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.     

Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer

The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa(3)- and ba(3)-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.     

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.     

Formation of stable nanodiscs by bihelical apolipoprotein A‐I mimetic peptide

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A‐I (apoA‐I) and phospholipids. Although peptide‐based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA‐I‐based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline‐punctuated bihelical amphipathic structure based on apoA‐I mimetic peptides. NSP formed α‐helical structure on 1‐palmitoyl‐2‐oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA‐I‐POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA‐I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK₂ complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.     

Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.     

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species.     

Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest.     

Molecular dynamics simulations of lipid nanodiscs

A lipid nanodisc is a discoidal lipid bilayer stabilized by proteins, peptides, or polymers on its edge. Nanodiscs have two important connections to structural biology. The first is associated with high-density lipoprotein (HDL), a particle with a variety of functionalities including lipid transport. Nascent HDL (nHDL) is a nanodisc stabilized by Apolipoprotein A-I (APOA1). Determining the structure of APOA1 and its mimetic peptides in nanodiscs is crucial to understanding pathologies related to HDL maturation and designing effective therapies. Secondly, nanodiscs offer non-detergent membrane-mimicking environments and greatly facilitate structural studies of membrane proteins. Although seemingly similar, natural and synthetic nanodiscs are different in that nHDL is heterogeneous in size, due to APOA1 elasticity, and gradually matures to become spherical. Synthetic nanodiscs, in contrast, should be homogenous, stable, and size-tunable. This report reviews previous molecular dynamics (MD) simulation studies of nanodiscs and illustrates convergence and accuracy issues using results from new multi-microsecond atomistic MD simulations. These new simulations reveal that APOA1 helices take 10–20 μs to rearrange on the nanodisc, while peptides take 2 μs to migrate from the disc surfaces to the edge. These systems can also become kinetically trapped depending on the initial conditions. For example, APOA1 was trapped in a biologically irrelevant conformation for the duration of a 10 μs trajectory; the peptides were similarly trapped for 5 μs. It therefore remains essential to validate MD simulations of these systems with experiments due to convergence and accuracy issues. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Advances in nanodisc platforms for membrane protein purification

Membrane scaffold protein nanodiscs (MSPNDs) are an invaluable tool for improving purified membrane protein (MP) stability and activity compared to traditional micellar methods, thus enabling an increase in high-resolution MP structures, particularly in concert with cryogenic electron microscopy (cryo-EM) approaches. In this review we highlight recent advances and breakthroughs in MSPND methodology and applications. We also introduce and discuss saposin–lipoprotein nanoparticles (salipros) and copolymer nanodiscs which have recently emerged as authentic MSPND alternatives. We compare the advantages and disadvantages of MSPNDs, salipros, and copolymer nanodisc technologies to highlight potential opportunities for using each platform for MP purification and characterization.     

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.     

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N‐terminal domain of anthrax lethal factor (LF_N) into lipid nanodiscs and analyzed the resulting complexes by negative‐stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (～43%). Three‐dimensional analysis of negatively stained single particles revealed the LF_N‐PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF_N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo‐EM LF_N‐PA nanodisc structures and use of advanced automated particle selection methods.     

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.     

Structure and energy-linked activities in reconstituted bacteriorhodopsin--yeast ATPase proteoliposomes.

Bacteriorhodopsin-F₁·F₀ (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F₁·F₀ is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F₁·F₀ by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [³²P]ATP formation where pyridine is present during first stage illumination.F₁·F₀ can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.     

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.     

Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition

The vesicle-micelle transition of egg phosphatidylcholine (PC) and sodium cholate was described by comparing cryo-transmission electron microscopic (cryo-TEM) images of the structures formed to the associated turbidity changes. These experiments were designed to identify the morphology of the intermediates between vesicles and small spheroidal mixed micelles. With increasing cholate concentration, the vesicular structures changed size and more multilamellar vesicles were seen. Between the apparent upper and lower phase boundaries, three structures were observed: open vesicles, large bilayer sheets (twenty to several hundred nanometers in diameter), and long (150-300 nm) flexible cylindrical micelles. The cylindrical micelles evolved from the edges of the bilayer sheets. At higher relative cholate concentration, the phase boundary was sharply defined by optical clarification of the egg PC-cholate mixtures. Cryo-TEM revealed only small spheroidal mixed micelles at this transition. These results provide the first direct evidence of the structural pathway or of molecular intermediates between a lamellar and a micellar state. Understanding these specific intermediates and the transitions between them is essential to developing reconstitution protocols and properly analyzing either activity or structural data obtained from cholate-dispersed membrane proteins.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.     

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.