Single-molecule Electrophysiology Research Articles

Complex functional phenotypes of NMDA receptor disease variants.

NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors' normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R side chain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.

Insertion state of modular protein nanopores into a membrane

In the past decade, significant progress has been made in the development of new protein nanopores. Despite these advancements, there is a pressing need for the creation of nanopores equipped with relatively large functional groups for the sampling of biomolecular events on their extramembranous side. Here, we designed, produced, and analyzed protein nanopores encompassing a robust truncation of a monomeric β-barrel membrane protein. An exogenous stably folded protein was anchored within the aqueous phase via a flexible peptide tether of varying length. We have extensively examined the pore-forming properties of these modular protein nanopores using protein engineering and single-molecule electrophysiology. This study revealed distinctions in the nanopore conductance and current fluctuations that arose from tethering the exogenous protein to either the N terminus or the C terminus. Remarkably, these nanopores insert into a planar lipid membrane with one specific conductance among a set of three substate conductance values. Moreover, we demonstrate that the occurrence probabilities of these insertion substates depend on the length of the peptide tether, the orientation of the exogenous protein with respect to the nanopore opening, and the molecular mass of tethered protein. In addition, the three conductance values remain unaltered by major changes in the composition of modular nanopores. The outcomes of this work serve as a platform for further developments in areas of protein engineering of transmembrane pores and biosensor technology.

Crowding Alters the Kinetics of Polypeptide-Protein Nanopore Interaction

Cellular crowding has wide-ranging significance on biomolecular processes inside the cell. In this study, the interaction of a charged polypeptide with a transmembrane protein pore was investigated using single-molecule electrophysiology under high concentrations of relatively large and neutral polyethylene glycols (PEG). Here, we show that the presence of high concentration PEGs at semi-dilute regime strengthens the affinity of polypeptide-pore interaction by increasing the association rate constant and decreasing the dissociation rate constant. this outcome is in agreement with the lower rate of diffusion in a crowded environment and with the higher depletion-attraction force between the polypeptide and PEG-free transmembrane pore.

Dynamic Plasticity of Mitochondrial VDAC2 Revealed by Single-Molecule Electrophysiology

Dynamic Plasticity of Mitochondrial VDAC2 Revealed by Single-Molecule Electrophysiology

Interactions of a Polypeptide with a Protein Nanopore Under Crowding Conditions.

Molecular crowding, a ubiquitous feature of the cellular environment, has significant implications in the kinetics and equilibrium of biopolymer interactions. In this study, a single charged polypeptide is exposed to competing forces that drive it into a transmembrane protein pore versus forces that pull it outside. Using single-molecule electrophysiology, we provide compelling experimental evidence that the kinetic details of the polypeptide-pore interactions are substantially affected by high concentrations of less-penetrating polyethylene glycols (PEGs). At a polymer concentration above a critical value, the presence of these neutral macromolecular crowders increases the rate constant of association but decreases the rate constant of dissociation, resulting in a stronger polypeptide-pore interaction. Moreover, a larger-molecular weight PEG exhibits a lower rate constant of association but a higher rate constant of dissociation than those values corresponding to a smaller-molecular weight PEG. These outcomes are in accord with a lower diffusion constant of the polypeptide and higher depletion-attraction forces between the polypeptide and transmembrane protein pore under crowding and confinement conditions.

Characterizing the Quantum-Confined Stark Effect in Semiconductor Quantum Dots and Nanorods for Single-Molecule Electrophysiology

We optimized the performance of quantum confined Stark effect QCSE based voltage nanosensors. A high throughput approach for single particle QCSE characterization was developed and utilized to screen a library of such nanosensors. Type II ZnSe CdS seeded nanorods were found to have the best performance among the different nanosensors evaluated in this work. The degree of correlation between intensity changes and spectral changes of the excitons emission under applied field was characterized. An upper limit for the temporal response of individual ZnSe CdS nanorods to voltage modulation was characterized by high throughput, high temporal resolution intensity measurements using a novel photon counting camera. The measured 3.5 us response time is limited by the voltage modulation electronics and represents about 30 times higher bandwidth than needed for recording an action potential in a neuron.

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores.

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ∼8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric β-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

Bacterial Outer Membrane Porins as Electrostatic Nanosieves: Exploring Transport Rules of Small Polar Molecules.

