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Abstract The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles. 

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE. 

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing. 

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. Encapsulation of these systems in synthetic membranes to form artificial cells can reintroduce features of the cellular membrane, including molecular containment and selective permeability, to modulate cell-free sensing capabilities. Here, we demonstrate robust and tunable performance of a transcriptionally regulated, cell-free riboswitch encapsulated in lipid membranes, allowing the detection of fluoride, an environmentally important molecule. Sensor response can be tuned by varying membrane composition, and encapsulation protects from sensor degradation, facilitating detection in real-world samples. These sensors can detect fluoride using two types of genetically encoded outputs, enabling detection of fluoride at the Environmental Protection Agency maximum contaminant level of 0.2 millimolars. This work demonstrates the capacity of bilayer membranes to confer tunable permeability to encapsulated, genetically encoded sensors and establishes the feasibility of artificial cell platforms to detect environmentally relevant small molecules. 

Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.