters and thermodynamic properties of these amphiphilic assemblies are characterized by a host of physical techniques. The vesicles are further shown to sustain model biochemical reactions.

The final part (Chapters 6) of the dissertation describes the formation of sponge phase droplets from fatty acyl galactopyranosylamides and non-ionic detergents. The droplets contain a dense bicontinuous network of bilayers and nanometric aqueous channels, which facilitates molecules to partition into them based on their size, polarity, and specific binding motifs. The sequestration of biomolecules can be programmed by doping the droplets with suitably functionalized amphiphiles. The droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled.

    Cover page: Applications of Lipid Assemblies in Artificial Cell Development
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2017)
    The assembly of artificial cells provides a novel strategy to reconstruct life's functions and shed light on how life emerged on Earth and possibly elsewhere. A major challenge to the development of artificial cells is the establishment of simple methodologies to mimic native membrane generation. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of highly ordered membrane architectures with defined functionality. This perspective will cover recent advances and the current state-of-the-art of minimal lipid architectures that can faithfully reconstruct the structure and function of living cells. Specifically, we will overview work related to the de novo formation and growth of biomimetic membranes. These studies give us a deeper understanding of the nature of living systems and bring new insights into the origin of cellular life.
      Cover page: De novo vesicle formation and growth: an integrative approach to artificial cells
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2019)
      All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require pre-existing membranes for function. Thus, the principle of membrane continuity creates a paradox when considering how the first biochemical membrane-synthesis machinery arose and has hampered efforts to develop simplified pathways for membrane generation in synthetic cells. Here, we develop a high-yielding strategy for de novo formation and growth of phospholipid membranes by repurposing a soluble enzyme FadD10 to form fatty acyl adenylates that react with amine-functionalized lysolipids to form phospholipids. Continuous supply of fresh precursors needed for lipid synthesis enables the growth of vesicles encapsulating FadD10. Using a minimal transcription/translation system, phospholipid vesicles are generated de novo in the presence of DNA encoding FadD10. Our findings suggest that alternate chemistries can produce and maintain synthetic phospholipid membranes and provides a strategy for generating membrane-based materials.
        Cover page: A minimal biochemical route towards de novo formation of synthetic phospholipid membranes
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2020)
        All living organisms synthesize phospholipids as the primary constituent of their cell membranes. Enzymatic synthesis of diacylphospholipids requires preexisting membrane-embedded enzymes. This limitation has led to models of early life in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate an enzyme-free synthesis of natural diacylphospholipids by transacylation in water, which is enabled by a combination of ion pairing and self-assembly between lysophospholipids and acyl donors. A variety of membrane-forming cellular phospholipids have been obtained in high yields. Membrane formation takes place in water from natural alkaline sources such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained. This high-yielding non-enzymatic synthesis of natural phospholipids in water opens up new routes for lipid synthesis in artificial cells and sheds light on the origin and evolution of cellular membranes.
          Cover page: Enzyme-free synthesis of natural phospholipids in water
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2020)
          Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.
            Cover page: Lipid sponge droplets as programmable synthetic organelles
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