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Sphingolipids are an important class of lipids found in mammalian cell membranes with important structural and signaling roles. They differ from another major group of lipids, the glycerophospholipids, in the connection of their hydrocarbon chains to their headgroups. In this study, a combination of electrochemical and structural methods has been used to elucidate the effect of this difference on sphingolipid behavior in an applied electric field.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes which link substrate release and opening of the cytoplasmic gate, and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

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.

The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original ‘Wuhan’ SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.

The dynamic nature of micellar nanostructures is employed to form a self-assembled Förster resonance energy transfer (FRET) nanoplatform for enhanced sensing of DNA. The platform consists of lipid oligonucleotide FRET probes incorporated into micellar scaffolds, where single recognition events result in fusion and fission of DNA mixed micelles, triggering the fluorescence response of multiple rather than a single FRET pair. In comparison to conventional FRET substrates where a single donor interacts with a single acceptor, the micellar multiplex FRET system showed ∼20- and ∼3-fold enhancements in the limit of detection and FRET efficiency, respectively. This supramolecular signal amplification approach could potentially be used to improve FRET-based diagnostic assays of nucleic acid and non-DNA based targets.

M13 bacteriophage is a well-established versatile nano-building block, which can be employed to produce novel self-assembled functional materials and devices. Sufficient production and scalability of the M13, often require a large quantity of the virus and thus, improved propagation methods characterised by high capacity and degree of purity are essential. Currently, the ‘gold-standard’ is represented by infecting Escherichia coli cultures, followed by precipitation with polyethylene glycol (PEG). However, this is considerably flawed by the accumulation of contaminant PEG inside the freshly produced stocks, potentially hampering the reactivity of the individual M13 filaments. Our study demonstrates the effectiveness of implementing an isoelectric precipitation procedure to reduce the residual PEG along with FT-IR spectroscopy as a rapid, convenient and effective analytic validation method to detect the presence of this contaminant in freshly prepared M13 stocks.

Graphical abstract, Over recent years, there has been a rapid development of membrane-mimetic systems to encapsulate and stabilize planar segments of phospholipid bilayers in solution. One such system has been the use of amphipathic copolymers to solubilize lipid bilayers into nanodiscs. The attractiveness of this system, in part, stems from the capability of these polymers to solubilize membrane proteins directly from the host cell membrane. The assumption has been that the native lipid annulus remains intact, with nanodiscs providing a snapshot of the lipid environment. Recent studies have provided evidence that phospholipids can exchange from the nanodiscs with either lipids at interfaces, or with other nanodiscs in bulk solution. Here we investigate kinetics of lipid exchange between three recently studied polymer-stabilized nanodiscs and supported lipid bilayers at the silicon-water interface. We show that lipid and polymer exchange occurs in all nanodiscs tested, although the rate and extent differs between different nanodisc types. Furthermore, we observe adsorption of nanodiscs to the supported lipid bilayer for one nanodisc system which used a polymer made using reversible addition-fragmentation chain transfer polymerization. These results have important implications in applications of polymer-stabilized nanodiscs, such as in the fabrication of solid-supported films containing membrane proteins.

Nucleic acid detection is an important part of our bio-detection arsenal, with the COVID-19 pandemic clearly demonstrating the importance to healthcare of rapid and efficient detection of specific pathogenic sequences. As part of the drive to establish new DNA detection methodologies and signal read-outs, here we show how linear dichroism (LD) spectroscopy can be used to produce a rapid and modular detection system for detecting quantities of DNA from both bacterial and viral pathogens. The LD sensing method exploits changes in fluid alignment of bionanoparticles (bacteriophage M13) engineered with DNA stands covalently attached to their surfaces, with the read-out signal induced by the formation of complementary duplexes between DNA targets and two M13 bionanoparticles. This new sandwich assay can detect pathogenic material down to picomolar levels in under 1 minute without amplification, as demonstrated by the successful sensing of DNA sequences from a plant virus (Potato virus Y) and an ampicillin resistance gene, ampR., A novel DNA sensing method based on LD spectroscopy and using bionanoparticle scaffolds is described, as demonstrated by the rapid detection of DNA strands associated with bacterial and viral pathogens.

