Membrane Proteins In Lipid Bilayers Research Articles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Advantages and potential limitations of applying AFM kymograph analysis to pharmaceutically relevant membrane proteins in lipid bilayers

Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. Torsional constraints can alleviate this potential pitfall. Confidence in detected transitions can be increased by adding conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution.

Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin?

Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid-protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca2+ transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by studying large membrane protein systems.

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Biophysical characterization of a series of novel non‐RAFT copolymers with improved buffer behavior

Synthetic amphipathic polymers present an invaluable tool for excision and liberation of superstructures of membrane proteins (MPs) and their surrounding annular membrane bilayer in donut‐shaped nanoparticles or discs. Styrene‐co‐maleic acid polymers (SMAs) have demonstrated most efficiency for the structural characterization of membrane proteins. However, some drawbacks such as sensitivity to acidic pH and high concentration of divalent cations, interference with the spectroscopic profiles of proteins, high polydispersity index (PDI) and nonspecific interaction with protein surface have hindered the ultimate optimal utilization of SMA polymers for functional characterization of MPs in lipid bilayer and hence for drug discovery purposes. We have developed multiple series of novel non‐RAFT amphipathic polymers that demonstrate improved behaviors in acidic pH and high concentration of calcium. Some of these unique amphipathic polymers display exemplary polydispersity (PDI ~1), strict alternation (a well‐defined sequence of comonomers) and distinct spectrophotometric profiles. We further characterized the application of these novel polymeric detergents for purification of a model integral beta‐barrel MP, PagP, from the outer membrane of E. coli.

PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers.

Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. Thiscan beachieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complementsboth X-ray crystallography andcryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

EPR Spectroscopic Studies of the Voltage Sensor Domain (Q1-VSD) of Human KCNQ1 Potassium Ion Channel in Lipid Bilayers

KCNQ1 is a voltage gated potassium ion channel expressed in several tissues of the body and modulated by another membrane protein KCNE1 (E1). Q1 is involved in the repolarization phase of cardiac action potential. Q1/E1 interaction slows down the activation kinetics required for proper channel and heart function. Mutations in Q1 can cause Long-QT syndrome resulting in atrial fibrillation, sudden infant death syndrome, cardiac arrhythmias and congenital deafness. Q1-VSD is important as the channel is activated by a change in voltage, which is sensed by the VSD. Around 40% of the >200 reported disease-related mutations arise from the amino acid substitutions in the VSD, making structural and functional studies of Q1-VSD important. The structure of a membrane protein varies from micelles to lipid bilayer environment. Site-directed spin labeling (SDSL) EPR is a powerful structural biology technique to study the structural dynamics and topology of membrane proteins in lipid bilayers. In this study, the structural dynamics and topology of several residues of Q1-VSD in the POPC/POPG vesicles were studied using CW-EPR line shape analysis and CW power saturation techniques. Spin labeled mutants F130C and F232C were found to be located inside the lipid bilayer, while Q147C and F222C were found to be solvent accessible. The study provides novel insights to the structural details of Q1-VSD reconstituted in POPC/POPG vesicles for more advanced structural and functional studies.

Automated Removal of Phospholipids from Membrane Proteins for H/D Exchange Mass Spectrometry Workflows.

Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling. Phospholipids were depleted from protein samples by the addition of zirconium oxide beads, which were subsequently removed by inline filtration using syringeless nanofilters. To demonstrate this method, single-pass transmembrane protein FcγRIIa (CD32a) expressed into liposomes was used. Successful depletion of phospholipids ensured optimal liquid-chromatography-mass-spectrometry performance, and measurement of peptides from the transmembrane domain of FcγRIIa indicated phospholipids associated with this region were either not present or did not shield the transmembrane domain from digestion by pepsin. Furthermore, amino acid sequence coverage provided by this method was suitable to enable future measurement of structural dynamics of ectodomain, transmembrane domain, and endodomain of FcγRIIa. Moreover, this method is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers, a notable advancement to facilitate understanding of membrane proteins, development of pharmaceuticals, and characterization for regulatory agencies.

Watching Three-Dimensional Movements of Single Membrane Proteins in Lipid Bilayers.

It is challenging to assess protein-membrane interactions because of the lack of appropriate tools to detect position changes of single proteins in the ∼4 nm range of biological membranes. We developed an assay recently, termed surface-induced fluorescence attenuation (SIFA). It is able to track both vertical and lateral dynamic motion of singly labeled membrane proteins in supported lipid bilayers. Similar to the FRET (fluorescence resonance energy transfer) principle, SIFA takes advantage of the energy transfer from a fluorophore to a light-absorbing surface to determine the distance at 2-8 nm away from the surface. By labeling a protein with a proper fluorophore and using graphene oxide as a two-dimensional quencher, we showed that SIFA is capable of monitoring three-dimensional movements of the fluorophore-labeled protein not only inside but also above the lipid bilayer atop the graphene oxide. Our data show that SIFA is a well-suited method to study the interplay between proteins and membranes.

