Membrane protein extraction and purification using partially-esterified SMA polymers

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

Conformational transitions of maleic acid segment drive pH induced changes in SMA polymer structure and solubility

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications – particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.

The infection of a bacterium by a phage starts with attachment to a receptor molecule on the host cell surface by the phage. Since receptor-phage interactions are crucial to successful infections, they are major determinants of phage host range and, by extension, of the broader effects that phages have on bacterial communities. Many receptor molecules, particularly membrane proteins, are difficult to isolate because their stability is supported by their native membrane environments. Styrene maleic acid lipid particles (SMALPs), a recent advance in membrane protein studies, are the result of membrane solubilizations by styrene maleic acid (SMA) copolymer chains. SMALPs thereby allow for isolation of membrane proteins while maintaining their native environment. Here, we explore SMALPs as a tool to isolate and study phage-receptor interactions. We show that SMALPs produced from taxonomically distant bacterial membranes allow for receptor-specific decrease of viable phage counts of several model phages that span the three largest phage families. After characterizing the effects of incubation time and SMALP concentration on the activity of three distinct phages, we present evidence that the interaction between two model phages and SMALPs is specific to bacterial species and the phage receptor molecule. These interactions additionally lead to DNA ejection by nearly all particles at high phage titers. We conclude that SMALPs are a potentially highly useful tool for phage-host interaction studies.IMPORTANCE Bacteriophages (viruses that infect bacteria or phages) impact every microbial community. All phage infections start with the binding of the viral particle to a specific receptor molecule on the host cell surface. Due to its importance in phage infections, this first step is of interest to many phage-related research and applications. However, many phage receptors are difficult to isolate. Styrene maleic acid lipid particles (SMALPs) are a recently developed approach to isolate membrane proteins in their native environment. In this study, we explore SMALPs as a tool to study phage-receptor interactions. We find that different phage species bind to SMALPs, while maintaining specificity to their receptor. We then characterize the time and concentration dependence of phage-SMALP interactions and furthermore show that they lead to genome ejection by the phage. The results presented here show that SMALPs are a useful tool for future studies of phage-receptor interactions.

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.

Extraction and purification of membrane proteins has for a long time represented a significant challenge. Polymer‐based extraction methods, like those using styrene maleic acid co‐polymers have provided a fertile approach to generate samples that include the local lipid environment surrounding the protein. However, the wide variety of different polymers now available provides a challenge to identify the optimal solution. In this study we develop and demonstrate a novel high‐throughput screening approach for rapid optimization of polymer solubilization agents and chromatography resins for membrane protein purification. Using this approach, we explore whether there are standard conditions that perform well for a range of membrane protein morphologies, sources and functions. These data show that no such standard conditions exist for either polymer solubilization agent or chromatography resin and that some combinations are rarely suitable for membrane protein purifications under these conditions, such as the use of TALON resin at a pH of 7.5 or SMALP300 in the Synthetic Nanodisc Screening Kit MINI kit. Instead, the use of the screening approach developed in this work is the best route to an optimal membrane protein preparation protocol.

Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

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

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo3 and can also be performed with complex membrane protein mixtures.

Effects of Styrene-Maleic Acid (SMA) Copolymer on the Photoactivation Mechanism of Rhodopsin

Amphipathic styrene-maleic acid (SMA) copolymers directly solubilize biomembranes into SMA-lipid particles, or SMALPs, that are often regarded as nanodiscs and hailed as a native membrane platform. The promising outlook of SMALPs inspires the discovery of many SMA-like copolymers that also solubilize biomembranes into putative nanodiscs, but a fundamental question remains on how much the SMALPs or SMALP analogues truly resemble the bilayer structure of nanodiscs. This unfortunate ambiguity undermines the utility of SMA or SMA-like copolymers in membrane biology because the structure and function of many membrane proteins depend critically on their surrounding matrices. Here, we report the structural heterogeneity of SMALPs revealed through fractionating SMALPs comprised of lipids and well-defined SMAs via size-exclusion chromatography followed by quantitative determination of the polymer-to-lipid (P/L) stoichiometric ratios in individual fractions. Through the lens of P/L stoichiometric ratios, different self-assembled polymer-lipid nanostructures are inferred, such as polymer-remodeled liposomes, polymer-encased nanodiscs, polymer-lipid mixed micelles, and lipid-doped polymer micellar aggregates. We attribute the structural heterogeneity of SMALPs to the microstructure variations amongst individual polymer chains that give rise to their polydisperse detergency. As an example, we demonstrate that SMAs with a similar S/MA ratio but different chain sizes participate preferentially in different polymer-lipid nanostructures. We further demonstrate that proteorhodopsin, a light-driven proton pump solubilized within the same SMALPs is distributed amongst different self-assembled nanostructures to display different photocycle kinetics. Our discovery challenges the native nanodisc notion of SMALPs or SMALP analogues and highlights the necessity to separate and identify the structurally dissimilar polymer-lipid particles in membrane biology studies.