Controllable higher-order assembly is a central aim of macromolecular chemistry. An essential challenge to developing these molecules is improving our understanding of the structures they adopt under different conditions. Here, we demonstrate how flow linear dichroism (LD) spectroscopy is used to provide insights into the solution structure of a chiral, self-assembled fibrillar foldamer. Poly(para-aryltriazole)s fold into different structures depending on the monomer geometry and variables such as solvent and ionic strength. LD spectroscopy provides a simple route to determine chromophore alignment in solution and is generally used on natural molecules or molecular assemblies such as DNA and M13 bacteriophage. In this contribution, we show that LD spectroscopy is a powerful tool in the observation of self-assembly processes of synthetic foldamers when complemented by circular dichroism, absorbance spectroscopy, and microscopy. To that end, poly(para-aryltriazole)s were aligned in a flow field under different solvent conditions. The extended aromatic structures in the foldamer give rise to a strong LD signal that changes in sign and in intensity with varying solvent conditions. A key advantage of LD is that it only detects the large assemblies, thus removing background due to monomers and small oligomers.

Controllable higher-order assembly is a central aim of macromolecular chemistry. An essential challenge to developing these molecules is improving our understanding of the structures they adopt under different conditions. Here, we demonstrate how flow linear dichroism (LD) spectroscopy is used to provide insights into the solution structure of a chiral, self-assembled fibrillar foldamer. Poly(para-aryltriazole)s fold into different structures depending on the monomer geometry and variables such as solvent and ionic strength. LD spectroscopy provides a simple route to determine chromophore alignment in solution and is generally used on natural molecules or molecular assemblies such as DNA and M13 bacteriophage. In this contribution, we show that LD spectroscopy is a powerful tool in the observation of self-assembly processes of synthetic foldamers when complemented by circular dichroism, absorbance spectroscopy, and microscopy. To that end, poly(para-aryltriazole)s were aligned in a flow field under different solvent conditions. The extended aromatic structures in the foldamer give rise to a strong LD signal that changes in sign and in intensity with varying solvent conditions. A key advantage of LD is that it only detects the large assemblies, thus removing background due to monomers and small oligomers.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins., Using the GPCR rhodopsin as an exemplar, SMA SMI and DIBMA constitute a ‘tool-kit’ of structurally-related solubilising polymers, with each providing different advantages for studying membrane proteins encapsulated in lipid particles.

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.

Membrane proteins such as receptors, ion channels and transport proteins are important drug targets. The structure-based study of membrane proteins is challenging, especially when the target protein contains both soluble and insoluble domains. Most membrane proteins are insoluble in aqueous solvent and embedded in the plasma membrane lipid bilayer, which significantly complicates biophysical studies. Poly(styrene-co-maleic acid) (SMA) and other polymer derivatives are increasingly common solubilisation agents, used to isolate membrane proteins stabilised in their native lipid environment in the total absence of detergent. Since the initial report of SMA-mediated solubilisation, and the formation of SMA lipid particles (SMALPs), this technique can directly isolate therapeutic targets from biological membranes, including G-protein coupled receptors (GPCRs). SMA now allows biophysical and structural analyses of membrane proteins in solution that was not previously possible. Here, we critically review several existing biophysical techniques compatible with SMALPs, with a focus on hydrodynamic analysis, microcalorimetric analysis and optical spectroscopic techniques.

