Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

BPROMPT:a consensus server for membrane protein prediction

Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT.

A statistical physics perspective on alignment-independent protein sequence comparison.

Motivation: Within bioinformatics, the textual alignment of amino acid sequences has long dominated the determination of similarity between proteins, with all that implies for shared structure, function, and evolutionary descent. Despite the relative success of modern-day sequence alignment algorithms, so-called alignment-free approaches offer a complementary means of determining and expressing similarity, with potential benefits in certain key applications, such as regression analysis of protein structure-function studies, where alignment-base similarity has performed poorly. Results: Here, we offer a fresh, statistical physics-based perspective focusing on the question of alignment-free comparison, in the process adapting results from “first passage probability distribution” to summarize statistics of ensemble averaged amino acid propensity values. In this paper, we introduce and elaborate this approach.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Protein modification and phospholipid oxidation

Phospholipid oxidation can generate reactive and electrophilic products that are capable of modifying proteins, especially at cysteine, lysine and histidine residues. Such lipoxidation reactions are known to alter protein structure and function, both with gain of function and loss of activity effects. As well as potential importance in the redox regulation of cell behaviour, lipoxidation products in plasma could also be useful biomarkers for stress conditions. Although studies with antibodies suggested the occurrence of lipoxidation adducts on ApoB-100, these products had not previously been characterized at a molecular level. We have developed new mass spectrometry-based approaches to detect and locate adducts of oxidized phospholipids in plasma proteins, as well as direct oxidation modifications of proteins, which avoid some of the problems typically encountered with database search engines leading to erroneous identifications of oxidative PTMs. This approach uses accurate mass extracted ion chromatograms (XICs) of fragment ions from peptides containing oxPTMs, and allows multiple modifications to be examined regardless of the protein that contains them. For example, a reporter ion at 184.074 Da/e corresponding to phosphocholine indicated the presence of oxidized phosphatidylcholine adducts, while 2 reporter ions at 100.078 and 82.025 Da/e were selective for allysine. ApoB-100-oxidized phospholipid adducts were detected even in healthy human samples, as well as LDL from patients with inflammatory disease. Lipidomic studies showed that more than 350 different species of lipid were present in LDL, and were altered in disease conditions. LDL clearly represents a very complex carrier system and one that offers a rich source of information about systemic conditions, with potential as indicators of oxidative damage in ageing or inflammatory diseases.

IUPHAR-DB:the IUPHAR database of G protein-coupled receptors and ion channels

The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. © 2008 The Author(s).

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.

The use of site-directed mutagenesis to study GPCRs

G protein coupled receptors (GPCRs) are highly flexible and dynamic proteins, which are able to interact with diverse ligands, effectors, and regulatory proteins. Site-directed mutagenesis (SDM) is a powerful tool for providing insight into how these proteins actually work, both in its own right and when used in conjunction with information provided by other techniques such as crystallography or molecular modelling. Mutagenesis has been used to identify and characterise a myriad of functionally important residues, motifs and domains within the GPCR architecture, and to identify aspects of similarity and differences between the major families of GPCRs. This chapter presents the necessary information for undertaking informative SDM of these proteins. Whilst this is relevant to protein structure/function studies in -general, specific pitfalls and protocols suited to investigating GPCRs in particular will be highlighted.

A predictor of membrane class:discriminating α-helical and β-barrel membrane proteins from non-membranous proteins

Accurate protein structure prediction remains an active objective of research in bioinformatics. Membrane proteins comprise approximately 20% of most genomes. They are, however, poorly tractable targets of experimental structure determination. Their analysis using bioinformatics thus makes an important contribution to their on-going study. Using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we have addressed the alignment-free discrimination of membrane from non-membrane proteins. The method successfully identifies prokaryotic and eukaryotic α-helical membrane proteins at 94.4% accuracy, β-barrel proteins at 72.4% accuracy, and distinguishes assorted non-membranous proteins with 85.9% accuracy. The method here is an important potential advance in the computational analysis of membrane protein structure. It represents a useful tool for the characterisation of membrane proteins with a wide variety of potential applications.

Identifiying human MHC supertypes using bioinformatic methods

Classification of MHC molecules into supertypes in terms of peptide-binding specificities is an important issue, with direct implications for the development of epitope-based vaccines with wide population coverage. In view of extremely high MHC polymorphism (948 class I and 633 class II HLA alleles) the experimental solution of this task is presently impossible. In this study, we describe a bioinformatics strategy for classifying MHC molecules into supertypes using information drawn solely from three-dimensional protein structure. Two chemometric techniques–hierarchical clustering and principal component analysis–were used independently on a set of 783 HLA class I molecules to identify supertypes based on structural similarities and molecular interaction fields calculated for the peptide binding site. Eight supertypes were defined: A2, A3, A24, B7, B27, B44, C1, and C4. The two techniques gave 77% consensus, i.e., 605 HLA class I alleles were classified in the same supertype by both methods. The proposed strategy allowed “supertype fingerprints” to be identified. Thus, the A2 supertype fingerprint is Tyr9/Phe9, Arg97, and His114 or Tyr116; the A3-Tyr9/Phe9/Ser9, Ile97/Met97 and Glu114 or Asp116; the A24-Ser9 and Met97; the B7-Asn63 and Leu81; the B27-Glu63 and Leu81; for B44-Ala81; the C1-Ser77; and the C4-Asn77. action fields calculated for the peptide binding site. Eight supertypes were defined: A2, A3, A24, B7, B27, B44, C1, and C4. The two techniques gave 77% consensus, i.e., 605 HLA class I alleles were classified in the same supertype by both methods. The proposed strategy allowed “supertype fingerprints” to be identified. Thus, the A2 supertype fingerprint is Tyr9/Phe9, Arg97, and His114 or Tyr116; the A3-Tyr9/Phe9/Ser9, Ile97/Met97 and Glu114 or Asp116; the A24-Ser9 and Met97; the B7-Asn63 and Leu81; the B27-Glu63 and Leu81; for B44-Ala81; the C1-Ser77; and the C4-Asn77.

Protein deposition at polymer surfaces:the bulk and surface structure-property effects of hydrogels

The primary objective of this research has been to investigate the interfacial phenomenon of protein adsorption in relation to the bulk and surface structure-property effect s of hydrogel polymers. In order to achieve this it was first necessary to characterise the bulk and surface properties of the hydrogels, with regard to the structural chemistry of their component monomers. The bulk properties of the hydrogels were established using equilibrium water content measurements, together with water-binding studies by differential scanning calorimetry (D.S.C.). Hamilton and captive air bubble-contact angle techniques were employed to characterise the hydrogel-water interface and from which by a mathematical derivation, the interfacial free energy (ðsw) and the surface free energy components (ð psv, ðdsv, ðsv) were obtained. From the adsorption studies using the radio labelled iodinated (125I) proteins of human serum albumin (H.S.A.) and human fibrinogen (H.Fb.), it was Found that multi-layered adsorption was occurring and that the rate and type of this adsorption was dependent on the physico-chemical behaviour of the adsorbing protein (and its bulk concentration in solution), together with the surface energetics of the adsorbent polymer. A potential method for the invitro evaluation of a material's 'biocompatibility' was also investigated, based on an empirically observed relationship between the adsorption of albumin and fibrinogen and the 'biocompatibility' of polymeric materials. Furthermore, some consideration was also given to the biocompatibility problem of proteinaceous deposit formation on hydrophilic soft' contact lenses and in addition a number of potential continual wear contact lens formulations now undergoing clinical trials,were characterised by the above techniques.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

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

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst 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 to 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 weight (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.