Membrane Scaffold Protein Research Articles

Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy

Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

Collisional lipid exchange among DIBMA-encapsulated nanodiscs (DIBMALPs)

Copolymers of diisobutylene/maleic acid (DIBMA), styrene/maleic acid (SMA), and styrene/maleimide (SMI) solubilise membrane proteins and surrounding lipids directly from artificial and biological membranes to form polymer-bounded nanodiscs. Although they preserve a lipid-bilayer core, these nanodiscs are much more dynamic than other membrane mimics, as we have recently demonstrated for two SMA variants. Here, we used time-resolved Förster resonance energy transfer spectroscopy to quantify the kinetics and unravel the mechanisms of lipid transfer among DIBMA-encapsulated nanodiscs (DIBMALPs), with particular emphasis on the role of Coulombic repulsion, which is modulated by ionic strength and nanodisc size. Our results show that DIBMALPs exchange lipids through both binary and ternary collisions on the timescale of seconds to minutes under typical experimental conditions. By contrast, nanodiscs bounded by SMA polymers exchange lipids considerably faster through binary collisions, whereas vesicles and nanodiscs based on membrane scaffold proteins exchange lipids much more slowly through lipid monomer diffusion. As DIBMALPs are polyanionic, collisional lipid transfer is substantially accelerated by screening of Coulombic repulsion caused by increasing ionic strength or nanodisc size. Analysis of the ionic-strength dependence in terms of an extended version of the Debye–Hückel equation suggests an effective DIBMALP charge number of z = −47, which is considerably larger than that previously determined for nanodiscs bounded by more hydrophobic SMA polymers.

Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc

Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.

A Split-Intein-Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins.

Phospholipid nanodiscs are a native-like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long-term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time-consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split-intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long-term stability and homogeneity, the presented small circular nanodiscs are suited for high-resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.

Supramolecular Organization of Apolipoprotein-A-I-Derived Peptides within Disc-like Arrangements

Apolipoprotein A-I is the major protein component of high-density lipoproteins and fulfils important functions in lipid metabolism. Its structure consists of a chain of tandem domains of amphipathic helices. Using this protein as a template membrane scaffolding protein, class A amphipathic helical peptides were designed to support the amphipathic helix theory and later as therapeutic tools in biomedicine. Here, we investigated the lipid interactions of two apolipoprotein-A-I-derived class A amphipathic peptides, 14A (Ac-DYLKA FYDKL KEAF-NH2) and 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2), including the disc-like supramolecular structures they form with phospholipids. Thus, the topologies of 14A and 18A in phospholipid bilayers have been determined by oriented solid-state NMR spectroscopy. Whereas at a peptide-to-lipid ratio of 2 mol% the peptides align parallel to the bilayer surface, at 7.5 mol% disc-like structures are formed that spontaneously orient in the magnetic field of the NMR spectrometer. From a comprehensive data set of four 15N- or 2H-labeled positions of 14A, a tilt angle, which deviates from perfectly in-planar by 14°, and a model for the peptidic rim structure have been obtained. The tilt and helical pitch angles are well suited to cover the hydrophobic chain region of the bilayer when two peptide helices form a head-to-tail dimer. Thus, the detailed topology found in this work agrees with the peptides forming the rim of nanodiscs in a double belt arrangement.

Reconstitution of P‐glycoprotein in Nanodiscs using the MSP1D1 Scaffold Protein for Biochemical Inhibitor Screens

Many cancers become multidrug resistant due to overexpression of the transmembrane efflux pump P‐glycoprotein (P‐gp). Compounds inhibiting P‐gp function may therefore have the potential to improve the effectiveness of chemotherapeutic drugs. Using in silico methods, we have been able to successfully identify a number of putative P‐gp inhibitor compounds. In cell culture assays, many of these compounds were shown to re‐sensitize cancers to chemotherapeutics. However, when tested with purified P‐gp, some of these compounds showed little effect on the catalytic activity of the isolated protein. The relatively high concentrations of detergents needed to solubilize P‐gp likely reduced the potency of the inhibitor compounds. To overcome this issue, purified P‐gp was inserted into nanostructures (nanodiscs) which much more closely resemble natural cell membrane. We and others have observed that this stabilizes P‐gp and stimulates its catalytic activity. However, previous assays of inhibitor compounds with medium size MSP1E3D1 nanodiscs still did not show clear effects on catalytic activity of the protein despite the fact that the inhibitors were effective in cell culture. We hypothesize that the previously used MSP1E3D1 nanodiscs still may have been too large, leaving hydrophobic space into which the inhibitors could partition, again reducing the effective concentration of the inhibitors. To improve on this protocol, we here show our efforts to use a shorter membrane scaffold protein, known as MSP1D1, for the assembly of smaller nanodiscs. After optimization of conditions for assembly and insertion, and tests for biological activity, we will assay our potential inhibitor hit compounds for their ability to affect catalytic ATP hydrolysis activities of P‐gp.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars Program, SMU Undergraduate Research Assistantships, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization of the Production of Covalently Circularized Nanodiscs and Their Characterization in Physiological Conditions.

