A synthetic method to assay polycystin channel biophysics.

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive states are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay the functional properties of the polycystin subfamily of transient receptor potential (TRP) channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes.

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.

Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

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.

Engineering transmembrane signal transduction in synthetic membranes using two-component systems.

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.

Rapid Generation of Therapeutic Nanoparticles Using Cell-Free Expression Systems.

The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules, such as binding proteins, is an important step in the design of therapeutic materials. However, this modification can be costly and time-consuming, requiring cellular hosts for protein expression and lengthy purification and conjugation steps to attach proteins to the surface of nanocarriers, which ultimately limits the development of effective protein-conjugated nanocarriers. Here, the use of cell-free protein synthesis systems to rapidly create protein-conjugated membrane-based nanocarriers is demonstrated. Using this approach, multiple types of functional binding proteins, including affibodies, computationally designed proteins, and scFvs, can be cell-free expressed and conjugated to liposomes in one-pot. The technique can be expanded further to other nanoparticles, including polymersomes and lipid nanoparticles, and is amenable to multiple conjugation strategies, including surface attachment to and integration into nanoparticle membranes. Leveraging these methods, rapid design of bispecific artificial antigen presenting cells and enhanced delivery of lipid nanoparticle cargo in vitro is demonstrated. It is envisioned that this workflow will enable the rapid generation of membrane-based delivery systems and bolster our ability to create cell-mimetic therapeutics.

Tuning Targeted Liposome Avidity to Cells via Lipid Phase Separation.

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickel-nitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on lipid nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.

Cell-Free Synthesis Goes Electric: Dual Optical and Electronic Biosensor via Direct Channel Integration into a Supported Membrane Electrode.

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.

Cell-free protein synthesis systems for vaccine design and production.

Vaccines are vital for protection against existing and emergent diseases. Current vaccine production strategies are limited by long production times, risky viral material, weak immunogenicity, and poor stability, ultimately restricting the safe or rapid production of vaccines for widespread utilization. Cell-free protein synthesis (CFPS) systems, which use extracted transcriptional and translational machinery from cells, are promising tools for vaccine production because they can rapidly produce proteins without the constraints of living cells, have a highly optimizable open system, and can be used for on-demand biomanufacturing. Here, we review how CFPS systems have been explored for the production of subunit, conjugate, virus-like particle (VLP), and membrane-augmented vaccines and as a tool in vaccine design. We also discuss efforts to address potential limitations with CFPS such as the presence of endotoxins, poor protein folding, reaction stability, and glycosylation to enable promising future vaccine design and production.

Engineered membrane receptors with customizable input and output functions.

Morsut et al. reported a synthetic receptor system, based on the natural Notch receptor, with customizable input and output functions. Their work on advanced receptor design expands the reach of synthetic receptor systems. Incorporating new protein design tools with better-understood membrane biophysics will create the next generation of engineered receptors.

Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles.

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.

PEO-b-PBD Diblock Copolymers Induce Packing Defects in Lipid/Hybrid Membranes and Improve Insertion Rates of Natively Folded Peptides.

Hybrid membranes assembled from biological lipids and synthetic polymers are a promising scaffold for the reconstitution and utilization of membrane proteins. Recent observations indicate that inclusion of small fractions of polymer in lipid membranes can improve protein folding and function, but the exact structural and physical changes a given polymer sequence imparts on a membrane often remain unclear. Here, we use all-atom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO-b-PBD diblock copolymers. We verified our computational model using new and existing experimental data and obtained a detailed picture of the polymer conformations in the lipid membrane that we can relate to changes in membrane elastic properties. We find that inclusion of low polymer fractions induces transient packing defects into the membrane. These packing defects act as insertion sites for two model peptides, and in this way, small amounts of polymer content in lipid membranes can lead to large increases in peptide insertion rates. Additionally, we report the peptide conformational space in both pure lipid and hybrid membranes. Both membranes support similar alpha helical peptide structures, exemplifying the biocompatibility of hybrid membranes.

Elucidating Design Principles for Engineering Cell-Derived Vesicles to Inhibit SARS-CoV-2 Infection.

The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade mAbs and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

Lipid Phase Separation in Vesicles Enhances TRAIL-Mediated Cytotoxicity.

Ligand spatial presentation and density play important roles in signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Here, we harness lipid phase separation to spatially control the protein presentation on lipid vesicles. We use this system to improve the cytotoxicity of TNF-related apoptosis inducing ligand (TRAIL), a therapeutic anticancer protein. Vesicles with phase-separated TRAIL presentation induce more cell death in Jurkat cancer cells than vesicles with uniformly presented TRAIL, and cytotoxicity is dependent on TRAIL density. We assess this relationship in other cancer cell lines and demonstrate that phase-separated vesicles with TRAIL only enhance cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, is less sensitive to TRAIL density. This work demonstrates a rapid and accessible method to control protein conjugation and density on vesicles that can be adopted to other nanoparticle systems to improve receptor signaling by nanoparticles.

Cell-Free Membrane Protein Expression into Hybrid Lipid/Polymer Vesicles.

Hybrid membranes comprised of diblock copolymers, and phospholipids have gained interest due to their unique properties that result from blending natural and synthetic components. The integration of membrane proteins into these synthetic membranes is an important step towards creating biomembrane systems for uses such as artificial cellular systems, biosensors, and drug delivery vehicles. Here, we outline a technique to create hybrid membranes composed of phospholipids and diblock copolymers. Next, we describe how membrane proteins can be co-translationally integrated into hybrid lipid/polymer membranes using a cell-free reaction. We then outline a method to monitor insertion and folding of a membrane-embedded channel protein into the hybrid membrane using a fluorescent-protein reporter and dye release assay, respectively. This method is expected to be applicable for a wide range of membrane proteins that do not require chaperones for co-translational integration into vesicles and provides a generalized protocol for expressing a membrane protein into a membrane mimetic.

Elucidating design principles for engineering cell-derived vesicles to inhibit SARS-CoV-2 infection.

The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

Vesicle-Based Sensors for Extracellular Potassium Detection.

INTRODUCTION: The design of sensors that can detect biological ions in situ remains challenging. While many fluorescent indicators exist that can provide a fast, easy readout, they are often nonspecific, particularly to ions with similar charge states. To address this issue, we developed a vesicle-based sensor that harnesses membrane channels to gate access of potassium (K+) ions to an encapsulated fluorescent indicator. METHODS: We assembled phospholipid vesicles that incorporated valinomycin, a K+ specific membrane transporter, and that encapsulated benzofuran isophthalate (PBFI), a K+ sensitive dye that nonspecifically fluoresces in the presence of other ions, like sodium (Na+). The specificity, kinetics, and reversibility of encapsulated PBFI fluorescence was determined in a plate reader and fluorimeter. The sensors were then added to E. coli bacterial cultures to evaluate K+ levels in media as a function of cell density. RESULTS: Vesicle sensors significantly improved specificity of K+ detection in the presence of a competing monovalent ion, sodium (Na+), and a divalent cation, calcium (Ca2+), relative to controls where the dye was free in solution. The sensor was able to report both increases and decreases in K+ concentration. Finally, we observed our vesicle sensors could detect changes in K+ concentration in bacterial cultures. CONCLUSION: Our data present a new platform for extracellular ion detection that harnesses ion-specific membrane transporters to improve the specificity of ion detection. By changing the membrane transporter and encapsulated sensor, our approach should be broadly useful for designing biological sensors that detect an array of biological analytes in traditionally hard-to-monitor environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00688-7.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.