Peptide-Mediated Membrane Transport of Macromolecular Cargo Driven by Membrane Asymmetry.

Pep-1 is a cell-penetrating peptide that represents a powerful strategy for delivering large, hydrophilic therapeutic molecules into cells. Model membranes, such as lipid vesicles and planar bilayers, have been useful for investigating the direct translocation of cell-penetrating peptides. Here, we present a droplet interface bilayer-based approach to quantify pep-1-mediated ß-galactosidase translocation. We found that ß-galactosidase translocation is driven only by the negative transmembrane potential resulting from the asymmetric bilayers. The asymmetric droplet interface bilayer method may be generally applicable for high-throughput screening of the efficacy of cell-penetrating peptides.

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers.

The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.

Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore.

Outer membrane protein G is a monomeric ß-barrel porin that has seven flexible loops on its extracellular side. Conformational changes of these labile loops induce gating spikes in current recordings that we exploited as the prime sensing element for protein detection. The gating characteristics, open probability, frequency, and current decrease, provide rich information for analyte identification. Here, we show that two antibiotin antibodies each induced a distinct gating pattern, which allowed them to be readily detected and simultaneously discriminated by a single OmpG nanopore in the presence of fetal bovine serum. Our results demonstrate the feasibility of directly profiling proteins in real-world samples with minimal or no sample pretreatment.

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers.

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer.

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.

Screening blockers against a potassium channel with a droplet interface bilayer array.

Droplet interface bilayers (DIBs) form between two lipid monolayer-encased aqueous droplets submerged in oil. Both major structural classes of membrane proteins, alpha-helix bundles and beta barrels, represented by channels and pores, respectively, spontaneously insert into DIBs when freshly expressed by cell-free transcription and translation. Electrodes embedded within the droplets allow the measurement of transmembrane ionic currents carried by individual channels and pores. On the basis of these findings, we have devised a chip-based approach for the rapid screening of blockers against ion channels. The technique is demonstrated here with the viral potassium channel, Kcv.

Asymmetric droplet interface bilayers.

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.

Building interconnected membrane networks.

Reconstituted replica cell membranes are easily created by contacting two lipid-monolayer-encased aqueous droplets under an oil phase. Called the droplet interface bilayer (DIB), this technique has been used to study a wide range of membrane processes. Importantly, this method is compatible with electrical measurements, meaning that membrane protein activities are easily observed in DIBs. By positioning droplets in two- and three-dimensional networks, sophisticated interconnected systems can be created that possess collective properties. The methods described here summarize the approaches used to create DIB networks and how to operate the devices that have been constructed so far.

Robust reagent addition and perfusion strategies for droplet-interface bilayers.

We have designed two novel devices which extend the applications for the droplet-interface bilayer (DIB) as a model membrane system. The add-chip allows successive reagent additions to one side of the lipid bilayer during an experiment while maintaining a simple setup with much lower volumes than in planar bilayer systems. The flow-chip is capable of multiple complete solution perfusions concurrently with electrophysiology measurements. Both devices preserve all of the key advantages that DIBs have relative to planar membranes, including low volume, leaflet asymmetry and the ability to separate the monolayers prior to further analysis of a droplet's contents. As a demonstration, we use these devices to monitor and quantitate molecular transport across DIBs.

Droplet networks with incorporated protein diodes show collective properties.

Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.

Droplet interface bilayers.

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

Functional bionetworks from nanoliter water droplets.

We form networks from aqueous droplets by submerging them in an oil/lipid mixture. When the droplets are joined together, the lipid monolayers surrounding them combine at the interface to form a robust lipid bilayer. Various protein channels and pores can incorporate into the droplet-interface bilayer (DIB), and the application of a potential with electrodes embedded within the droplets allows ionic currents to be driven across the interface and measured. By joining droplets in linear or branched geometries, functional bionetworks can be created. Although the interfaces between neighboring droplets comprise only single lipid bilayers, the structures of the networks are long-lived and robust. Indeed, a single droplet can be "surgically" excised from a network and replaced with a new droplet without rupturing adjacent DIBs. Networks of droplets can be powered with internal "biobatteries" that use ion gradients or the light-driven proton pump bacteriorhodopsin. Besides their interest as coupled protocells, the droplets can be used as devices for ultrastable bilayer recording with greatly reduced electrolyte volume, which will permit their use in rapid screening applications.

Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling.

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. Such DIB networks might be used as model systems for the study of membrane-based biological phenomena. In this study, we develop and experimentally validate an electrical modeling approach for DIB networks by applying it to describe the current flow through a simple network containing protein pores and blocking molecules. We demonstrate the use of SPICE (Simulation Program with Integrated Circuit Emphasis) for simulating the electrical behavior of DIB networks. The modular and scalable nature of DIB networks should enable a straightforward extension of the analysis presented in this paper to large, complex networks.

Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording.

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

Microfluidic tools for studying the specific binding, adsorption, and displacement of proteins at interfaces.

A combination of temperature and concentration gradient microfluidic devices were employed to study the mechanistic details of biomacromolecule interactions at oxide interfaces. These lab-on-a-chip techniques allowed high-throughput, multiplexed data collection using only nanoliters of analyte. The three examples presented demonstrate rapid data acquisition relative to standard methods. First, we show ligand-receptor binding data for multivalent binding between membrane-bound ligands and incoming aqueous proteins with several binding pockets. A model is described for obtaining both the first and second dissociation constant for the reaction. The second example employs temperature gradient microfluidics to study the thermoresponsive properties of polymers and proteins. Both the folding mechanism and subsequent formation of an aqueous two-phase system were followed. Finally, these microfluidic techniques were combined with fluorescence microscopy and nonlinear optical spectroscopy to elucidate the mechanism of fibrinogen displacement from silica surfaces. This combination of methods enabled both direct and indirect observation of protein conformational changes.

Direct introduction of single protein channels and pores into lipid bilayers.

We have developed a mechanical method for inserting single pores and channels into lipid bilayers. A hand-operated hydrogel probe, coated with a layer of proteins, is mechanically engaged with the lipid bilayer. The two major classes of membrane proteins (beta barrels and alpha-helix bundles) that can be inserted, thereby demonstrating the wide applicability of the approach. Recordings from the proteins show that they retain electrical properties that are the same as those of proteins inserted from solution. Protein-loaded probes can be used repeatedly, allowing individual pores to be rapidly screened one at a time. The method has implications for fundamental studies of cell membranes, array fabrication, and chemical screening.

Light activated patterning of dye-labeled molecules on surfaces.

This Communication describes a novel method for patterning proteins, ligands, or other organic/inorganic species onto solid substrates. The process works by linking the moiety of interest to a fluorophore that can be subsequently photobleached and bound to the interface. The technique can be performed in aqueous solution and allows several species to be addressed onto the surface simultaneously by using different frequencies of light. Because the method can be used with fluorophores excited by visible light, shorter and more damaging wavelengths can be avoided.

Patterning enzymes inside microfluidic channels via photoattachment chemistry.

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.

Creating fluid and air-stable solid supported lipid bilayers.

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.

Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density.

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.