Charged Biological Membranes Repel Large Neutral Molecules by Surface Dielectrophoresis and Counterion Pressure.

Macromolecular crowding is the usual condition of cells. The implications of the crowded cellular environment for protein stability and folding, protein-protein interactions, and intracellular transport drive a growing interest in quantifying the effects of crowding. While the properties of crowded solutions have been extensively studied, less attention has been paid to the interaction of crowders with the cellular boundaries, i.e., membranes. However, membranes are key components of cells and most subcellular organelles, playing a central role in regulating protein channel and receptor functions by recruiting and binding charged and neutral solutes. While membrane interactions with charged solutes are dominated by electrostatic forces, here we show that significant charge-induced forces also exist between membranes and neutral solutes. Using neutron reflectometry measurements and molecular dynamics simulations of poly(ethylene glycol) (PEG) polymers of different molecular weights near charged and neutral membranes, we demonstrate the roles of surface dielectrophoresis and counterion pressure in repelling PEG from charged membrane surfaces. The resulting depletion zone is expected to have consequences for drug design and delivery, the activity of proteins near membrane surfaces, and the transport of small molecules along the membrane surface.

The Single Residue K12 Governs the Exceptional Voltage Sensitivity of Mitochondrial Voltage-Dependent Anion Channel Gating.

The voltage-dependent anion channel (VDAC) is a ß-barrel channel of the mitochondrial outer membrane (MOM) that passively transports ions, metabolites, polypeptides, and single-stranded DNA. VDAC responds to a transmembrane potential by "gating," i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport, while enhancing calcium transport. Voltage gating is a universal property of ß-barrel channels, but VDAC gating is anomalously sensitive to transmembrane potential. Here, we show that a single residue in the pore interior, K12, is responsible for most of VDAC's voltage sensitivity. Using the analysis of over 40 µs of atomistic molecular dynamics (MD) simulations, we explore correlations between motions of charged residues inside the VDAC pore and geometric deformations of the ß-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the ß-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 Å indicates a similar architecture of the K12E mutant to the wild type; however, 60 µs of atomistic MD simulations using the K12E mutant show restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that ß-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.

Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Voltage-activated complexation of α-synuclein with three diverse ß-barrel channels: VDAC, MspA, and α-hemolysin.

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes-its anion selectivity and voltage gating behavior-have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized ß-barrel channels-VDAC, MspA, and α-hemolysin-that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering.

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.

Regulation of Mitochondrial Respiration by VDAC Is Enhanced by Membrane-Bound Inhibitors with Disordered Polyanionic C-Terminal Domains.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC's sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics-reduced metabolite flux and increased calcium flux-are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.

Exploring lipid-dependent conformations of membrane-bound α-synuclein with the VDAC nanopore.

Regulation of VDAC by α-synuclein (αSyn) is a rich and instructive example of protein-protein interactions catalyzed by a lipid membrane surface. αSyn, a peripheral membrane protein involved in Parkinson's disease pathology, is known to bind to membranes in a transient manner. αSyn's negatively charged C-terminal domain is then available to be electromechanically trapped by the VDAC ß-barrel, a process that is observed in vitro as the reversible reduction of ion flow through a single voltage-biased VDAC nanopore. Binding of αSyn to the lipid bilayer is a prerequisite of the channel-protein interaction; surprisingly, however, we find that the strength of αSyn binding to the membrane does not correlate in any simple way with its efficiency of blocking VDAC, suggesting that the lipid-dependent conformations of the membrane-bound αSyn control the interaction. Quantitative models of the free energy landscape governing the capture and release processes allow us to discriminate between several αSyn (sub-) conformations on the membrane surface. These results, combined with known structural features of αSyn on anionic lipid membranes, point to a model in which the lipid composition determines the fraction of αSyn molecules for which the charged C terminal domain is constrained to be close, but not tightly bound, to the membrane surface and thus readily captured by the VDAC nanopore. We speculate that changes in the mitochondrial membrane lipid composition may be key regulators of the αSyn-VDAC interaction and consequently of VDAC-facilitated transport of ions and metabolites in and out of mitochondria and, i.e. mitochondrial metabolism.

pH dependent electrical properties of the inner- and outer- leaflets of biomimetic cell membranes.

