Single Membrane Protein Research Articles

Nonlinear compliance of NompC gating spring and its implication in mechanotransduction.

Cytoskeleton-tethered mechanosensitive channels (MSCs) utilize compliant proteins or protein domains called gating springs to convert mechanical stimuli into electric signals, enabling sound and touch sensation and proprioception. The mechanical properties of these gating springs, however, remain elusive. Here, we explored the mechanical properties of the homotetrameric NompC complex containing long ankyrin-repeat domains (ARDs). We developed a toehold-mediated strand displacement approach to tether single membrane proteins, allowing us to exert force on them and precisely measure their absolute extension using optical tweezers. Our findings revealed that each ARD has a low stiffness of ~0.7 pN/nm and begins to unfold stepwise at ~7 pN, leading to nonlinear compliance. Our calculations indicate that this nonlinear compliance may help regulate NompC's sensitivity, dynamic range, and kinetics to detect mechanical stimuli. Overall, our research highlights the importance of a compliant and unfolding-refolding gating spring in facilitating a graded response of MSC ion transduction across a wide spectrum of mechanical stimuli.

Intermembrane space-localized TbTim15 is an essential subunit of the single mitochondrial inner membrane protein translocase of trypanosomes.

All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm−2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm−2) in dense cytosolic tubulin datasets.

Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.

Aberrant topologies of bacterial membrane proteins revealed by high sensitivity fluorescence labelling

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins.

Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 hr at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane homodimer for 9 hr at 12 pN and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling analysis, respectively. The Kramers rate framework yields a considerably low-speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.

Nanoconfinement-Enhanced Electrochemiluminescence for in Situ Imaging of Single Biomolecules.

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications.

Single-molecule investigation of ligand-activation mechanisms for membrane proteins in cell-derived nanovesicles.

Molecular recognition of ligands for specific binding sites in membrane proteins is a cornerstone of biological signaling processes. Despite structural and functional evidence often painting a relatively clear picture of the binding sites, the mechanism by which ligand binding to multiple active sites confers a change in protein activity is in many cases still poorly understood. This is due in part to the fact that individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we describe an approach for resolving individual binding events of fluorescently labeled ligands at single membrane proteins in cell-derived nanovesicles. The approach involves simple transient transfection, does not use detergent solubilization so that proteins are maintained throughout in their native lipid bilayer, and provides solution access to either intracellular or extracellular binding domains. Furthermore, we describe a fully automated approach to idealization of fluorescence time series describing the binding dynamics at many molecules imaged simultaneously. As an exemplar, we apply these approaches to cyclic nucleotide binding to TAX-4 ion channels critical for sensory transduction and discuss mechanistic insights from kinetic modeling of their binding dynamics. This approach is broadly applicable to studies of binding dynamics for membrane proteins with extracellular or intracellular domains in native cell membrane.

Nanopore-enabled macroscopic alignment of 700 kDa membrane protein complex: Characterization by pulsed EPR

The Photosystem II (PSII) catalytic cycle involves proton transfer as well as rearrangements of hydrogen bonding of sidechains and cofactors and their reorientation. While thought to be critically important for the function, some of the changes including protonation states of the cofactors and metal cluster ligands are inaccessible by x-ray crystallography but can be addressed in EPR experiments with macroscopically aligned PSII embedded in lipid membranes. Previously, we reported on aligning lipid bilayers within anodic aluminum oxide nanopores (AAO) 40 to 90 nm in diameter. The method provides for lipid alignment within 2-3o mosaic spread and worked well for single domain membrane proteins. Here, we successfully address a challenge of aligning a much larger membrane protein complex - ca. 700-kDa dimer of PSII. This was accomplished by developing AAO substrates with 300-400-nm pore diameter and 80 μm in length and forming nanotubular PSII bilayer inside the pores by self-assembly directly from aqueous suspensions. After the deposition, the nanoporous substrates acquired dark green color indicative of PSII complexes in high density. AAO strips were exposed to ambient light for 10 min at room temperature to generate YD⦁ radical species. The shock frozen samples retained macroscopic alignment of PSII as revealed by EPR. The angle dependent hyperfine interactions with 3- and 5-ring protons were measured in 2D HYSCORE experiments at two orthogonal sample orientations in magnetic field, B0. The two spectra exhibited well-pronounced differences caused by intensity redistributions among the cross-peaks arising from different orientations of hyperfine tensors vs. B0 and provided angular restrains for the tensor orientation with respect to the bilayer normal. Spectral resolution was also improved by decreasing HYSCORE peak crowding from the macroscopic alignment.

Simultaneous single photon counting and ion channel recording from free-standing lipid bilayers on a microstructured array.

