Sheereen Majd

Controlling protein translocation through nanopores with bio-inspired fluid walls

Synthetic nanopores have been used to study individual biomolecules in high thoroughput but their performance as sensors does not match biological ion channels. Controlling the translocation times of single-molecule analytes and their non-specific interaction with pore walls remain a challenge. Inspired by the olfactory sensilla of the insect antenna, here we show that coating nanopores with fluid bilayer lipids allows the pore diameters to be fine-tuned in sub-nanometre increments. Incorporation of mobile ligands in the lipid conferred specificity and slowed down the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. The lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of translocation time, volume, charge, shape and ligand affinity, different proteins were identified.

Applications of biological pores in nanomedicine, sensing, and nanoelectronics.

Biological protein pores and pore-forming peptides can generate a pathway for the flux of ions and other charged or polar molecules across cellular membranes. In nature, these nanopores have diverse and essential functions that range from maintaining cell homeostasis and participating in cell signaling to activating or killing cells. The combination of the nanoscale dimensions and sophisticated - often regulated - functionality of these biological pores make them particularly attractive for the growing field of nanobiotechnology. Applications range from single-molecule sensing to drug delivery and targeted killing of malignant cells. Potential future applications may include the use of nanopores for single strand DNA sequencing and for generating bio-inspired, and possibly, biocompatible visual detection systems and batteries. This article reviews the current state of applications of pore-forming peptides and proteins in nanomedicine, sensing, and nanoelectronics.

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Significance This study focuses on the design of highly permeable artificial water channels for the use in membrane-based separation materials. A platform was developed for the systematic characterization of the single-channel water conduction of artificial channels, which is based on permeability measurement by stopped-flow light-scattering experiments and single-molecule counting by fluorescence correlation spectroscopy. With this platform the water conduction of the redesigned peptide-appended pillar[5]arene channels was found to be similar to that of aquaporins, natural water channel proteins, and their synthetic analogs, carbon nanotubes, which is an order of magnitude higher than that of first-generation artificial water channels. The channel can also self-assemble into arrays in membranes, opening the possibility for materials that can be used in engineering applications such as liquid and gas separations. Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10−14 cm3/s or 3.5(±1.0) × 108 water molecules per s, which is in the range of AQPs (3.4∼40.3 × 108 water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 108 water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼107 water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 105 pores per μm2) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 103 pores per μm2). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.

Gramicidin Pores Report the Activity of Membrane-Active Enzymes

Phospholipases constitute a ubiquitous class of membrane-active enzymes that play a key role in cellular signaling, proliferation, and membrane trafficking. Aberrant phospholipase activity is implicated in a range of diseases including cancer, inflammation, and myocardial disease. Characterization of these enzymes is therefore important, both for improving the understanding of phospholipase catalysis and for accelerating pharmaceutical and biotechnological applications. This paper describes a novel approach to monitor, in situ and in real-time, the activity of phospholipase D (PLD) and phospholipase C (PLC) on planar lipid bilayers. This method is based on lipase-induced changes in the electrical charge of lipid bilayers and on the concomitant change in ion concentration near lipid membranes. The approach reports these changes in local ion concentration by a measurable change in the single channel ion conductance through pores of the ion channel-forming peptide gramicidin A. This enzyme assay takes advantage of the amplification characteristics of gramicidin pores to sense the activity of picomolar to nanomolar concentrations of membrane-active enzymes without requiring labeled substrates or products. The resulting method proceeds on lipid bilayers without the need for detergents, quantifies enzyme activity on native lipid substrates within minutes, and provides unique access to both leaflets of well-defined lipid bilayers; this method also makes it possible to generate planar lipid bilayers with transverse lipid asymmetry.

A Review of Patterned Organic Bioelectronic Materials and their Biomedical Applications

Organic electronic materials are rapidly emerging as superior replacements for a number of conventional electronic materials, such as metals and semiconductors. Conducting polymers, carbon nanotubes, graphenes, organic light-emitting diodes, and diamond films fabricated via chemical vapor deposition are the most popular organic bioelectronic materials that are currently under active research and development. Besides the capability to translate biological signals to electrical signals or vice versa, organic bioelectronic materials entail greater biocompatibility and biodegradability compared to conventional electronic materials, which makes them more suitable for biomedical applications. When patterned, these materials bring about numerous capabilities to perform various tasks in a more-sophisticated and high-throughput manner. Here, we provide an overview of the unique properties of organic bioelectronic materials, different strategies applied to pattern these materials, and finally their applications in the field of biomedical engineering, particularly biosensing, cell and tissue engineering, actuators, and drug delivery.

Generating arrays with high content and minimal consumption of functional membrane proteins.

This paper introduces a widely accessible and straightforward technique for fabricating membrane protein arrays. This technique employs topographically patterned agarose gels to deliver various membrane preparations to glass substrates in a rapid and parallel fashion. It can fabricate more than 30 identical copies of a membrane protein array while requiring only femtomoles of protein. Taking advantage of on-stamp preconcentration, it is able to pattern arrays of multilayered membrane spots with more than 20-fold increased content of membrane proteins compared to existing methods.

A Simple and Versatile Method for the Formation of Arrays of Giant Vesicles with Controlled Size and Composition

A simple and versatile method for the Formation of Arrays of Giant Vesicles with Controlled Size and Composition. The ability of this technique to generate arrays of giant liposomes from a wide range of membrane lipids and protein compositions is demonstrated. The resulting vesicles are utilized for studying protein activity, lipid-protein interactions, and protein-protein interactions.

Hydrogel‐Mediated Direct Patterning of Conducting Polymer Films with Multiple Surface Chemistries

A new methodology for selective electropolymerization of conducting polymer films using wet hydrogel stamps is presented. The ability of this simple method to generate patterned films of conducting polymers with multiple surface chemistries in a one-step process and to incorporate fragile biomolecules in these films is demonstrated.

A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.