Composition and asymmetry of lipid membranes provide a means for regulation of trans-membrane permeability of ions and small molecules. The pH dependence of these processes plays an important role in the functioning and survival of cells. In this work, we study the pH dependence of membrane electrical resistance and capacitance using electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR) and neutron reflectometry (NR) measurements of biomimetic tethered bilayer lipid membranes (tBLMs). tBLMs were prepared with single-component phospholipid compositions, as well as mixtures of phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and cholesterol) that mimic the inner- and outer- leaflets of plasma cell membranes. We found that all studied tBLMs have a resistance maximum at pHs near the pKas of the phospholipids. SPR and NR indicated that surface concentration of phospholipids and the thickness of the hydrophobic part of the membrane did not change versus pH. We postulate that these maxima are the result of protonation of the phosphate oxygen of the phospholipids and that hydronium ions play a major role in the conductance at pHs < pKas while sodium ions play the major role at pHs > pKas. An additional sharp resistance maximum of the PE tBLMs found at pH 5.9 and most likely represents the phosphatidylethanolamine's isoelectric point. The data show the key roles of the characteristic parts of phospholipid molecules: terminal group (choline, carboxyl, amine), phosphate, glycerol and ester oxygens on the permeability and selectivity of ions through the membrane. The interactions between these groups lead to significant differences in the electrical properties of biomimetic models of inner- and outer- leaflets of the plasma cell membranes.

Charge Effects Provide Ångström-Level Control of Lipid Bilayer Morphology on Titanium Dioxide Surfaces.

Interfaces between molecular organic architectures and oxidic substrates are a central feature of biosensors and applications of biomimetics in science and technology. For phospholipid bilayers, the large range of pH- and ionic strength-dependent surface charge densities adopted by titanium dioxide and other oxidic surfaces leads to a rich landscape of phenomena that provides exquisite control of membrane interactions with such substrates. Using neutron reflectometry measurements, we report sharp, reversible transitions that occur between closely surface-associated and weakly coupled states. We show that these states arise from a complex interplay of the tunable length scale of electrostatic interactions with the length scale arising from other forces that are independent of solution conditions. A generalized free energy potential, with its inputs only derived from established measurements of surface and bilayer properties, quantitatively describes these and previously reported observations concerning the unbinding of bilayers from supporting substrates.

Tunable Electromechanical Nanopore Trap Reveals Populations of Peripheral Membrane Protein Binding Conformations.

We demonstrate that a naturally occurring nanopore, the voltage-dependent anion channel (VDAC) of the mitochondrion, can be used to electromechanically trap and interrogate proteins bound to a lipid surface at the single-molecule level. Electromechanically probing α-synuclein (αSyn), an intrinsically disordered neuronal protein intimately associated with Parkinson's pathology, reveals wide variation in the time required for individual proteins to unbind from the same membrane surface. The observed distributions of unbinding times span up to 3 orders of magnitude and depend strongly on the lipid composition of the membrane; surprisingly, lipid membranes to which αSyn binds weakly are most likely to contain subpopulations in which electromechanically driven unbinding is very slow. We conclude that unbinding of αSyn from the membrane surface depends not only on membrane binding affinity but also on the conformation adopted by an individual αSyn molecule on the membrane surface.

VDAC Gating Thermodynamics, but Not Gating Kinetics, Are Virtually Temperature Independent.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and an archetypical ß-barrel channel. Here, we study the effects of temperature on VDAC channels reconstituted in planar lipid membranes at the single- and multichannel levels within the 20°C to 40°C range. The temperature dependence of conductance measured on a single channel in 1 M KCl shows an increase characterized by a 10°C temperature coefficient Q10 = 1.22 ± 0.02, which exceeds that of the bathing electrolyte solution conductivity, Q10 = 1.17 ± 0.01. The rates of voltage-induced channel transition between the open and closed states measured on multichannel membranes also show statistically significant increases, with temperatures that are consistent with activation energy barriers of ∼10 ± 3 kcal/mol. At the same time, the gating thermodynamics, as characterized by the gating charge and voltage of equipartitioning, does not display any measurable temperature dependence. The two parameters stay within 3.2 ± 0.2 elementary charges and 30 ± 2 mV, respectively. Thus, whereas the channel kinetics, specifically its conductance and rates of gating response to voltage steps, demonstrates a clear increase with temperature, the conformational voltage-dependent equilibria are virtually insensitive to temperature. These results, which may be a general feature of ß-barrel channel gating, suggest either an entropy-driven gating mechanism or a role for enthalpy-entropy compensation.

Information gain from isotopic contrast variation in neutron reflectometry on protein-membrane complex structures.

A framework is applied to quantify information gain from neutron or X-ray reflectometry experiments [Treece, Kienzle, Hoogerheide, Majkrzak, Lösche & Heinrich (2019). J. Appl. Cryst. 52, 47-59], in an in-depth investigation into the design of scattering contrast in biological and soft-matter surface architectures. To focus the experimental design on regions of interest, the marginalization of the information gain with respect to a subset of model parameters describing the structure is implemented. Surface architectures of increasing complexity from a simple model system to a protein-lipid membrane complex are simulated. The information gain from virtual surface scattering experiments is quantified as a function of the scattering length density of molecular components of the architecture and the surrounding aqueous bulk solvent. It is concluded that the information gain is mostly determined by the local scattering contrast of a feature of interest with its immediate molecular environment, and experimental design should primarily focus on this region. The overall signal-to-noise ratio of the measured reflectivity modulates the information gain globally and is a second factor to be taken into consideration.

