Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies

Engineered Antibody and Monobody Domains with T Cell Receptor-Like Selectivity for Tumor Associated Peptide-MHC Antigens

Monoclonal antibody (mAb)-based therapeutics have established themselves as meaningful components of the treatment paradigm for a variety of tumors. However, since the approval of rituximab in 1997 as the first mAb-based therapy for cancer, there has been a paucity of novel, validated cancer targets for therapeutic intervention by mAbs. In effect, numerous challenges lie in the discovery of suitable extracellular or transmembrane antigens that permit the differentiation of tumor from healthy tissue. The adaptive immune system, though, mediates recognition of foreign antigens derived from the intracellular proteome by T cell receptor (TCR) binding to peptide-loaded major histocompatibility complex (pMHC) molecules. Because cancer is associated with large-scale alterations in the genome, there are a vast number of novel epitopes presented to the adaptive immune system. Although natural TCRs have exquisite functionality in distinguishing these foreign epitopes, and several tumor-reactive TCRs have, in fact, been characterized, the molecules themselves are poorly developable as therapeutic candidates. Thus, in order to enable TCR-like binding of a broader class of protein agents, this study explores the transfer of TCR binding domains to other mAb-based scaffolds, including the fibronectin-derived Fn3 and the IgG-derived 4D5 scaffolds. By using a combination of rational design and directed evolution to guide binding domain transfer, evidence for TCR-like binding was demonstrated for several engineered molecules. In addition to conferring binding functionality, the grafted TCR domains had a deleterious effect on the biophysical properties of these inherently robust protein scaffolds. Thus, this work provides novel insight into the objective of developing mAb-based agents with TCR-like binding specificity for pMHC antigens, informing future efforts to target the abundance of intracellular tumor epitopes

IMPROvER : the Integral Membrane Protein Stability Selector

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of alpha -helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/.Peer reviewe

Technology development for the over-expression, purification and crystallisation of human membrane proteins