Transport of molecules through biological membranes is a fundamental process in biology, facilitated by selective channels and general pores. The architecture of some outer membrane pores in Gram-negative bacteria, common to other eukaryotic pores, suggests them as prototypes of electrostatically regulated nanosieve devices. In this study, we sensed the internal electrostatics of the two most abundant outer membrane channels of Escherichia coli, using norfloxacin as a dipolar probe in single molecule electrophysiology. The voltage dependence of the association rate constant of norfloxacin interacting with these nanochannels follows an exponential trend, unexpected for neutral molecules. We combined electrophysiology, channel mutagenesis, and enhanced sampling molecular dynamics simulations to explain this molecular mechanism. Voltage and temperature dependent ion current measurements allowed us to quantify the transversal electric field inside the channel as well as the distance where the applied potential drops. Finally, we proposed a general model for transport of polar molecules through these electrostatic nanosieves. Our model helps to further understand the basis for permeability in Gram-negative pathogens, contributing to fill in the innovation gap that has limited the discovery of effective antibiotics in the last 20 years.

Functional Reconstitution of V-ATPase Proteolipid Ring into a Planar Lipid Membrane

In this work, we show the first purification and functional characterization of the proteolipid ring (c-ring) complex of the membrane-bound Vo sector of the eukaryotic vacuolar H+-ATPase (V-ATPase) from the yeast Saccharomyces cerevisiae using techniques of membrane biochemistry, structural biology, and single-molecule electrophysiology. Our work was motivated by, and builds on, a large body of literature spanning more than three decades that suggested that Vo, besides its proton pumping function in holo V-ATPase, has other, so-called “non-canonical” functions in essential processes, such as membrane fusion and neurotransmitter release. However, direct evidence that Vo, and specifically, its core proteolipid ring (c-ring) complex, possesses the required properties to fulfill these non-canonical functions is still lacking. Our results presented here show that the c-ring forms a large-conductance transmembrane pore, which inserted into planar lipid bilayers with a preferred orientation. Moreover, using single-particle cryo-electron microscopy, we demonstrate that the purified c-ring forms dimers through an interaction mediated by the cytoplasmic loops of the c subunits. Taken together, these findings provide the first direct attestation in support of the proposed non-canonical functions of the V-ATPase Vo sector.1 This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.). 1. Couoh-Cardel, S.; Hsueh, Y. C.; Wilkens, S.; Movileanu, L., Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Scientific reports 2016, 6, 24774.

Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore.

The vacuolar H+ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase’s membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits’ cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ~8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.

Global redesign of a native β-barrel scaffold

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded β-barrel protein into a large-conductance protein pore. Here, we show that this redesigned β-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric β-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation.

The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

Investigation of Cu2+ Binding to Human and Rat Amyloid Fragments Aβ (1–16) with a Protein Nanopore

Recent evidence shows that metal coordination by amyloid beta peptides (Aβ) determines structural alterations of peptides, and His-13 from Aβ is crucial for Cu(2+) binding. This study used the truncated, more soluble Aβ1-16 isoforms derived from human and rat amyloid peptides to explore their interaction with Cu(2+) by employing the membrane-immobilized α-hemolysin (α-HL) protein as a nanoscopic probe in conjunction with single-molecule electrophysiology techniques. Unexpectedly, the experimental data suggest that unlike the case of the human Aβ1-16 peptide, Cu(2+) complexation by its rat counterpart leads to an augmented association and dissociation kinetics of the peptide reversible interaction with the protein pore, as compared to the Cu(2+)-free peptide. Single-molecule electrophysiology data reveal that both human and rat Cu(2+)-complexed Aβ peptides induce a higher degree of current flow obstruction through the α-HL pore, as compared to the Cu(2+)-free peptides. It is suggested that morphology changes brought by Cu(2+) binding to such amyloidic fragments depend crucially upon the presence of the His-13 residue on the primary sequence of such peptide fragments, and the α-HL protein-based approach provides unique opportunities and challenges to probing metal-induced folding of peptides.

