Fluid Lipid Bilayer Research Articles

Construction of tethered bilayer lipid membrane with oriented membrane proteins on surface modified mica substrate

Incorporating membrane proteins, which are major targets of drug discovery, into artificial planar lipid bilayer membranes is important. From the observation at the high spatiotemporal resolution in the microscopic region, the intermolecular interactions between lipids and membrane proteins can be revealed. It leads to elucidation of the reaction mechanisms occurring in biological membranes. However, when a lipid membrane supported on a solid substrate (SLB) is used, the following points are interfering with the research. The first point is the structural change due to the contact between the membrane protein and the underlying substrate. The other is disorder of the membrane protein orientation in the SLB. Research on the incorporation of membrane proteins has been done in the field of both physical and biological chemistry, but few reports have achieved detailed measurements with placing emphasis on the plasticity and diversity of membrane proteins. In this study, the tethered bilayer lipid membrane (tBLM) was formed on the atomically flat self-assembled monolayer modified surface which specifically binds to the membrane proteins in the membrane. The fluidity of artificial lipid bilayer and orientation of membrane proteins are observed by the epi-fluorescence microscopy and atomic force microscopy, respectively. The consequent decreased interaction between the substrate and lipid membrane and limited observation for the specific orientation of membrane proteins suggest that it was possible to reproduce the same environment as the biological membrane even in the artificial lipid bilayer membrane. The present tBLM system should be useful for the creation of basic technologies for structural, functional, and dynamical analysis of membrane proteins. It also leads to the understanding the molecular structure of biological membrane which include the various membrane proteins such as ion channels, transporters, pumps and enzymes with addition of more complex structural properties such as the underlying membrane scaffolds, asymmetric lipid composition and domains.

Hereditary spherocytosis and allied disorders

Hereditary spherocytosis and allied disorders

Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore.

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Unraveling the Role of Monoolein in Fluidity and Dynamical Response of a Mixed Cationic Lipid Bilayer.

The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperatures when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation in the lipids, so that the membranes return to a more fluidic state. To probe how introduction of unsaturation leads to reduced membrane fluidity, a model cationic lipid dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behavior of DODAB have been monitored by quasielastic neutron scattering and time-resolved fluorescence measurements. In the coagel phase, fluidity of the lipid bilayer increases significantly in the presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330 K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in the mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperatures (below the phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.

Abstract B058: Rapid and controlled T-cell expansion using scaffolds that mimic antigen-presenting cells

Abstract Current approaches to expand T-cells ex vivo for therapeutic applications are limited by low expansion rates and often result in unpredictable expansion products of varying phenotype and function. Key attributes, such as a balanced ratio of CD4-to-CD8 T-cells, or a high central memory T-cell fraction are known to correlate with robust and durable therapeutic responses but remain difficult to reproducibly achieve. Strategies that allow one to precisely control the phenotype of expanded T-cell products may thus potentiate the effects of CAR-T-cell therapies, and understanding how T-cells respond to stimulation is a crucial step to this end. Recently, we described a 3D expansion system that mimics natural antigen presenting cells (APCs) and promotes greater polyclonal expansion of primary human T-cells than commercial expansion beads (Dynabeads). These biodegradable, APC-mimetic scaffolds (APC-ms) are composed of fluid lipid bilayers supported on mesoporous silica micro-rods, which enables precise control over the spatial organization of membrane-bound T-cell receptor (TCR) stimulatory and costimulatory cues (i.e., anti-CD3/anti-CD28). Here, we investigated the activation and memory signature of primary human T-cells over time in response to APC-ms with different densities of activating surface cues compared to Dynabead stimulation. Higher surface cue densities of anti-CD3/anti-CD28 promoted greater proportions of central memory T-cells, with a parallel increase in the CD4-to-CD8 T-cell ratio. T-cells stimulated with Dynabeads exhibited higher expression of CD25 and CD69 that peaked earlier than standard APC-ms formulations, suggesting that APC-ms promotes relatively more transient T-cell activation than Dynabeads, potentially mitigating overstimulation and exhaustion. Overall, these data demonstrate that APC-ms can be used to rapidly expand primary human T-cells and tune their phenotypic attributes (e.g., CD4-to-CD8 ratio and memory subpopulations). This technology has the potential to improve the production of T-cells for adoptive T-cell-based therapies. Citation Format: David K.Y. Zhang, Anna Vaynrub, David J. Mooney. Rapid and controlled T-cell expansion using scaffolds that mimic antigen-presenting cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B058.

Glucose Can Protect Membranes against Dehydration Damage by Inducing a Glassy Membrane State at Low Hydrations.