Currently, the field of mammalian membrane protein structural biology is in its infancy. Existing technologies and experiences have shown that it is possible to obtain the structures of mammalian membrane proteins if sufficient work and thought has been invested. However, there is still an urgent need to develop new methodologies and approaches to improve all aspects of this important area of biological research. Here, a series of novel technologies for the overproduction, purification and crystallisation of human membrane proteins are described which have been tested with a representative member from each of the G-protein coupled receptor (adenosine 2a receptor (A2aR)) and membrane enzyme (sterol isomerase (SI)) superfamilies. The methylotrophic yeast Pichia pastoris is an excellent host cell for the overproduction of recombinant proteins including membrane proteins of mammalian origin. However, the commercially available expression vectors are far from what is required to maximise the production levels as well as simplify the detergent extraction and purification of human membrane proteins. Here, a series of related expression constructs were made that had different combinations of tags at both ends of the recombinant protein. The final optimised expression vectors had a C3 protease-iLOV-biotin acceptor-His10 (CLBH) tag fused to the C-terminus of the recombinant protein. The -CLBH vectors gave high level production of both test proteins (one Nin – hSI; one Nout – hA2aR) that could be rapidly purified to homogeneity using a generic protocol. The position of the His10 tag did not affect the expression level of the recombinant protein. In contrast, fusion of the biotin acceptor domain to the C-terminus of the recombinant protein increased its expression by a factor of between 2-4. The biotin acceptor domain could also be fully biotinylated in vitro using recombinantly expressed biotin ligase allowing purification/immobilisation of the target protein with streptavidin beads. Removal of the expression/ purification tags from the recombinant proteins with C3 protease occurred more efficiently than when TEV protease was used. An optimised protocol was developed that gave maximal production of our target proteins in fermenter culture at an induction temperature of 22°C. Care was taken to find a methanol feed rate that gave the highest levels of protein production without causing the accumulation of excess methanol in the culture (which is known to be toxic to the yeast). Using this protocol it was possible to make both hSI and hA2aR with a production level >10 mg of recombinant protein per litre of culture. As most MPs are colourless, target protein identification is usually performed by methods such as radioligand binding and/or Western blotting. However, these techniques can be time-consuming, use a lot of protein and do not give any information on the aggregation state of the protein in detergent solution. Previously, it has been shown that the processes of identifying and analysing membrane proteins in detergent solution can be accelerated by attaching green fluorescent protein to the C-terminus of the recombinant MP. Here, the potential of the recently described iLOV fluorescence tag for membrane protein applications was assessed. iLOV was shown to be an useful tool for optimising processes such as yeast clonal selection, protein production in fermenter culture, detergent and construct screening as well as tracking recombinant MPs through the purification process. Of note, the iLOV tag allowed a direct assessment of the stability and dispersity state of both target MPs in a range of detergents by fluorescence size exclusion chromatography (FSEC). Using this approach, it was shown that wild-type hA2aR solubilised using a combination of dodecyl-βDmaltoside (DDM) and cholesteryl-hemisuccinate (CHS) aggregated during purification on a Ni2+ column. Furthermore, it was shown that the hA2aR agonistconformationally-fixed mutant Rag23 is stable in DDM without any CHS present. Moreover, Rag23 was found to be monodisperse in a series of short-chain detergents (decyl-βD-maltoside, nonyl-βD-maltoside (NM) and β-octylglucoside) suggesting that this mutant is well-suited to structural studies. SI was remarkably robust in short chain detergents demonstrating a reasonable level of stability in the short chain detergent NM. The FSEC experiments showed that wild-type SI has considerably higher intrinsic stability than native hA2aR suggesting that membrane enzymes will prove to be more amenable to structural analysis than GPCRs. Rag23 and SI were both purified to homogeneity in a simple four-step procedure: i) Ni2+ purification, ii) cleavage with C3 protease, iii) reverse Ni2+ purification and iv) gel-filtration chromatography. A buffer/salt screen was devised that allowedthose conditions where SI had maximal thermostability in detergent-solution to be identified. SI was found to have greatest stability in sodium phosphate buffer at acidic pH. Using this information, it was possible to purify monodisperse SI in DM suggesting that this protein may make an excellent candidate for structural studies too. Crystallisation trials with SI were performed using the commercially available sparse matrix screen MemSys/MemStart. In addition, a lipidic-sponge phase sparse-matrix crystallisation screen that was developed in collaboration with Prof. Richard Neutze (University of Chalmers, Sweden) was tested using SI. Cholesterol could be incorporated into all of the sponges that make up the screen upto a concentration of 10%. (This is important as the activity of many mammalian membrane proteins is cholesterol-dependent). To date, no diffracting crystals of SI have been obtained with either the conventional or lipidic-sponge phase crystallisation approaches. In short, a series of novel technologies/methodologies have been developed that will act as a platform for future efforts to solve the structures of a wide-range of human membrane proteins

From existing data to novel hypotheses : design and application of structure-based Molecular Class Specific Information Systems

