Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Lipid bilayers are widely employed as a model system to investigate interactions between cells and their environment. Supported lipid bilayers (SLB) with integrated transmembrane proteins are emerging as a preferred platform for sensing applications. Challenges lie in the generation of SLB on surfaces which allow transduction of signals for characterization of lipid bilayer and incorporated transmembrane proteins. For the first time, the formation of SLBs is shown on films of the conducting polymer, poly(3,4‐ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), using traditional methods for characterizing lipid bilayer quality and function (QCM‐D, FRAP) combined with impedance spectroscopy. Further, partial formation of SLBs on PEDOT:PSS based organic electrochemical transistors (OECTs) is successfully demonstrated, as well as the ability to integrate and sense the ion pore α‐hemolysin, confirming the sensitivity of the OECT as a transducer of biological membrane function. This work represents a highly promising first step toward the use of such OECTs for functional readout of transmembrane proteins in their native environment.

The bacterial outer membrane (OM) is a barrier containing membrane proteins and liposaccharides that fulfill crucial functions for Gram-negative bacteria. With the advent of drug-resistant bacteria, it is necessary to understand the functional role of this membrane and its constituents to enable novel drug designs. Here we report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). We characterize the formation of OM-SBs using quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. We show that the orientation of proteins in the OM-SB matches the native bacterial membrane, preserving the characteristic asymmetry of these membranes. As a demonstration of the utility of the OM-SB platform, we quantitatively measure antibiotic interactions between OM-SBs and polymyxin B, a cationic peptide used to treat Gram-negative infections. This data enriches understanding of the antibacterial mechanism of polymyxin B, including disruption kinetics and changes in membrane mechanical properties. Combining OM-SBs with microfluidics will enable higher throughput screening of antibiotics. With a broader view, we envision that a molecularly complete membrane-scaffold could be useful for cell-free applications employing engineered membrane proteins in bacterial membranes for myriad technological purposes.

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Lipid–protein interactions are essential for modulating membrane protein structures and biological functions in the cell plasma membrane. In this review we describe the salient features of classical and emerging methodologies for studying protein–lipid interactions and their limitations.

Vaccines and antivirals to combat dengue, Zika, and other flavivirus pathogens present a major, unmet medical need. Vaccine development has been severely challenged by the antigenic diversity of these viruses and the propensity of non-neutralizing, cross-reactive antibodies to facilitate cellular infection and increase disease severity. As an alternative, direct-acting antivirals targeting the flavivirus envelope protein, E, have the potential to act via an analogous mode of action without the risk of antibody-dependent enhancement of infection and disease. We previously discovered that structurally diverse small molecule inhibitors of the dengue virus E protein exhibit varying levels of antiviral activity against other flaviviruses in cell culture. Here we demonstrate that the broad-spectrum activity of several cyanohydrazones against dengue, Zika, and Japanese encephalitis viruses is due to specific inhibition of E-mediated membrane fusion during viral entry and provide proof of concept for pharmacological inhibition of E as an antiviral strategy in vivo.

A new method is presented to separate, enrich, and sort membrane-bound biomolecules based on their affinity for different coexisting lipid phases in a supported lipid bilayer using a two-dimensional, continuous extraction procedure. Analogous to classic liquid-liquid phase extraction, we created two distinct lipid phases in our planar membrane system: a liquid-ordered (l(o)) phase and a liquid-disordered (l(d)) phase arranged in parallel stripes inside a microfluidic device. Membrane-bound biomolecules in an adjacent supported lipid bilayer are convected in plane along the microfluidic channel and brought into contact with a different lipid phase using hydrodynamic force. A mixture of two lipid species, a glycolipid and a phospholipid, with known affinities for the two lipid phases employed here are used to demonstrate continuous extraction of the lipid-microdomain preferring glycolipid to the lo phase, while the phospholipid remains primarily in the ld phase. In this demonstration, we characterize the performance of this affinity-based separation device by building models to describe the velocity profile and transport in the two-phase coexistent membrane. We then characterize the impact of residence time on the extraction yield of each species. This new procedure sorts membrane species on the basis of chemical properties and affinities for specific lipid phases within a membrane environment near physiological conditions, critical for extending this method to the separation of lipid-linked proteins and transmembrane proteins while minimizing denaturation. This platform could facilitate the separation and identification of lipid membrane domain residents, or the characterization of changes in membrane affinity due to post-translational modifications or environmental conditions.

The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.