The physical effects of small sugars on membranes have been studied for decades, primarily because of their membrane stabilization in cold or dehydrated environments. We studied the effects of up to 20 mol% glucose in bilayers made of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at low hydration by combining X-ray diffraction and Molecular Dynamics (MD) simulations. In agreement with previous studies, we observe membrane thinning at low and membrane thickening at high sugar concentrations. Glucose was found to preferentially localize to the outer head region of phospholipid bilayers at all concentrations, and partitioning of sugar in the membranes was found to monotonically increase with increasing sugar concentration. While the number of gauche defects in the lipid acyl tails and the lipid packing in the presence of sugar resembled values of a fluid lipid bilayer, tail dynamics, as assessed by autocorrelation of the carbon atoms in the phospholipid tails, were slowed down significantly with increasing glucose content. Thus, our findings suggest that sugar leads to a a disordered, glassy state of the hydrophobic membrane core. The non-monotonic effect of glucose on membrane thickness was found to be an effect of fluidification at low concentrations and decreased interdigitation in the higher sugar concentration regime.

Nanoscale Probing of Informational Polymers with Nanopores. Applications to Amyloidogenic Fragments, Peptides, and DNA-PNA Hybrids.

The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or β-barrel moiety. Second, we lay out the principles of an approach dubbed "nanopore tweezing", enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA-DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.

Formation and Characteristics of Lipid-Blended Block Copolymer Bilayers on a Solid Support Investigated by Quartz Crystal Microbalance and Atomic Force Microscopy.

Liposomes grafted with polymer have long been used in drug delivery applications, and block copolymersomes have emerged as attractive and more robust alternatives for both drug delivery and artificial organelle applications. Hybrid membranes that could combine the respective advantages of fluid lipid and robust polymer bilayers are an attractive and enticing alternative. The properties of membranes in amphiphile vesicles are challenging to study and many applications benefit from surface-based access to the membrane. We therefore explore the self-assembly and mechanical properties of supported hybrid bilayers (SHBs) composed of polybutadiene- block-poly(ethylene oxide) block copolymers and zwitterionic phosphatidylcholine lipids on SiO2 supports. Quartz crystal microbalance with dissipation monitoring (QCM-D) measurements show that formation of SHB on SiO2 by vesicle fusion depends on the mass fractions of lipids and block copolymers. Atomic force microscopy was used to study the microscopic mixing of lipids in the SHB to reveal that lipid-phase separation is not observed in SHBs. Force spectroscopy was performed to extract information about thickness and mechanical properties of the hybrid membranes. SHBs are shown to combine the properties of lipid membranes and polymer brushes, and the tip force required to rupture the membrane decreases and the bilayer thickness increases as the block copolymer fraction is increased.

Spectroscopic Ellipsometry of fluid and gel phase lipid bilayers in hydrated conditions.

The biological membranes play a crucial role in the various biological processes due to their characteristic physical properties. The parameters such as membrane composition, thickness undulations, and the influence of external stimuli play a crucial role in the phase state behavior of biological membranes. The supported lipid bilayer (SLBs) systems closely represent cell membranes and are often studied to understand their behavior. In the current study, we have implemented spectroscopic Ellipsometry (SE) technique to explore the optical properties of various single component lipid bilayers having different phase state (fluid and gel) under hydrated or native conditions. The measurement of ellipsometric angles ψ and Δ were obtained from SE and were utilized to realize an explicit phase state dependence. Further, we obtained the thickness (t) and refractive index (n) by modeling of these SE results. These results were found to be inter-related to the phase state and lateral organization. The bilayer thickness is further verified against values obtained from coarse-grained molecular dynamics simulations. The proposed method was tested on various lipid bilayers under their native phase state at room temperature and was found to have a correlation with AFM measurements.

Modulation of Intestinal Transport and Absorption of Topotecan, a BCRP Substrate, by Various Pharmaceutical Excipients and Their Inhibitory Mechanisms of BCRP Transporter

Breast cancer resistance protein transporter (ABCG2/BCRP) is highly expressed on the intestinal epithelial membrane and has a significant impact on the oral absorption of topotecan. In this study, we examined 6 pharmaceutical excipients including BL-9EX, Brij97, Cremophor EL, Labrasol, Pluronic F68, and Tween 20 for their BCRP inhibitory effects. A bidirectional transport study using Caco-2 cells demonstrated that Tween 20 and Cremophor EL significantly increased the absorptive transport of topotecan, while simultaneously decreasing secretory transport. Interestingly, Labrasol selectively increased absorptive transport, whereas Pluronic F68 selectively decreased the secretory transport, of topotecan. Further investigation using an in situ closed loop experiment showed that 0.05% (w/v) Tween 20 and Cremophor EL significantly increased the intestinal absorption of topotecan in rats. An LDH assay demonstrated that 0.05% (w/v) Tween 20 and Cremophor EL did not cause significant damage to intestinal epithelial membranes. Furthermore, we examined the absorption-enhancing mechanisms of these excipients and found that Cremophor EL, Tween 20, and Labrasol increased the membrane fluidity of the inner lipid bilayers of the intestine. Therefore, this might be one of the most important mechanisms for inhibition of BCRP function by these excipients in the intestine.