Kinetic and Energetic Insights into the Gating of a Single Protein Channel

Gating of a beta-barrel membrane protein is a fundamentally important and ubiquitous process in membrane biology. Here, we systematically examined details of the kinetics and energetics of the current fluctuations within the OccK1 protein, a family member of outer membrane carboxylate channels from Pseudomonsa Aeruginosa. Temperature-dependent, single-molecule electrophysiology analysis of the native and loop-deletion OccK1 proteins demonstrated the distinctive nature of two energetic barriers: an enthalpic barrier of the large-current amplitude, infrequent O1 to O2 transitions and an entropic barrier of the low-current amplitude, highly frequent O2 to O3 transitions. Changes in temperature produced asymmetric kinetic and energetic modifications that caused a switch of the most probable open sub-state, O2 to O3, at the lowest examined temperature of 4 0C. Our approach might be used in the future to obtain a semi-quantitative assessment of the discrete fluctuation dynamics in other beta-barrel protein channels of the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts.Acknowledgments: This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M. and R01 GM085785, B.v.d.).

Pseudomonas Aerginosa Outer Membrane Carboxylate Channels Examined at the Single-Molecule Level Reveals Conserved Selectivity within each Subfamily

Pseudomonas aerginosa is a Gram-negative bacterium, which employs unique outer membrane proteins for the uptake of small, water-soluble molecules. Here, we explore the two subfamilies of the outer membrane carboxylate channels, OccK and OccD, using single-molecule electrophysiology to probe the specific signature of each of the members of these two families. The seven member OccK family displays a broad range of unitary conductance values which includes low (∼40−100 pS) and medium (∼100−380 pS) conductance. These values are broader than what was expected from structural studies alone. It was also found that the OccK subfamily displays conserved anion selectivity, coinciding with the location of a net pool of positive charges within the channels' constriction sites. Single-channel activities of 6 members of the OccD subfamily also displayed a broad range of unitary conductance values between 20 and 670 pS. Single-channel electrophysiology studies indicated that the OccD subfamily members are all cation selective. Together these findings lead to a better understanding of the diversity within the outer membrane carboxylate channels of Pseudomonas aerginosa.

Protein Nanopore-Based, Single-Molecule Exploration of Copper Binding to an Antimicrobial-Derived, Histidine-Containing Chimera Peptide

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.

An Outer Membrane Protein Undergoes Enthalpy- and Entropy-Driven Transitions

β-Barrel membrane proteins often fluctuate among various open substates, yet the nature of these transitions is not fully understood. Using temperature-dependent, single-molecule electrophysiology analysis, along with rational protein design, we show that OccK1, a member of the outer membrane carboxylate channel from Pseudomonas aeruginosa, features a discrete gating dynamics comprising both enthalpy-driven and entropy-driven current transitions. OccK1 was chosen for the analysis of these transitions, because it is a monomeric transmembrane β-barrel of a known high-resolution crystal structure and displays three distinguishable, time-resolvable open substates. Native and loop-deletion OccK1 proteins showed substantial changes in the activation enthalpies and entropies of the channel transitions, but modest alterations in the equilibrium free energies, confirming that the system never departs from equilibrium. Moreover, some current fluctuations of OccK1 indicated a counterintuitive, negative activation enthalpy, which was compensated by a significant decrease in the activation entropy. Temperature scanning of the single-channel properties of OccK1 exhibited a thermally induced switch of the energetically most favorable open substate at the lowest examined temperature of 4 °C. Therefore, such a semiquantitative assessment of the current fluctuation dynamics not only demonstrates the complexity of channel gating but also reveals distinct functional traits of a β-barrel outer membrane protein under different temperature circumstances.

Meet Me on the Other Side: Trans-Bilayer Modulation of a Model Voltage-Gated Ion Channel Activity by Membrane Electrostatics Asymmetry

While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin - a model voltage-gated ion channel - was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.

Mapping Heat Exchange in an Allosteric Protein

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.

Mechanism of DNA Transport Through Pores

The transport of electrically charged macromolecules such as DNA through narrow pores is a fundamental process in life. When polymer molecules are forced to navigate through pores, their transport is controlled by entropic barriers that accompany their conformational changes. During the past decade, exciting results have emerged from single-molecule electrophysiology experiments. Specifically, the passage of single-stranded DNA/RNA through alpha-hemolysin pores and double-stranded DNA through solid-state nanopores has been investigated. By a combination of these results with the entropic barrier theory of polymer transport and macromolecular simulations, an understanding of the mechanism of DNA transport through pores has emerged.