Effect of a post-translational modification mimic on protein translocation through a nanopore.

Post-translational modifications (PTMs) of proteins are recognized as crucial components of cell signaling pathways through modulating folding, altering stability, changing interactions with ligands, and, therefore, serving multiple regulatory functions. PTMs occur as covalent modifications of the protein's amino acid side chains or the length and composition of their termini. Here we study the functional consequences of PTMs for α-synuclein (αSyn) interactions with the nanopore of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. PTMs were mimicked by a divalent Alexa Fluor 488 sidechain attached separately at two positions on the αSyn C-terminus. Using single-channel reconstitution into planar lipid membranes, we find that such modifications change interactions drastically in both efficiency of VDAC inhibition by αSyn and its translocation through the VDAC nanopore. Analysis of the on/off kinetics in terms of an interaction "quasipotential" allows the positions of the C-terminal modifications to be determined with an accuracy of about three residues. Moreover, our results uncover a previously unobserved mechanism by which cytosolic proteins control ß-barrel channels and thus a new regulatory function for PTMs.

Accurate background correction in neutron reflectometry studies of soft condensed matter films in contact with fluid reservoirs.

Neutron reflectometry (NR) is a powerful method for looking at the structures of multilayered thin films, including biomolecules on surfaces, particularly proteins at lipid interfaces. The spatial resolution of the film structure obtained through an NR experiment is limited by the maximum wavevector transfer at which the reflectivity can be measured. This maximum is in turn determined primarily by the scattering background, e.g. from incoherent scattering from a liquid reservoir or inelastic scattering from cell materials. Thus, reduction of scattering background is an important part of improving the spatial resolution attainable in NR measurements. Here, the background field generated by scattering from a thin liquid reservoir on a monochromatic reflectometer is measured and calculated. It is shown that background subtraction utilizing the entire background field improves data modeling and reduces experimental uncertainties associated with localized background subtraction.

Advances in Translational Nanotechnology: Challenges and Opportunities.

The burgeoning field of nanotechnology aims to create and deploy nanoscale structures, devices, and systems with novel, size-dependent properties and functions. The nanotechnology revolution has sparked radically new technologies and strategies across all scientific disciplines, with nanotechnology now applied to virtually every area of research and development in the US and globally. NanoFlorida was founded to create a forum for scientific exchange, promote networking among nanoscientists, encourage collaborative research efforts across institutions, forge strong industry-academia partnerships in nanoscience, and showcase the contributions of students and trainees in nanotechnology fields. The 2019 NanoFlorida International Conference expanded this vision to emphasize national and international participation, with a focus on advances made in translating nanotechnology. This review highlights notable research in the areas of engineering especially in optics, photonics and plasmonics and electronics; biomedical devices, nano-biotechnology, nanotherapeutics including both experimental nanotherapies and nanovaccines; nano-diagnostics and -theranostics; nano-enabled drug discovery platforms; tissue engineering, bioprinting, and environmental nanotechnology, as well as challenges and directions for future research.

Protein Translocation Activity in Surface-Supported Lipid Bilayers.

Surface-supported lipid bilayers are used widely throughout the nanoscience community as cellular membrane mimics. For example, they are frequently employed in single-molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding. However, in AFM as well as in other surface-sensing techniques, the close proximity of the supporting surface raises questions about preservation of the biochemical activity. Employing the model translocase from the general secretory (Sec) system of Escherichia coli, here we quantify the activity via two biochemical assays in surface-supported bilayers. The first assesses ATP hydrolysis and the second assesses polypeptide translocation across the membrane via protection from added protease. Hydrolysis assays revealed distinct levels of activation ranging from medium (translocase-activated) to high (translocation-associated) that were similar to traditional solution experiments and further identified an adenosine triphosphatase population exhibiting characteristics of conformational hysteresis. Translocation assays revealed turn over numbers that were comparable to solution but with a 10-fold reduction in apparent rate constant. Despite differences in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topographic complexity of the underlying surface. Rough glass coverslips were favored over atomically flat mica, likely due to differences in frictional coupling between the translocating polypeptide and surface. Neutron reflectometry and AFM corroborated the biochemical measurements and provided structural characterization of the submembrane space and upper surface of the bilayer. Overall, the translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.