Lipid nanodiscs are widely used platforms for studying membrane proteins in a near-native environment. Lipid nanodiscs made with membrane scaffold proteins (MSPs) in the linear form have been well studied. Recently, a new kind of nanodisc made with MSPs in the circular form, referred to as covalently circularized nanodiscs (cNDs), has been reported to have some possible advantages in various applications. Given the potential of nanodisc technology, researchers in the field are very interested in learning more about this new kind of nanodisc, such as its reproducibility, production yield, and the possible pros and cons of using it. However, research on these issues is lacking. Here, we report a new study on nanodiscs made with circular MSPs, which are produced from a method different from the previously reported method. We show that our novel production method, detergent-assisted sortase-mediated ligation, can effectively avoid high-molecular-weight byproducts and also significantly improve the yield of the target proteins up to around 80% for larger circular MSP constructs. In terms of the application of circular MSPs, we demonstrate that they can be used to assemble nanodiscs using both synthetic lipids and native lipid extract as the source of lipids. We also show that bacteriorhodopsin can be successfully incorporated into this new kind of cND. Moreover, we found that cNDs have improved stability against both heat and high-concentration-induced aggregations, making them more beneficial for related applications.

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

Reconstitution of Voltage-Activated Potassium Channel into Phospholipid Bilayer

Voltage-activated ion channels are expressed in the membranes of excitable cells, and open and close in response to changes in membrane voltage. Physiologically, they play diverse roles, from the generation of action potentials to triggering the release of neurotransmitters at synapses. To study the biochemical properties, these ion channels are usually purified and studied under detergent-solubilized condition, but many ion channels behave differently in detergent when compared to their native membrane environment. One recently introduced technique to overcome this issue is using lipid bilayer nanodiscs. Nanodiscs are a few nanometer-sized hockey-puck-shaped lipid bilayers solubilized by engineered apolipoprotein, membrane scaffold protein (MSP). MSP is comprised of short repeats of amphipathic alpha helies that surround the hydrophobic acyl chains of phospholipids. To investigate the structure and biochemical properties of voltage-activated potassium (Kv) channels in a membrane environment, we reconstituted a Kv channel into phospholipid bilayer nanodiscs using phospholipid and MSP. We first attempted to assemble empty nandiscs (nanodisc without Kv) to determine the optimal ratio between lipid and MSP that provides the best yields. Using these conditions, we reconstituted Kv channels into nanodiscs, and show that they elute as a single peak in gel-filtration chromatography. SDS-PAGE result indicates that the preparation contains each subunit corresponding to the Kv and MSP, confirming successful reconstitution of the Kv into nanodiscs. We also show that a voltage-sensor toxin can bind to the Kv channel in nanodiscs, confirming that the architecture of the Kv channel is well maintained in nanodiscs.

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

Emerging evidence suggests that Sigma1 (SIGMAR1, also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy.Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR.