Fluorescence microscopy and, in particular, single molecule optical spectroscopy is of great potential value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine such high-resolution optical measurements with high-resolution voltage clamp electrical recordings that can provide direct information on single ion channel gating a block by drugs and analytes. Here, we report on the use of a novel chip-based, 2 × 2 arrangement of microelectrode cavities with borosilicate glass optical windows which facilitates optical access on an inverted microscope with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes, while controlling membrane voltage and recording currents using individual micropatterned, ring-shaped Ag/AgCl-electrodes to perform time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities. Single channel activity induced by the fluorescently labelled pore-forming peptide ceratotoxin A was simultaneously acquired. This device allows for rapid formation of four membranes that are simultaneously monitored electrically using a four-channel amplifier and can be sequentially optically addressed, greatly reducing the time needed for successful experimentation. During our experiments, we noted autofluorescence of the borosilicate to be a limiting factor for wide-field low-intensity fluorescence recording, but this can likely be circumvented by using quartz glass instead. In summary, the novel device increases the likelihood of realizing the long standing ambition to correlate structural and functional dynamics of single membrane proteins on the single molecule level.

Enhanced electrochemiluminescence imaging of single cell membrane proteins based on Co3O4 nanozyme catalysis.

The development of enhanced strategies with excellent biocompatibility is critical for electrochemiluminescence (ECL) imaging of single cells. Here, we report an ECL imaging technique for a single cell membrane protein based on a Co3O4 nanozyme catalytic enhancement strategy. Due to the remarkable catalytic performance of Co3O4 nanozymes, H2O2 can be efficiently decomposed into reactive oxygen radicals, and the reaction with L012 was enhanced, resulting in stronger ECL emission. The anti-carcinoembryonic antigen (CEA) was coupled with nanozyme particles to construct a probe that specifically recognized the overexpressed CEA on the MCF-7 cell membrane. According to the locally enhanced visualized luminescence, the rapid ECL imaging of a single cell membrane protein was eventually realized. Accordingly, Co3O4 nanozymes with highly efficient activity will provide new insights into ECL imaging analysis of more biological small molecules and proteins.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

Selective inhibition and quantification of endogenous cAMP binding sites in supported native membranes for single-molecule studies

Single membrane proteins are often studied in solubilized or reconstituted form to avoid endogenous background signals. However, such an artificial experimental environment, generated by the required purification protocols, might alter functional properties of the proteins investigated. Here we demonstrate on the example of the HCN2 pacemaker channel in supported membranes, that the endogenous background can be reduced by a suitable inhibitor protocol. Additionally, quantitative insight into the density of endogenous cAMP binding sites in the membrane can be obtained.

EPR deer distance measurements on KCNE3 in proteoliposomes

KCNE3 is a single pass membrane protein involved in modulating the function and trafficking of variety of voltage gated potassium ion channels. A three dimensional structure of KCNE3 was recently determined using a solution NMR spectroscopy in isotropic DHPC/DMPG bicelles by the Sanders group. However, the structural topology and dynamic properties of KCNE3 are not fully understood in its native like environment. Double electron electron resonance (DEER), an advanced electron paramagnetic resonance (EPR) spectroscopic technique, is a very powerful and rapidly growing structure biology tool to study structural dynamics of a membrane protein by measuring distances within the range of 20-80 Å between two spin-labeled sites.

Liquid-phase scanning electron microscopy for single membrane protein imaging

Liquid-phase electron microscopy is highly desirable for observing biological samples in their native liquid state at high resolution. We developed liquid imaging approaches for biological cells using scanning electron microscopy. Novel approaches included scanning transmission electron imaging using a liquid-cell apparatus (LC-STEM), as well as correlative cathodoluminescence and electron microscopy (CCLEM) imaging. LC-STEM enabled imaging at a ∼2 nm resolution and excellent contrast for the precise recognition of localization, distribution, and configuration of individually labeled membrane proteins on the native cells in solution. CCLEM improved the resolution of fluorescent images down to 10 nm. Liquid SEM technologies will bring unique and wide applications to the study of the structure and function of cells and membrane proteins in their near-native states at the monomolecular level.

Single Biomolecule Imaging by Electrochemiluminescence.

Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides.

Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Investigation of Structural Dynamics of the KCNE3 in a Lipid Bilayer Membrane using Site-Directed Spin Labeling EPR Spectroscopy

KCNE3, a single pass membrane protein, is responsible for controlling the function of multiple potassium channels, one of which is the KCNQ1 channel. The binding of KCNE3 with KCNQ1 is vital in transepithelial potassium ion recycling which has been associated with disorders such as cystic fibrosis. Although KCNE3 has great biological significance, its structural and dynamic information in a native like environment is poorly understood. In this study, we have utilized site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopic technique to investigate structural dynamics of KCNE3 in lipid bilayers. KCNE3 was overexpressed and purified in LMPG detergent micelles. MTSL spin label was introduced followed by the incorporation of the protein into POPC/POPG lipid bilayer. Continuous wave (CW)-EPR spectral lineshape analysis was performed on several spin labeling sites including transmembrane domain and extracellular domain of KCNE3 in LMPG detergent micelles and POPC/POPG lipid bilayes to obtain the spin label sidechain mobility and the rotational correlation times. Our EPR data indicated that the nitroxide spin label sidechains in liposomes have slower motion for the sites in the transmembrane domain of KCNE3 when compared to that in the extracellular region of KCNE3. Our results are consistent with the previously published solution NMR structure of KCNE3.

Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Structural Studies.

Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.