As the active component of many biological systems, proteins are of great interest to life scientists. Proteins are used in a large number of different applications such as the production of precursors and compounds, for bioremediation, as drug targets, to diagnose patients suffering from genetic disorders, etc. Many research projects have therefore focused on the characterization of proteins and on improving the understanding of the functional and mechanistic properties of proteins. Studies have examined folding mechanisms, reaction mechanisms, stability under stress, effects of mutations, etc. All these research projects have resulted in an enormous amount of available data in lots of different formats that are difficult to retrieve, combine, and use efficiently. The main topic of this thesis is the 3DM platform that was developed to generate Molecular Class Specific Information Systems (3DM systems) for protein superfamilies. These superfamily systems can be used to collect and interlink heterogeneous data sets based on structure based multiple sequence alignments. 3DM systems can be used to integrate protein, structure, mutation, reaction, conservation, correlation, contact, and many other types of data. Data is visualized using websites, directly in protein structures using YASARA, and in literature using Utopia Documents. 3DM systems contain a number of modules that can be used to analyze superfamily characteristics namely Comulator for correlated mutation analyses, Mutator for mutation retrieval, and Validator for mutant pathogenicity prediction. To be able to determine the characteristics of subsets of proteins and to be able to compare the characteristics of different subsets a powerful filtering mechanism is available. 3DM systems can be used as a central knowledge base for projects in protein engineering, DNA diagnostics, and drug design. The scientific and technical background of the 3DM platform is described in the first two chapters. Chapter 1 describes the scientific background, starting with an overview of the foundations of the 3DM platform. Alignment methods and tools for both structure and sequence alignments, and the techniques used in the 3DM modules are described in detail. Alternative methods are also described with the advantages and disadvantages of the various strategies. Chapter 2 contains a technical description of the implementation of the 3DM platform and the 3DM modules. A schematic overview of the database used to store the data is provided together with a description of the various tables and the steps required to create new 3DM systems. The techniques used in the Comulator, Mutator and Validator modules of the 3DM platforms are discussed in more detail. Chapter 3 contains a concise overview of the 3DM platform, its capabilities, and the results of protein engineering projects using 3DM systems. Thirteen 3DM systems were generated for superfamilies such as the PEPM/ICL and Nuclear Receptors. These systems are available online for further examination. Protein engineering studies aimed at optimizing substrate specificity, enzyme activity, or thermostability were designed targeting proteins from these superfamilies. Preliminary results of drug design and DNA diagnostics projects are also included to highlight the diversity of projects 3DM systems can be applied to. Project HOPE: a biomedical tool to predict the effect of a mutation on the structure of a protein is described in chapter 4. Project HOPE is developed at the Radboud University Nijmegen Medical Center under supervision of H. Venselaar. Project HOPE employs webservices to optimally reuse existing databases and computing facilities. After selection of a mutant in a protein, data is collected from various sources such as UniProt and PISA. A homology model is created to determine features such as contacts and side-chain accessibility directly in the structure. Using a decision tree, the available data is evaluated to predict the effects of the mutation on the protein. Chapter 5 describes Comulator: the 3DM module for correlated mutation analyses. Two positions in an alignment correlate when they co-evolve, that is they mutate simultaneously or not at all. Comulator uses a statistical coupling algorithm to calculate correlated mutation analyses. Correlated mutations are visualized using heatmaps, or directly in protein structures using YASARA. Analyses of correlated mutations in various superfamilies showed that positions that correlate are often found in networks and that the positions in these networks often share a common function. Using these networks, mutants were predicted to increase the specificity or activity of proteins. Mutational studies confirmed that correlated mutation analyses are a valuable tool for rational design of proteins. Mutator, the text mining tool used to incorporate mutations into 3DM systems is described in chapter 6. Mutator was designed to automatically retrieve mutations from literature and store these mutations in a 3DM system. A PubMed search using keywords from the 3DM system is used to preselect articles of interest. These articles are retrieved from the internet, converted to text, and parsed for mutations. Mutations are then grounded to proteins and stored in a 3DM database. Mutation retrieval was tested on the alpha-amylase superfamily as this superfamily contains the enzyme involved in Fabry’s disease: an x linked lysosomal storage disease. Compared to existing mutant databases, such as the HGMD and SwissProt, Mutator retrieved 30% more mutations from literature. A major problem in DNA diagnostics is the differentiation between natural variants and pathogenic mutations. To distinguish between pathogenic mutations and natural variation in proteins the Validator modules was added to 3DM. Validator uses the data available in a 3DM system to predict the pathogenicity of a mutant using, for example, the residue conservation of the mutants alignment position, side-chain accessibility of the mutant in the structure, and the number of mutations found in literature for the alignment position. Mutator and Validator can be used to study mutants found in disorder related genes. Although these tools are not the definitive solution for DNA diagnostics they can hopefully be used to increase our understanding of the molecular basis of disorders. Chapter 7 and 8 describe applied research projects using 3DM systems containg proteins of potential commercial interest. A 3DM system for the a/b-beta hydrolases superfamily is described in chapter 7. This superfamily consists of almost 20,000 proteins with a diverse range of functions. Superfamily alignments were generated for the common beta-barrel fold shared by all superfamily members, and for five distinct subtypes within the superfamily. Due to the size and functional diversity of the superfamily, there is a lot of potential for industrial application of superfamily members. Chapter 8 describes a study focusing on a sucrose phosphorylase enzyme from the a-amylase superfamily. This enzyme can be potentially used in an industrial setting for the transfer of glucose to a wide variety of molecules. The aim of the study was to increase the stability of the protein at higher temperatures. A combination of rational design using a 3DM system, and in-depth study of the protein structure, led to a series of mutations that resulted in more than doubling the half-life of the protein at 60°C. 3DM systems have been successfully applied in a wide range of protein engineering and DNA diagnostics studies. Currently, 3DM systems are applied most successfully in project studying a single protein family or monogenetic disorder. In the future, we hope to be able to apply 3DM to more complex scenarios such as enzyme factories and polygenetic disorders by combining multiple 3DM systems for interacting proteins.</p