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation., Graphical abstract Unlabelled Image, Highlights • Partially-esterified SMA polymers are effective for solubilisation of membrane proteins from different expression systems. • Proteins in partially-esterified SMALPs can be purified by Ni2+ affinity chromatography, but yield is lower with SMA 1440. • Partial esterification of SMA does not overcome the sensitivity to divalent cations. • SMA1440 is more sensitive to magnesium ions than SMA 2000.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co2+ and Zn2+ resulted in an increased sensitivity.

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.\ud \ud

We report a rapid isothermal method for detecting SARS-CoV-2, the virus responsible for COVID-19. The procedure uses a novel reverse transcriptase-free (RTF) approach for converting RNA into DNA, which triggers a rapid amplification 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 of a sample of patient SARS-CoV-2 RNA in under 5 minutes.

Simple Summary The atomic structure of a biological macromolecule determines its function. Discovering how one or more amino acid chains fold and interact to form a protein complex is critical, from understanding the most fundamental cellular processes to developing new therapies. However, this is far from a straightforward task, especially when studying a membrane protein. The functional link between the oligomeric state and complex composition of the proteins involved in the active mitochondrial transport of cytosolic pyruvate is a decades-old question but remains urgent. We present a brief historical review beginning with the identification of the so-called mitochondrial pyruvate carrier (MPC) proteins, followed by a rigorous conceptual analysis of technical approaches in more recent biochemical studies that seek to isolate and reconstitute the functional MPC complex(es) in vitro. We correlate these studies with early kinetic observations and current experimental and computational knowledge to assess their main contributions, identify gaps, resolve ambiguities, and better define the research goal. Abstract The molecular identity of the mitochondrial pyruvate carrier (MPC) was presented in 2012, forty years after the active transport of cytosolic pyruvate into the mitochondrial matrix was first demonstrated. An impressive amount of in vivo and in vitro studies has since revealed an unexpected interplay between one, two, or even three protein subunits defining different functional MPC assemblies in a metabolic-specific context. These have clear implications in cell homeostasis and disease, and on the development of future therapies. Despite intensive efforts by different research groups using state-of-the-art computational tools and experimental techniques, MPCs’ structure-based mechanism remains elusive. Here, we review the current state of knowledge concerning MPCs’ molecular structures by examining both earlier and recent studies and presenting novel data to identify the regulatory, structural, and core transport activities to each of the known MPC subunits. We also discuss the potential application of cryogenic electron microscopy (cryo-EM) studies of MPC reconstituted into nanodiscs of synthetic copolymers for solving human MPC2.

G-protein-coupled receptor (GPCR) drug research is presently hindered by the technical challenges associated with generating purified receptors. Consequently, the application of critical modern discovery technologies has been limited, and the vast untapped opportunity for new GPCR-directed medicines is not being realised. A simple but transformative solution is to purify receptors without removing them from their native phospholipid environment by using polymer lipid particle (PoLiPa) technology, with reagents such as styrene-maleic acid co-polymer (SMA). Compared with contemporary detergent-based and stabilising mutagenesis methods, the PoLiPa approach is simple and generic and, therefore, offers huge advantages, with the potential to revolutionise GPCR research by facilitating the availability of the purified receptors that are required for structural biology, biophysical, and panning technologies.

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.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Polymer stabilized nanodiscs are self-assembled structures composed of a polymer belt that wraps around a segment of lipid bilayer, and as such are capable of encapsulating membrane proteins directly from the cell membrane. To date, most studies on these nanodiscs have used poly(styrene- co-maleic acid) (SMA) with the term SMA-lipid particles (SMALPs) coined to describe them. In this study, we have determined the physical and thermodynamic properties of such nanodiscs made with two different SMA copolymers. These include a widely used and commercially available statistical poly(styrene- co-maleic acid) copolymer (coSMA) and a reversible addition-fragmentation chain transfer synthesized copolymer with narrow molecular weight distribution and alternating styrene and maleic acid groups with a polystyrene tail, (altSMA). We define phase diagrams for each polymer, and show that, regardless of polymer topological structure, self-assembly is driven by the free energy change associated with the polymers. We also show that nanodisc size is polymer dependent, but can be modified by varying polymer concentration. The thermal stability of each nanodisc type is similar, and both can effectively solubilize proteins from the E. coli membrane. These data show the potential for the development of different SMA polymers with controllable properties to produce nanodiscs that can be optimized for specific applications and will enable more optimized and widespread use of the SMA-based nanodiscs in membrane protein research.

