Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs

An important step in the production of inflammatory mediators of the leukotriene family is the Ca²⁺ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca²⁺ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.     

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies

Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag. The resulting integral membrane protein was incorporated into nanodiscs. Binding of the nanodisc-inserted histidine-tagged protein to a monoclonal anti-pentahistidine antibody was quantified using surface plasmon resonance (SPR) experiments. For the first time, a membrane-inserted transmembrane protein was employed as analyte while the antibody served as ligand immobilized on the sensor chip surface. SPR experiments were conducted in single-cycle mode. We demonstrate that the nanodisc-incorporated membrane protein showed nearly identical affinity toward the antibody as did the soluble decahistidine-tagged ubiquitin studied in a comparative experiment. Advantages of the new experimental setup and potential applications are discussed.     

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.     

Optimization of the design and preparation of nanoscale phospholipid bilayers for its application to solution NMR

Despite arduous efforts and recent technological developments structural investigation of integral membrane proteins remains a challenge. The primary deterrents include difficulties with their expression, low inherent solubility, and problems associated with existing membrane mimicking systems. A relatively new class of membrane mimetics, nanodiscs, is emerging as a promising alternative. Although nanodiscs have been proven successful for several biophysical applications, they yet remain to become the system of preferred choice for structure determination. We have hereby made nanodiscs more suitable for solution NMR applications by reducing the diameter of the self‐assembly complex to its potential limit. We achieved a noticeable improvement in the quality of NMR spectra obtained for the transmembrane and cytoplasmic domains of integrin α_IIb incorporated into these smaller discs rendering them susceptible for a thorough structural investigation. In addition, we also present an on‐column method for a rapid, efficient, single‐step preparation of protein incorporated nanodiscs at high concentrations. These discs have been fully characterized by transmission electron microscopy, dynamic light scattering, and differential scanning calorimetry. Proteins 2013; 81:1222–1231. © 2013 Wiley Periodicals, Inc.     

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H⁺-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H⁺-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H⁺-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.     

Nanodiscs versus macrodiscs for NMR of membrane proteins

It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.     

Modifying styrene-maleic acid co-polymer for studying lipid nanodiscs

Recently, styrene-maleic acid copolymer lipid nanodiscs have become an increasingly popular tool for the study of membrane proteins. In the work we report here, we have developed a novel method for the efficient preparation of labeled nanodiscs, under chemically mild conditions, by modification of the hydrolyzed styrene-maleic acid copolymer. This protocol is designed to be easily accessible to biochemistry laboratories. We use this procedure to prepare various fluorescent nanodiscs labeled with different fluorophores. By studying the development of Förster resonance energy transfer, we demonstrate the rapid exchange of co-polymer between nanodiscs. This demonstration, in conjunction of previous work, indicates that the lipid nanodiscs prepared using this polymer are very dynamic structures with rapid exchange of the different components.     

Structure and function of proteins in membranes and nanodiscs

The field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. This has led to conceptual advances including biological membrane:protein assemblies or “memteins” as the fundamental functional units of biological membranes. Tools including cryo-electron microscopy and X-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. Challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. In this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. Ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets.     

Nanodisc-based co-immunoprecipitation for mass spectrometric identification of membrane-interacting proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.     

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.     

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.     

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Nanodiscs are self-assembled ∼50-nm² patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.     

The lipid environment of Escherichia coli Aquaporin Z

In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W₁₄A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting.     

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.     

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

Discovering how membrane proteins recognize signals and passage molecules remains challenging. Life depends on compartmentalizing these processes into dynamic lipid bilayers that are technically difficult to work with. Several polymers have proven adept at separating the responsible machines intact for detailed analysis of their structures and interactions. Styrene maleic acid (SMA) co-polymers efficiently solubilize membranes into native nanodiscs and, unlike amphipols and membrane scaffold proteins, require no potentially destabilizing detergents. Here we review progress with the SMA lipid particle (SMALP) system and its impacts including three dimensional structures and biochemical functions of peripheral and transmembrane proteins. Polymers systems are emerging to tackle the remaining challenges for wider use and future applications including in membrane proteomics, structural biology of transient or unstable states, and discovery of ligand and drug-like molecules specific for native lipid-bound states.     

Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X‐ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR‐based investigations of membrane proteins in a natural bilayer environment.     

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aβ40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aβ40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20?μM) and nanodisc size (<10?nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aβ40 aggregation and were also found to be suitable to trap Aβ40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aβ40 conformation upon 4F binding through electrostatic and π–π interactions. Specifically, the 4F peptide was found to interfere with the central β-sheet-forming residues of Aβ40 through substantial hydrogen, π–π, and π–alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aβ40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aβ40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aβ40 intermediates for high-resolution structural and neurobiological studies.     

Solution-Based Single-Molecule FRET Studies of K+ Channel Gating in a Lipid Bilayer

Ion channels are dynamic multimeric proteins that often undergo multiple unsynchronized structural movements as they switch between their open and closed states. Such structural changes are difficult to measure within the context of a native lipid bilayer and have often been monitored via macroscopic changes in Förster resonance energy transfer (FRET) between probes attached to different parts of the protein. However, the resolution of this approach is limited by ensemble averaging of structurally heterogeneous subpopulations. These problems can be overcome by measurement of FRET in single molecules, but this presents many challenges, in particular the ability to control labeling of subunits within a multimeric protein with acceptor and donor fluorophores, as well as the requirement to image large numbers of individual molecules in a membrane environment. To address these challenges, we randomly labeled tetrameric KirBac1.1 potassium channels, reconstituted them into lipid nanodiscs, and performed single-molecule FRET confocal microscopy with alternating-laser excitation as the channels diffused in solution. These solution-based single-molecule FRET measurements of a multimeric ion channel in a lipid bilayer have allowed us to probe the structural changes that occur upon channel activation and inhibition. Our results provide direct evidence of the twist-to-shrink movement of the helix bundle crossing during channel gating and demonstrate how this method might be applied to real-time structural studies of ion channel gating.     

Cell-free synthesis and reconstitution of Bax in nanodiscs: Comparison between wild-type Bax and a constitutively active mutant

Bax is a major player in the mitochondrial pathway of apoptosis, by making the Outer Mitochondrial Membrane (OMM) permeable to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not yield Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed biochemical properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes. Both Bax variants were then reconstituted in nanodiscs. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer.     

Non-ionic detergent assists formation of supercharged nanodiscs and insertion of membrane proteins

Nanodiscs are used to stabilize membrane proteins in a lipid environment and enable investigations of the function and structure of these. Membrane proteins are often only available in small amounts, and thus the stability and ease of use of the nanodiscs are essential. We have recently explored circularizing and supercharging membrane scaffolding proteins (MSPs) for nanodisc formation and found increased temporal stability at elevated temperatures. In the present study, we investigate six different supercharged MSPs and their ability to form nanodiscs: three covalently circularized and the three non-circularized, linear versions. Using standard reconstitution protocols using cholate as the reconstitution detergent, we found that two of the linear constructs formed multiple lipid-protein species, whereas adding n-Dodecyl-B-D-maltoside (DDM) with the cholate in the reconstitution gave rise to single-species nanodisc formation for these MSPs. For all MSPs, the formed nanodiscs were analyzed by small-angle X-ray scattering (SAXS), which showed similar structures for each MSP, respectively, suggesting that the structures of the formed nanodiscs are independent of the initial DDM content, as long as cholate is present. Lastly, we incorporated the membrane protein proteorhodopsin into the supercharged nanodiscs and observed a considerable increase in incorporation yield with the addition of DDM. For the three circularized MSPs, a single major species appeared in the size exclusion chromatography (SEC) chromatogram, suggesting monodisperse nanodiscs with proteorhodopsin incorporated, which is in strong contrast to the samples without DDM showing almost no incorporation and high polydispersity.