Methods in protein engineering and screening : from rational design to directed evolution and beyond

Humans have engineered organisms for thousands of years. The domestication of plants and animals are early examples of how humans have changed genotypes of organisms for prized traits that persist today. The genotype to phenotype linkage is often the result of the expression or performance of proteins found within an organism. The modern era of biological engineering has seen the emergence of two fundamentally distinct philosophies on how best to alter the genetic code of an organism or sequence of a particular protein for desired phenotypic outcomes. Rational design and directed evolution are often seen as two competing methodologies working towards the same goal—the engineering of proteins and ultimately organisms. Here, I attempt to bridge the gap between the two engineering strategies and argue that a combination is needed in many cases. Starting with the rationalization of directed evolution experiments using software tools, we begin to understand the predictive power and limitations of rational computational approaches. We then explore the computational resurfacing of an enzyme and discover an unpredicted thermoswitchable phenotype. Next, we rationally design a number of fusion proteins, which allow us to create novel point-of-care diagnostics. We then turn our attention to the directed evolution of a number of DNA polymerases with novel functions. The amalgamation of rational design and directed evolution are then extended to a eukaryotic organism, which enables us to engineer more complex proteins and gives us the ability to create novel drug screening and discovery platforms.Biochemistr

ECUT: Energy Conversion and Utilization Technologies program. Biocatalysis project

The Biocatalysis Project is a mission-oriented, applied research and exploratory development activity directed toward resolution of the major generic technical barriers that impede the development of biologically catalyzed commercial chemical production. The approach toward achieving project objectives involves an integrated participation of Universities, Industrial Companies and Government Research Laboratories. The Project's technical activities were organized into three work elements: molecular modeling and applied genetics; bioprocess engineering; and bioprocess design and assessment

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

Biochemische und funktionelle Charakterisierung zellfrei exprimierter G-Protein-gekoppelter Rezeptoren des humanen Endothelin-Systems