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.

Graphene, since its successful exfoliation and characterisation has been continuously drawing extensive research interests due to its potential for a broad range of applications ranging from energy, microelectronics, through polymer fillers and sensors to environmental and biomedical devices. Exploitation of its unique chemical and physical properties for the manufacturing of functional materials, requires careful structural control and scaling-up into three-dimensional morphologies. Here, a facile method is established to create and control the bottom-up self-assembly of graphene oxide nano-sheets via unprecedented integration with a highly versatile bio-ingredient, the filamentous bacteriophage M13, into hierarchical, three-dimensional, porous sponges of GraPhage13. This study explores the interplay of the GraPhage13 structure formation and studies the mechanisms that give rise to the controllable self-assembly. The straightforward fabrication of robust hierarchical micro-nano-architectures further lays a platform for applications in energy storage and conversion, catalysis and sensing.

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.

The reversible photocontrol of an enzyme governing blood coagulation is demonstrated. The thrombin binding aptamer (TBA), was rendered photochromic by modification with two anthracene groups. Light-triggered anthracene photodimerisation distorts its structure, inhibiting binding of the enzyme thrombin, which in turn triggers catalysis and the resulting clotting process.

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form "SMA Lipid Particles" (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. "SMILPs" thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

Membrane proteins remain a somewhat enigmatic group of biomolecules. On the one hand they mediate some of the most important processes in biology with molecular mechanisms that are often elegantly complex. On the other hand they are exceptionally challenging to produce, making studies of membrane protein structure and function among the most difficult projects undertaken by biochemists. The central issue with studies of a membrane protein has been the need to extract them from their native lipid environment before purification and production of a homogenous sample. Historical approaches have utilized detergent solubilisation but these often lead to a sample with low activity and stability. In the past 15 years a new approach that focuses on preserving the local lipid environment surrounding the membrane proteins has been developed. The latest, and perhaps most complete, incarnation of this method has been the use of polymers based on styrene maleic acid (SMA) to stabilise nanoscale discs of lipid that contain membrane proteins. In this review we examine the range of SMA-related polymers that have now been shown to have utility in the production of membrane proteins. We discuss the differences between the polymers and attempt to discover rules and trends that explain their behavior.

The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor α-1-antitrypsin result from point mutations in its gene. In addition, α-2-macroglobulin competes with α-1-antitrypsin for proteinases, and the α-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of α-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either α-1-antitrypsin or α-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 μm into the tissue before becoming sequestered. We have shown that proteinases sequestered to α-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with α-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all α-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to α-2-macroglobulin where it retains proteolytic activity.

Fluorescence detection typically enhances sensitivity and selectivity for fluorescent analytes. The potential for combining fluorescence detection with flow orientation of the sample in the normal configuration of linear dichroism experiments is explored in this work by measuring the fluorescence emitted from flow-orientated DNA-bound ligands and M13 bacteriophage. Data for ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenyindole are presented. The theoretical basis of the technique is also presented for instruments running in both the fixed direct-current mode, which is the normal operation mode of circular dichroism spectropolarimeters, and also in fixed high-tension voltage mode. The role of the stray light reaching the detector that results in a spectral shape in fixed direct current mode that resembles the shape of a linear dichroism spectrum, rather than the expected reduced linear dichroism, is also explored.