1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins

Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is emerging as a unique method for the atomic resolution structure determination of native membrane proteins in lipid bilayers. Although 13C-detected ssNMR experiments continue to play a major role, recent technological developments have made it possible to carry out 1H-detected experiments, boosting both sensitivity and resolution. Here, we describe a new set of 1H-detected hybrid pulse sequences that combine through-bond and through-space correlation elements into single experiments, enabling the simultaneous detection of rigid and dynamic domains of membrane proteins. As proof-of-principle, we applied these new pulse sequences to the membrane protein phospholamban (PLN) reconstituted in lipid bilayers under moderate MAS conditions. The cross-polarization (CP) based elements enabled the detection of the relatively immobile residues of PLN in the transmembrane domain using through-space correlations; whereas the most dynamic region, which is in equilibrium between folded and unfolded states, was mapped by through-bond INEPT-based elements. These new 1H-detected experiments will enable one to detect not only the most populated (ground) states of biomacromolecules, but also sparsely populated high-energy (excited) states for a complete characterization of protein free energy landscapes.

Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers.

The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Investigations of the Structure and Orientation of Membrane Proteins in Oriented Lipid Bilayers

Oriented SRCD spectroscopy is a challenging but revealing tool for investigating the structure of membrane proteins in lipid bilayers. Using the B23 beamtime at Diamond Light Source, Oxfordshire, UK, we have been developing methods for the preparation and measurement of oriented SRCD samples to characterise the structure of integral membrane proteins and their orientation with respect to the lipid bilayer. In the work presented here we outline two methods for the preparation of oriented membrane proteins. The first relies on mechanical orientation obtained by macroscopically aligning bilayers on a Suprasil quartz support. The second exploits magnetic fields to align bicelle membrane mimetics with the membrane director either parallel or perpendicular to the direction of propagation of the light beam. The merits and drawbacks of these two methods for the determination of membrane protein orientation will be discussed.As a model membrane protein for these measurements we are using the transmembrane domain (TMD) of a putative glycosyltransferase Fukutin-I (FkI), whose mislocalization has been linked to the onset of Fukuyama Muscular Dytrophy. The localisation of Fk-I in the late ER/Golgi is thought to be mediated through changes in oligomeric state and structure induced by the surrounding lipids. Using oriented samples for both SRCD and solid-state NMR we are beginning to understand how the distinct lipid composition within the late ER/Golgi may govern the retention of this protein in these compartments.

NMR Restrained Protein Structure Calculations in Implicit Water/Membrane Environments

The aqueous and lipid membrane environments that make up the intra- and extra-cellular compartments of biological organisms are critical for supporting the structural and functional integrity of both soluble proteins and membrane proteins. NMR spectroscopy enables characterization of the structures of both types of proteins in their respective, near-native environments. However, most NMR structures are not calculated in physically realistic environments and de novo structure calculations in explicit solvent or explicit lipids are computationally expensive. Traditionally, NMR structures are calculated with a simplified repulsive term to prevent atom clashing. Such treatment accelerates the calculation but sacrifices non-bonded interactions. To facilitate NMR structure calculations in natural environments we are developing a computationally practical implicit solvent and membrane potential for Xplor-NIH. Here we show that this potential affords significant improvements in the conformational quality, accuracy and precision of the calculated structures. Further, it provides correct embedding of membrane proteins in lipid bilayers, as well as physically meaningful insights about residue-residue and protein-membrane interactions. We also describe extensions of the potential to bicelle and micelle sample environments as well as various membrane systems.This research was supported by grants from the National Institutes of Health (GM 110658; GM 100265; EB002031; CA 179087; CA 030199).

An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors

The study of mass-limited biological samples by magic angle spinning (MAS) solid-state NMR spectroscopy critically relies upon the high-yield transfer of material from a biological preparation into the MAS rotor. This issue is particularly important for maintaining biological activity and hydration of semi-solid samples such as membrane proteins in lipid bilayers, pharmaceutical formulations, microcrystalline proteins and protein fibrils. Here we present protocols and designs for rotor-packing devices specifically suited for packing hydrated samples into Pencil-style 1.6mm, 3.2mm standard, and 3.2mm limited speed MAS rotors. The devices are modular and therefore readily adaptable to other rotor and/or ultracentrifugation tube geometries.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F- ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl-/H+ antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known.

NMR of Membrane Proteins: Beyond Crystals.

Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current capabilities and future potential of NMR for membrane protein structural biology and ligand discovery.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

The in vitro influence of external electrostatic field on the lipid-protein interactions in membranes of rat erythrocytes

Purpose – The purpose of this paper is to investigate the in vitro influence of 200 kV/m electrostatic fields (ESF) on the microviscosity, viscosity and polarity of rat erythrocyte membranes for revealing the possible changes in lipid-protein interactions due to the field influence. Design/methodology/approach – The investigation of the parameters of erythrocyte membranes and their ghosts, particularly, their microviscosity, the amount and immersion degree of membrane proteins in lipid bilayer, polarity in depth of membrane and its viscosity carried out by the spectrofluorimetric method using the hydrophobic fluorescent probe pyrene. Findings – The carried out investigations shown that the in vitro influence of ESF changes membrane microviscosity, the quantity of membrane peripheral proteins and their immersion degree in the lipid bilayer, if the ghosts have prepared from erythrocytes previously exposed in the field. The analysis of the same parameters for previously prepared erythrocyte ghosts exposed to the 200 kV/m ESF during an hour did not reveal any changes. Originality/value – Data obtained and their comparison with the results of the previous works allow authors to conclude that the in vitro influence of ESF leads to the changes in the lipid-protein interactions in erythrocyte membranes.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.