Highlights of This Issue

Replication stress is emerging as an important mechanism for inhibitors of the DNA damage response. In this study, Parsels and colleagues defined the contribution of replication stress resulting from nucleotide depletion and PARP1 trapping to radiosensitization by the combination of WEE1 and PARP inhibitors, respectively, in KRAS-mutant NSCLC. They found that the enhanced radiosensitization from combined WEE1 and PARP inhibitors required PARP1 trapping, a dominant mechanism relative to nucleotide depletion. This study highlights the importance of replication stress to the therapeutic activity of WEE1 and PARP inhibitors and that PARP trapping potency is an important consideration in the choice of PARP inhibitor for combination therapy.YAP1, a target of the Hippo pathway, is regarded as an oncogene in most human cancers. However, YAP1 co-operates with p73 in hematopoietic cells and plays a tumor suppressor role. Maruyama and colleagues conducted a novel transcription-based functional screening and identified candidate YAP1 activators (n = 47) from a small-molecule chemical compound library (n = 18,606). These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator induced apoptosis in human multiple myeloma and chronic myeloid leukemia cells. Hence, YAP1 activation may be useful at suppressing hematological cancers.Immune checkpoint inhibitor antibodies that block PD-L1/PD-1 interactions are promising therapeutic agents; however, only a small percentage of cancer patients benefit from these agents. Small-molecule modulators of immune checkpoints and tumor microenvironment (TME) offer several advantages that complement current therapies, and may expand the population that responds to PD-L1/PD-1 targeted therapies. Herein, a novel mechanism is discovered by which small-molecule modulators of Sigma1, a unique ligand-operated integral membrane scaffolding protein, can be used to trigger PD-L1 degradation in cancer cells by selective autophagy. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies regulating tumor microenvironment.Precision oncology is currently limited in scope when using genomic biomarkers to guide prescription of molecularly targeted medications. In this study, Ding and colleagues applied state-of-the-art machine learning algorithms to create drug sensitivity prediction models using data from large pharmacogenomics studies, e.g., the Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE). The resulting models can accurately predict the efficacy of a wide array of molecularly targeted and non-specific chemotherapy drugs. Translated to the clinic, similar models have the potential to improve treatment outcomes for a significant population of patients currently receiving ineffective therapy.

The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

Background/Aims: The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

Time-course and degradation rate of membrane scaffold protein (MSP1D1) during recombinant production

Membrane scaffold proteins (MSPs) are synthetic derivatives of apolipoprotein A-I, a major protein component of human high-density lipoprotein complexes. The most common among these is the variant MSP1D1, which has been in the focus of research on membrane mimetics in the past. As such, the amphipathic MSP1D1 has the ability to self-assemble in the presence of synthetic phospholipids into discoidal nanoparticles, so called nanodiscs. The recombinant production of MSP is exclusively reported using a standard laboratory expression system of the pET family. However, strong variations in both yield and achieved concentration as well as complications related to unspecific degradation are commonly reported. In addition, the time-course of recombinant protein as well as specific protein yields have not yet been quantified conclusively. In this study, the time-course of MSP1D1 concentration was investigated in a standard pET expression system in terms of quantification of production and degradation rates in comparison to a reference protein (eGFP).

Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.

Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.

Endotoxin-free purification of recombinant membrane scaffold protein expressed in Escherichia coli

Abstract Membrane scaffold protein (MSP) is a versatile protein that can be used to study diverse membrane proteins. MSP is strongly expressed in E. coli; however, applications of MSP in in vivo studies remain limited because of contamination with large amounts of endotoxins. Endotoxins cannot be easily removed from MSP following standard purification protocols for His6-tagged proteins, washing with detergents, or Q-Sepharose anion exchange chromatography, regardless of whether the expression host is E. coli BL21(DE3) or ClearColi BL21(DE3). Furthermore, the concentrations of MSP-bound endotoxins were not reduced during nanodisc formation, such that the assembled nanodiscs still contained significant amounts of endotoxins. We hypothesized that the structural properties of MSP that are responsible for membrane scaffolding mediated the strong binding between MSP and the endotoxins. We showed that partial denaturation of MSP with 2 M urea effectively disrupted MSP-endotoxin interactions. MSP-bound endotoxins were successfully removed via Q-Sepharose chromatography following urea treatment. The combined treatment with urea and Q-Sepharose resulted in ∼80-fold reduction in the specific endotoxin level relative to that of conventional Ni-NTA chromatography combined with detergent treatment. The low endotoxin level of 2.0 EU/mg MSP obtained in this study makes it suitable for applications in animal studies.

Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation.