Methylocella silvestris, an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N-oxide (TMAO) as a sole nitrogen source, however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker-exchange mutagenesisenabled the identification of several genes involved in TMAO metabolism, including Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of anN-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO [RIGHTWARDS ARROW] 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestrisand eukaryotic TMAO demethylases have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km= 3.3 mM; Vmax=21.7 nmolmin-1mg-1) and only catalyses demethylation of TMAO and a structural homologue, dimethyldodecylamine N-oxide. Our study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M. silvestris.

Xanthene dyes are commonly used to label proteins in order to probe their location and activity using fluorescence spectroscopy and microscopy. However, fundamental properties such as the polarizations of transitions for many of the dyes have not been available. In this paper we report the use of recently developed oxidised polyethylene (PEOX) stretched film linear dichroism to determine the transition polarizations of xanthene, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyronin Y, pyronin B, fluorescein, and rhodamine 6G. The effect of the formation of higher order structures is also discussed when they occur. The dyes (except xanthene) all have an intense long-axis polarized transition in the region of 500 nm. They also have long-axis (//) polarized transitions from about 280 nm downwards in wavelength. There are suggestions of a weak short axis (⊥) polarized transition in the region of 320–350 nm in each case.

Hyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time–temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a Tm of approximately 106°C and could be described by a one-step irreversible model. The activation energy at 121°C was found to be 316kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121°C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs.

Metabolite channeling by a dynamic metabolon The specialized metabolite dhurrin breaks down into cyanide when plant cell walls have been chewed, deterring insect pests. Laursen et al. found that the enzymes that synthesize dhurrin in sorghum assemble as a metabolon in lipid membranes (see the Perspective by Dsatmaichi and Facchini). The dynamic nature of metabolon assembly and disassembly provides dhurrin on an as-needed basis. Membrane-anchored cytochrome P450s cooperated with a soluble glucosyltransferase to channel intermediates toward efficient dhurrin production. Science , this issue p. 890 ; see also p. 829

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

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.

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.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a ‘molecular pastry cutter’ to ‘solubilize’ GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR–SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Inhibition of the Fts family of proteins causes the growth of long filamentous cells, indicating that they play some role in cell division. FtsZ polymerizes into protofilaments and assembles into the Z-ring at the future site of the septum of cell division. We analyze the rigidity of GTP-bound FtsZ protofilaments by using cryoelectron microscopy to sample their bending fluctuations. We find that the FtsZ-GTP filament rigidity is κ = 4.7±1.0×10 − 27κ=4.7±1.0×10−27 Nm2, with a corresponding thermal persistence length of lp = 1.15±0.25lp=1.15±0.25μm, much higher than previous estimates. In conjunction with other model studies, our new higher estimate for FtsZ rigidity suggests that contraction of the Z-ring may generate sufficient force to facilitate cell division. The good agreement between the measured mode amplitudes and that predicted by equipartition of energy supports our use of a simple mechanical model for FtsZ fibers. The study also provides evidence that the fibers have no intrinsic global or local curvatures, such as might be caused by partial hydrolysis of the GTP.

Biomolecular detection has for a long time depended on a relatively small number of established methodologies. Many of these depend on the detection of a ligand-antibody complex using some kind of optical technique, e.g., chemiluminescence. Before this measurement can be made, the ligand-antibody complex generally has to be separated from bulk contaminants. This process involves the implementation of a heterogeneous assay format involving immobilization of the antibody onto a solid support to enable washing of the unbound ligand. This approach has a number of inherent issues including being slow and complex and requiring the use of expensive reagents. In this paper, we demonstrate how we have harnessed a biologically inspired nanoparticle to provide the basis for a homogeneous assay which requires no immobilization. The method relies on using fluid shear flow to align a fiber-like nanoparticle formed from a filamentous virus, M13, combined with a ligand-specific antibody. This renders the particle visible to linear dichroic spectroscopy, which provides an easily interpretable signal. The addition of the target ligand (in this case Escherichia coli O157) leads to the formation of a nanoparticle-ligand particle that is unable to align, leading to the perturbation of the linear dichroism signal.