ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their ...large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.
Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired ...metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.
Significance The study of membrane proteins is often hampered by their tendency to misfold when extracted by detergent. Here, we explore a detergent-free approach to isolating membrane proteins while ...retaining their native lipid environment, making use of an amphipathic polymer that solubilizes intact membrane patches in the form of nanodiscs. Using a potassium channel as a model protein, we show that these “native nanodiscs” are highly thermostable particles that are suitable for spectroscopic studies, allowing structural characterization of the protein in its native environment and direct analysis of the lipids in its immediate surroundings. We also demonstrate that the channel can be reconstituted from nanodiscs into planar lipid bilayers for functional characterization, thus making native nanodiscs an excellent alternative to detergent solubilization.
A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli , using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or “native nanodiscs.” Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid–protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine ...receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4–7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more “native” like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.
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•The first sub nm single particle reconstruction of a membrane protein using a SMALP scaffold•The ability to maintain a native like environment for structural studies holds great promise.•The resulting map is consistent with high resolution crystal structures and other EM derived maps.
Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from ...its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.
The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with ...conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.
To truly understand the mechanisms behind the supramolecular self-assembly of nanocomponents, the characterisation of their surface properties is crucial. M13 emerged as a practical nanocomponent for ...bio-nanoassemblies of functional materials and devices, and its popularity is increasing as time goes by. The investigation performed in this study provides important information about the surface charge and the surface area of M13 determined through the comparison of structural data and the measurement of
ζ
-potential at pH ranging between 2 and 11. The developed methodologies along with the experimental findings can be subsequently exploited as a novel and convenient prediction tool of the total charge of modified versions of M13. This, in turn, will facilitate the design of the self-assembly strategies which would combine the virus building block with other micro and nano components
via
intermolecular interactions.
Characterisation of the external surface of bacteriophage M13 using PVIII protein structural data and measuring
ζ
-potential of the entire virus.
ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples ...for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC-SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866-SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866-SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866-SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.
Polymer-based lipid nanoparticles like styrene-maleic acid lipid particles have revolutionized the study of membrane proteins. More recently, alternative polymers such as ...poly(diisobutylene-alt-maleic acid) (DIBMA) have been used in this field. DIBMA is commonly synthesized via conventional radical copolymerization. In order to study the influence of its chain length on lipid nanodisc formation and membrane protein extraction, we synthesized DIBMA with molar masses varying from 1.2–12 kDa via RAFT-mediated polymerization. For molar masses in the range of 3–7 kDa, the rate of lipid nanodisc formation was the highest and similar to those of poly(styrene-co-maleic acid) (SMA) and commercially available DIBMA. ZipA solubilization efficiency was significantly higher than for commercially available DIBMA and similar to SMA (circa 75%). Furthermore, RAFT-made DIBMA with a molar mass of 1.2–3.9 kDa showed a much cleaner separation on SDS–PAGE, without the smearing that is typically seen for SMA and commercially available DIBMA.
Significance
We report a rapid COVID-19 assay that gives a sample-to-signal time of under 10 min. The current gold-standard COVID-19 assay uses PCR, where strands of DNA are copied (amplified) many ...times to generate a read-out signal. However, as the virus genome is RNA, first conversion into DNA is required using reverse transcription (RT) before amplification. While just as sensitive, our assay is faster because 1) we have designed a method for generating DNA (the trigger strand) from RNA, bypassing the lengthy RT step, and 2) a quicker amplification process than PCR, called exponential amplification reaction (EXPAR), is used to amplify the trigger. This methodology could ultimately be applied to any RNA-based assay, including the detection of other infectious agents.
A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription–free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.
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