Tyrosine phosphorylation of membrane receptors and scaffold proteins followed by recruitment of SH2 domain-containing adaptor proteins constitutes a central mechanism of intracellular signal transduction. During early T-cell receptor (TCR) activation, phosphorylation of linker for activation of T cells (LAT) leading to recruitment of adaptor proteins, including Grb2, is one prototypical example. LAT contains multiple modifiable sites, and this multivalency may provide additional layers of regulation, although this is not well understood. Here, we quantitatively analyze the effects of multivalent phosphorylation of LAT by reconstituting the initial reactions of the TCR signaling pathway on supported membranes. Results from a series of LAT constructs with combinatorial mutations of tyrosine residues reveal a previously unidentified allosteric mechanism in which the binding affinity of LAT:Grb2 depends on the phosphorylation at remote tyrosine sites. Additionally, we find that LAT:Grb2 binding affinity is altered by membrane localization. This allostery mainly regulates the kinetic on-rate, not off-rate, of LAT:Grb2 interactions. LAT is an intrinsically disordered protein, and these data suggest that phosphorylation changes the overall ensemble of configurations to modulate the accessibility of other phosphorylated sites to Grb2. Using Grb2 as a phosphorylation reporter, we further monitored LAT phosphorylation by TCR ζ chain-recruited ZAP-70, which suggests a weakly processive catalysis on membranes. Taken together, these results suggest that signal transmission through LAT is strongly gated and requires multiple phosphorylation events before efficient signal transmission is achieved.

Polymer Nanodiscs: Discoidal Amphiphilic Block Copolymer Membranes as a New Platform for Membrane Proteins

Lipid nanodiscs are playing increasingly important roles in studies of the structure and function of membrane proteins. Development of lipid nanodiscs as a membrane-protein-supporting platform, or a drug targeting and delivery vehicle in general, is undermined by the fluidic and labile nature of lipid bilayers. Here, we report the discovery of polymer nanodiscs, i.e., discoidal amphiphilic block copolymer membrane patches encased within membrane scaffold proteins, as a novel two-dimensional nanomembrane that maintains the advantages of lipid nanodiscs while addressing their weaknesses. Using MsbA, a bacterial ATP-binding cassette transporter as a membrane protein prototype, we show that the protein can be reconstituted into the polymer nanodiscs in an active state. As with lipid nanodiscs, reconstitution of detergent-solubilized MsbA into the polymer nanodiscs significantly enhances its activity. In contrast to lipid nanodiscs that undergo time- and temperature-dependent structural changes, the polymer nanodiscs experience negligible structural evolution under similar environmental stresses, revealing a critically important property for the development of nanodisc-based characterization methodologies or biotechnologies. We expect that the higher mechanical and chemical stability of block copolymer membranes and their chemical versatility for adaptation will open new opportunities for applications built upon diverse membrane protein functions, or involved with drug targeting and delivery.

Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry.

Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.

Self-assembly of nanoscale particles with biosurfactants and membrane scaffold proteins

Nanodiscs are membrane mimetics which may be used as tools for biochemical and biophysical studies of a variety of membrane proteins. These nanoscale structures are composed of a phospholipid bilayer held together by an amphipathic membrane scaffold protein (MSP). In the past, nanodiscs were successfully assembled with membrane scaffold protein 1D1 and 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine with a homogeneous diameter of ∼10nm. In this study, the formation of nanoscale particles from MSP1D1 and rhamnolipid biosurfactants is investigated. Different protein to lipid ratios of 1:80, 1:90 and 1:100 were used for the assembly reaction, which were consecutively separated, purified and analyzed by size-exclusion chromatography (SEC) and dynamic light scattering (DLS). Size distributions were measured to determine homogeneity and confirm size dimensions. In this study, first evidence is presented on the formation of nanoscale particles with rhamnolipid biosurfactants and membrane scaffold proteins.

SecY-SecA fusion protein retains the ability to mediate protein transport.

In bacteria, the membrane protein complex SecY/E/G and SecA ATPase are essential for protein translocation. About 30% of newly synthesized proteins in the cytosol are targeted to and translocated across the cytoplasmic membrane by the Sec factors. Although a number of single-molecule analyses and structural studies, including the crystal structure of SecYEG complexed with SecA, have been published, the underlying molecular mechanisms and the functional oligomer states remain elusive. In this study, we constructed a fusion protein SecY-SecA, which induces the formation of the SecY-A/SecE/SecG complex (SecYAEG), to enable investigation of the molecular mechanisms by advanced single-molecule analyses. SecYAEG-reconstituted liposomes were found to possess protein translocation activity in vitro and form stable intermediates capable of the translocation using a mutant substrate protein. We additionally found that one unit of SecYAEG complex embedded into a nanodisc, using membrane scaffold proteins, interacts strongly with the substrate. The isolated SecYAEG-reconstituted nanodisc is a promising tool for investigation of the molecular mechanisms by which a single unit of Sec machinery mediates protein translocation.