Formation of Single Nanopores with Diameters of 20-50 nm in Silicon Nitride Membranes Using Laser-Assisted Controlled Breakdown.

Nanopores with diameters from 20 to 50 nm in silicon nitride (SiN x) windows are useful for single-molecule studies of globular macromolecules. While controlled breakdown (CBD) is gaining popularity as a method for fabricating nanopores with reproducible size control and broad accessibility, attempts to fabricate large nanopores with diameters exceeding ∼20 nm via breakdown often result in undesirable formation of multiple nanopores in SiN x membranes. To reduce the probability of producing multiple pores, we combined two strategies: laser-assisted breakdown and controlled pore enlargement by limiting the applied voltage. Based on laser power-dependent increases in nanopore conductance upon illumination and on the absence of an effect of ionic strength on the ratio between the nanopore conductance before and after laser illumination, we suggest that the increased rate of controlled breakdown results from laser-induced heating. Moreover, we demonstrate that conductance values before and after coating the nanopores with a fluid lipid bilayer can indicate fabrication of a single nanopore versus multiple nanopores. Complementary flux measurements of Ca2+ through the nanopore typically confirmed assessments of single or multiple nanopores that we obtained using the coating method. Finally, we show that thermal annealing of CBD pores significantly increased the success rate of coating and reduced the current noise before and after lipid coating. We characterize the geometry of these nanopores by analyzing individual resistive pulses produced by translocations of spherical proteins and demonstrate the usefulness of these nanopores for estimating the approximate molecular shape of IgG proteins.

Lipid- and Cholesterol-Mediated Time-Scale-Specific Modulation of the Outer Membrane Protein X Dynamics in Lipid Bilayers.

Membrane protein function fundamentally depends on lipid-bilayer fluidity and the composition of the biological membrane. Although dynamic interdependencies between membrane proteins and the surrounding lipids are suspected, a detailed description is still missing. To uncover lipid-modulated membrane protein backbone dynamics, time-scale-specific NMR relaxation experiments with residue-resolution were recorded. The data revealed that lipid order, modified either biochemically or biophysically, changes the dynamics of the immersed membrane protein in a specific and time-scale-dependent manner. A temperature-dependent dynamics analysis furthermore suggests a direct coupling between lipid and protein dynamics in the picosecond-nanosecond, microsecond, and millisecond time scales, caused by the lipid's trans-gauche isomerization, the segmental and rotational motion of lipids, and the fluidity of the lipid phase, respectively. These observations provide evidence of a direct modulatory capability of the membrane to regulate protein function through lipid dynamics ranging from picoseconds to milliseconds.

P-314 - Electron-transfer reactions within lipid bilayers take place in a medium whose averaged dielectric constant is higher than commonly assumed

Biochemical reactions occurring within lipid bilayers take place in a heterogeneous medium. A fluid lipid bilayer is a highly stratified structure with a distinct trans-bilayer molecular profile, e.g. the fluidity and polarity gradients, which in turn may influence the roles of biomembranes as selective barriers or functional arrays. Still, the analysis of electron transfer and redox reactions taking place in membranes is based in a theory that assumes the solvent as a continuum and whose dielectric constant is analogous to that of apolar methylenic solvents. We present the measurement of equivalent dielectric constants and their thermal behavior, for POPC and mixed POPC/cholesterol bilayers, using the pyrene (Py) Ham Effect. To do so, the emission spectra must exclude experimental artifacts from Py fluorescence in liposomes. Given that Py locates in the ordered section of bilayers, it probes dielectric constants averaged transversally in space (the long axis of Py, 9.2 A) and laterally in time (due to Py lateral diffusion during its lifetime 150 ns). At 20 °C, POPC bilayers display a dielectric constant analogous to 1-propanol which is much higher than that of an apolar solvent. Also, POPC bilayers exhibit higher dielectric constant than the mixtures at high cholesterol content.

Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'.

Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer.

