Postdoctoral Researcher, University of California, San Diego
Development of bottom-up approaches for the construction of functional artificial cell membranes
The fabrication of artificial cells from purely synthetic components provides a novel methodology to reconstruct life’s functions within unnatural materials. Artificial cells have the potential to shed light on biological processes such as gene expression and energy transduction, as well as organize chemical reactions in nanoscale compartments. Remarkably, they also offer a unique opportunity to study how life emerged on Earth and possibly elsewhere. Therefore, there has been an increasing interest to develop novel strategies for the incorporation and/or integration of biological components in synthetic capsules to facilitate signaling responses, drug delivery, encapsulation and extended expression of biomolecules. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of highly ordered membrane architectures with defined functionality. Bottom-up methodologies that assemble artificial cells from synthetic membranes are well suited for a range of applications, such as integrating functionalized vesicles with biological machinery and creating hybrid minimal cells using nonbiological chassis. My research is focused on the use of biomimetic bottom-up approaches to spontaneously generate and remodel phospholipid membranes from water-soluble starting materials. The orthogonality, high reaction rate, and biocompatibility of these methodologies are key features that make them a powerful option for the efficient encapsulation of relevant biomolecules. Additionally, my research explores the suitability of such bioorthogonal coupling reactions for driving the in situ formation of phospholipid membranes and concomitant spontaneous reconstitution of a variety of membrane proteins, with retention of functionality. These studies give us a deeper understanding of the nature of living systems that could bring new insights into the origin of cellular life, and provide novel synthetic chassis for advancing synthetic biology
Abstract: Cell transmembrane receptors play a key role in the detection of environmental stimuli and control of intracellular communication. G protein-coupled receptors constitute the largest transmembrane protein family involved in cell signaling. However, current methods for their functional reconstitution in biomimetic membranes remain both challenging and limited in scope. Herein, we describe the spontaneous reconstitution of adenosine A2A receptor (A2AR) during the de novo formation of synthetic liposomes via native chemical ligation. The approach takes advantage of a nonenzymatic and chemoselective method to rapidly generate A2AR embedded phospholiposomes from receptor solubilized in n-dodecyl-β-d-maltoside analogs. In situ lipid synthesis for protein reconstitution technology proceeds in the absence of dialysis and/or detergent absorbents, and A2AR assimilation into synthetic liposomes can be visualized by microscopy and probed by radio-ligand binding.
Pub.: 07 Mar '17, Pinned: 29 Jun '17
Abstract: A major challenge for the construction of artificial lipid membranes is the development of simple and robust methods for mimicking natural phospholipid membrane generation. Here we describe a nonenzymatic and chemoselective approach that relies on histidine ligation to form phospholipids de novo from water-soluble amphiphilic precursors. The resulting phospholipids can spontaneously self-assemble into micron-sized vesicles and encapsulate biomacromolecules.
Pub.: 18 Oct '16, Pinned: 29 Jun '17
Abstract: Cell membranes have a vast repertoire of phospholipid species whose structures can be dynamically modified by enzymatic remodeling of acyl chains and polar head groups. Lipid remodeling plays important roles in membrane biology and dysregulation can lead to disease. Although there have been tremendous advances in creating artificial membranes to model the properties of native membranes, a major obstacle has been developing straightforward methods to mimic lipid membrane remodeling. Stable liposomes are typically kinetically trapped and are not prone to exchanging diacylphospholipids. Here, we show that reversible chemoselective reactions can be harnessed to achieve nonenzymatic spontaneous remodeling of phospholipids in synthetic membranes. Our approach relies on transthioesterification/acyl shift reactions that occur spontaneously and reversibly between tertiary amides and thioesters. We demonstrate exchange and remodeling of both lipid acyl chains and head groups. Using our synthetic model system we demonstrate the ability of spontaneous phospholipid remodeling to trigger changes in vesicle spatial organization, composition, and morphology as well as recruit proteins that can affect vesicle curvature. Membranes capable of chemically exchanging lipid fragments could be used to help further understand the specific roles of lipid structure remodeling in biological membranes.
Pub.: 20 Jul '16, Pinned: 29 Jun '17
Abstract: Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.
Pub.: 01 Sep '15, Pinned: 29 Jun '17
Abstract: There has been increasing interest in utilizing bottom-up approaches to develop synthetic cells. A popular methodology is the integration of functionalized synthetic membranes with biological systems, producing "hybrid" artificial cells. This Concept article covers recent advances and the current state-of-the-art of such hybrid systems. Specifically, we describe minimal supramolecular constructs that faithfully mimic the structure and/or function of living cells, often by controlling the assembly of highly ordered membrane architectures with defined functionality. These studies give us a deeper understanding of the nature of living systems, bring new insights into the origin of cellular life, and provide novel synthetic chassis for advancing synthetic biology.
Pub.: 08 Jul '15, Pinned: 29 Jun '17
Abstract: Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non-enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water-soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self-assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.
Pub.: 28 Oct '14, Pinned: 29 Jun '17
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