Insights into membrane interactions and their therapeutic potential.

Recent research into membrane interactions has uncovered a diverse range of therapeutic opportunities through the bioengineering of human and non-human macromolecules. Although the majority of this research is focussed on fundamental developments, emerging studies are showcasing promising new technologies to combat conditions such as cancer, Alzheimer's and inflammatory and immune-based disease, utilising the alteration of bacteriophage, adenovirus, bacterial toxins, type 6 secretion systems, annexins, mitochondrial antiviral signalling proteins and bacterial nano-syringes. To advance the field further, each of these opportunities need to be better understood, and the therapeutic models need to be further optimised. Here, we summarise the knowledge and insights into several membrane interactions and detail their current and potential uses therapeutically.

The function of BK channels extracted and purified within SMALPs.

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of ß and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and ß1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of ß1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified ß1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

Biological insights from SMA-extracted proteins.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein-lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein-protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Biophysical analysis of lipidic nanoparticles.

Biological nanoparticles include liposomes, extracellular vesicle and lipid-based discoidal systems. When studying such particles, there are several key parameters of interest, including particle size and concentration. Measuring these characteristics can be of particular importance in the research laboratory or when producing such particles as biotherapeutics. This article briefly describes the major types of lipid-containing nanoparticles and the techniques that can be used to study them. Such methodologies include electron microscopy, atomic force microscopy, dynamic light scattering, nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing and microfluidic resistive pulse sensing. Whilst no technique is perfect for the analysis of all nanoparticles, this article provides advantages and disadvantages of each, highlighting the latest developments in the field. Finally, we demonstrate the use of microfluidic resistive pulse sensing for the analysis of biological nanoparticles.

Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts.

The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.

Expression and purification of recombinant G protein-coupled receptors: A review.

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines.

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment.

The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein.

Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation. Understanding how solubilization and stabilization are intimately linked is key to facilitate MP isolation toward fundamental structural and functional research as well as drug discovery applications. How to manage the tour de force of destabilizing the lipid bilayer and stabilizing MP at the same time is the holy grail of successful isolation and investigation of such a delicate and fascinating class of proteins.

An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1).

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Encapsulated membrane proteins: A simplified system for molecular simulation.

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.

The synthesis of recombinant membrane proteins in yeast for structural studies.

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

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.

Overcoming bottlenecks in the membrane protein structural biology pipeline.

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

Detergent-free purification of ABC (ATP-binding-cassette) transporters.

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.

Classifying tetraspanins: A universal system for numbering residues and a proposal for naming structural motifs and subfamilies.

All tetraspanins have four transmembrane domains (TMs). The large extracellular loop (LEL) that connects the third and fourth TMs contains multiple secondary structures together with the family's signature Cys-Cys-Gly motif. These intriguing membrane proteins are involved in diverse and incompletely understood cellular processes including cell adhesion, tissue differentiation, immune cell maturation and host-parasite interactions. Here we present a classification system that accurately describes the position of each amino acid within its primary sequence based on both sequence and topological conservation of the TMs and LEL. This builds on the numbering systems that have been used in the G protein-coupled receptor (GPCR) field for nearly three decades and which have aided the understanding of GPCR structure/activity relationships and ligand interactions. The high-resolution structures of the tetraspanins CD81, CD9, CD53 and Tspan15 were used to validate the structural relevance of our new tetraspanin classification system. Modelling of all tetraspanin LELs highlighted flexibility in LEL disulfide bonding across the family and suggests that the structural arrangement of tetraspanin LELs is more complex than previously thought. We therefore propose a new subfamily naming system that addresses this added complexity and facilitates the systematic classification of human tetraspanins, shedding light on all structural motifs within the family. We anticipate that our universal tetraspanin classification system will enable progress in defining how sequence and structure inform function.

Membrane Protein Production in Insect Cells.

Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.

Heterologous Expression of Membrane Proteins in E. coli.

Over the decades, the bacterium Escherichia coli (E. coli) has become the cornerstone of recombinant protein production, used for heterologous synthesis of a variety of membrane proteins. Due to its rapid growth to high densities in cheap media, and its ease of manipulation and handling, E. coli is an excellent host cell for a range of membrane protein targets. Furthermore, its genetic tractability allows for a variety of gene constructs to be screened for optimal expression conditions, resulting in relatively high yields of membrane protein in a short amount of time. Here, we describe the general workflow for the production of membrane proteins in E. coli. The protocols we provide show how the gene of interest is modified, transferred to an expression vector and host, and how membrane protein yields can be optimized and analyzed. The examples we illustrate are well suited for scientists who are starting their journey into the world of membrane protein production.

Membrane Protein Production in the Yeast P. pastoris.

The first crystal structures of recombinant mammalian membrane proteins were solved using high-quality protein that had been produced in yeast cells. One of these, the rat Kv1.2 voltage-gated potassium channel, was synthesized in Pichia pastoris. Since then, this yeast species has remained a consistently popular choice of host for synthesizing eukaryotic membrane proteins because it is quick, easy, and cheap to culture and is capable of posttranslational modification. Very recent structures of recombinant membrane proteins produced in P. pastoris include a series of X-ray crystallography structures of the human vitamin K epoxide reductase and a cryo-electron microscopy structure of the TMEM206 proton-activated chloride channel from pufferfish. P. pastoris has also been used to structurally and functionally characterize a range of membrane proteins including tetraspanins, aquaporins, and G protein-coupled receptors. This chapter provides an overview of the methodological approaches underpinning these successes.