Previously, we proposed a liposomal formulation of melphalan (Mlph)-a chemotherapeutic alkylating agent-incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM1, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86-90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM1 or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

Results Regarding the Resolving of Membrane-Protein Complexes using Neutron Reflection in Molecular Dynamics

Detailed characterization of membrane-associated proteins on fluid lipid bilayers is a challenge despite the availability of many biophysical techniques. Neutron reflectivity (NR) is a method that provides sub-nanometer resolution of such structures in biommetic environments. Analysis of NR data provides density distribution of protein along the membrane normal and coupling this data with complementing structural information from crystallography or NMR can reveal a full atomistic description of the protein-membrane complex. For systems in which this information is not available, because of disordered regions or ensembles of conformational states, additional work is required. Molecular dynamics (MD) simulations can provide the necessary structural information, but long equilibration times and sampling is an issue. Here we show a method to incorporate NR data into MD simulations to steer simulations toward configurations which reproduce experimental results. Biasing potentials are calculated from the one-dimensional densities derived in NR and evolve as the MD trajectory progresses. The bias directs the molecule toward the configurations that NR suggests and grows weaker as these conformations are adopted. Instead of rigidly confining the structure, the system is permitted to fluctuate about these configurations. We demonstrate applications to peptides and larger proteins with disordered regions.

A Fluid Membrane Enhances the Velocity of Cargo Transport by Small Teams of Kinesin-1

Kinesin-1 (hereafter referred to as kinesin) is a major microtubule-based motor protein for plus-end-directed intracellular transport in live cells. While the single-molecule functions of kinesin are well characterized, the physiologically relevant transport of membranous cargos by small teams of kinesins remains poorly understood. A key experimental challenge remains in the quantitative control of the number of motors driving transport. Here we utilized motile fraction to overcome this challenge and experimentally accessed transport by a single kinesin through the physiologically relevant transport by a small team of kinesins. We used a fluid lipid bilayer to model the cellular membrane in vitro and employed optical trapping to quantify the transport of membrane-enclosed cargos versus traditional membrane-free cargos under identical conditions. We found that coupling motors via a fluid membrane significantly enhances the velocity of cargo transport by small teams of kinesins. Importantly, enclosing a cargo in a fluid lipid membrane did not impact single-kinesin transport, indicating that membrane-dependent velocity enhancement for team-based transport arises from altered interactions between kinesins. Our study demonstrates that membrane-based coupling between motors is a key determinant of kinesin-based transport. Enhanced velocity may be critical for fast delivery of cargos in live cells.

Nanopore-Based, Multi-Parametric Characterization of Single, Unlabeled Proteins in Solution

Most methods for characterizing proteins - including electron microscopy, NMR spectroscopy, and X-Ray crystallography - require either some sort of physical modification such as purifying, drying, freezing, or crystallizing proteins, or they require chemical modification such as fluorescently labeling or immobilizing proteins. Resistive pulse-based nanopore sensing of proteins is emerging as a powerful analysis technique; it has the potential to overcome these requirements while characterizing individual, unlabeled proteins in aqueous solution. We recently used lipid-coated synthetic nanopores to determine the volume, shape, charge, dipole moment, and rotational diffusion coefficient of single proteins that we tethered to the fluid lipid bilayer to slow their passage through the pore. Here, we compare two different non-adsorptive nanopore coatings combined with high bandwidth recordings to determine the shape and volume of freely translocating proteins in solution. The difficulty of this approach lies in the lateral diffusion of untethered proteins transiting a nanopore, which facilitates position-dependent, asymmetric disruptions in the electric field as proteins approach the nanopore walls. These ‘off-axis’ effects pose a challenge to multi-parametric protein characterization because they distort the observed resistive pulse magnitudes, leading to analysis artifacts. Using lipid-coated nanopores to minimize non-specific protein adhesion, we mitigated these off-axis effects by reducing the residence time of proteins at the pore wall. This approach made it possible to estimate shape and volume values for nine different native proteins free in solution. In contrast, pores coated with the detergent Tween-20 were not suitable for protein shape and volume characterization likely because of significant adhesion to the pore wall and concomitant off-axis effects. By examining unlabeled proteins in an aqueous sample on a single molecule level in lipid bilayer-coated nanopores, we enable new applications for nanopore-based protein sensing, especially those involving transient protein complexes or macromolecules.

Scaffolds that mimic antigen-presenting cells enable ex vivo expansion of primary T cells.

Therapeutic ex vivo T cell expansion is limited by low rates, and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28 and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to ten-fold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a much greater magnitude than autologous monocyte-derived dendritic cells after two weeks. APC-ms support over 5-fold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.