G-protein coupled receptors (GPCRs) are the key players in signal perception and transduction and one of the currently most important class of drug targets. An example of high pharmacological relevance is the human endothelin (ET) system comprising two rhodopsin-like GPCRs, the endothelin A (ETA) and the endothelin B (ETB) receptor. Both receptors are major modulators in cardiovascular regulation and show striking diversities in biological responses affecting vasoconstriction and blood pressure regulation as well as many other physiological processes. Numerous disorders are associated with ET dysfunction and ET antagonism is considered an efficient treatment of diseases like heart failure, hypertension, diabetes, artherosclerosis and even cancer. This study exemplifies strategies and approaches for the preparative scale synthesis of GPCRs in individual cell-free (CF) systems based on E. coli, a newly emerging and promising technique for the production of even very difficult membrane proteins. The preparation of high quality samples in sufficient amounts is still a major bottleneck for the structural determination of the ET receptors. Heterologous overexpression has been a challenge now for decades but extensive studies with conventional cell-based systems had only limited success. A central milestone of this study was the development of efficient preparative scale expression protocols of the ETA receptor in qualities sufficient for structural analysis by using individual CF systems. Newly designed optimization strategies, the implementation of a variety of CF expression modes and the development of specific quality control assays finally resulted in the production of several milligrams of ETA receptor per one millilitre of reaction mixture. The versatility of CF expression was extensively used to modulate GPCR sample quality by modification of the solubilization environment with detergents and lipids in a variety of combinations at different stages of the production process. Downstream processing procedures of CF synthesized GPCRs were systematically optimized and sample properties were analysed with respect to homogeneity, protein stability and receptor ligand binding competence. Evaluation was accomplished by an array of complementary and specifically modified techniques. Depending on its hydrophobic environment, CF production of the ETA receptor resulted in non-aggregated, monodisperse forms with sufficient long-term stability and high degrees of secondary structure thermostability. The obtained results document the CF production of the ETA receptor in two different modes as an example of a class A GPCR in ligand-binding competent and non-aggregated form in quantities sufficient for structural approaches. The presented strategy could serve as basic guideline for the production of related receptors in similar systems.G-Protein gekoppelte Rezeptoren (GPCRs), bilden mit etwa 3 % die größte Klasse an Zelloberflächenrezeptoren im menschlichen Proteom. Aufgrund ihrer bedeutenden Funktion bei der Verarbeitung vielfältiger Umweltsignale und bei der Steuerung physiologischer Abläufe im menschlichen Organismus gehören GPCRs mit rund 50 % zu den wichtigsten Angriffspunkten heutiger Arzneimittel. Ein Beispiel mit hoher pharmakologischer Relevanz ist das humane Endothelin- (ET) System, das aus zwei Rhodopsin-ähnlichen GPCRs, dem Endothelin A (ETA) und dem Endothelin B Rezeptor (ETB) besteht. Beide Rezeptoren gehören zu den wichtigsten Modulatoren des kardiovaskulären Systems. Es beeinflusst Gefäßverengung und Blutdruckregulation und spielt zudem eine essentielle Rolle bei Zellproliferation und Mitogenese. Viele bekannte Zivilisationskrankheiten sind mit endothelialer Fehlfunktion assoziiert. ET-Rezeptor-Antagonismus wird daher als potente Methode zur Behandlung von Herzinsuffizienz, Bluthochdruck, Diabetes, Atherosklerose und sogar von Krebs aufgefasst. Die Produktion von Proteinproben hoher Qualität in ausreichenden Mengen ist eine unabdingbare Voraussetzung zur strukturellen Aufklärung von GPCRs. Die heterologe Überexpression zur Herstellung der ET-Rezeptoren in konventionellen zell basierenden Systemen hatte allerdings bisher nur eingeschränkten Erfolg. Diese Arbeit konzentrierte sich daher auf die Anwendung alternativer Technologien, die auf selbst hergestellten zellfreien Expressionssystemen als hochkompetitive Option zur Produktion selbst schwieriger Membranproteine basieren. Das Hauptziel der vorliegenden Dissertation bestand in der Prozessentwicklung zur Etablierung individueller und effizienter zellfreier Syntheseprotokolle zur präparativen Produktion von GPCRs am Beispiel des humanen ET-Rezeptoms. Der Schwerpunkt wurde auf die zellfreie Produktion und Qualitätskontrolle des ETA Rezeptors gelegt, da hierzu noch keinerlei primäre Erkenntnisse vorlagen. Die erstellten Produktionsprozesse sowie die weitere Aufarbeitung des Rezeptors wurden systematisch im Detail optimiert. Ein wichtiger und neuartiger Ansatz war in diesem Hinblick die gezielte Modifikation der direkten Translationsumgebung des ETA Rezeptors, eine Möglichkeit die durch den charakteristischen Aufbau der zellfreien Reaktion geboten wird und bisher einzigartig für diese Technologie ist. Die Qualität der produzierten Proteine wurde nach schrittweisen Reaktionsmodifikationen und in Abhängigkeit der eingesetzten Expressionsmodi sowie von zugesetzten hydrophoben Additiven im Hinblick auf eine Reihe komplementärer Kriterien wie Homogenität, Stabilität und Ligandenbindungs-Kompetenz analysiert. Die spezifisch erarbeiteten Optimierungs¬strategien im Verbund mit den individuell auf das Protein abgestimmten und neu etablierten Methoden zur Qualitätskontrolle ermöglichten die Produktion des ETA Rezeptors in routinemäßigen Mengen von etwa 2 Milligramm pro Millilitern Reaktionsansatz. Grundlegende Prozessparameter ergaben sich durch Qualitätsanalysen von ETA Proben nach deren Synthese in verschiedenen zellfreien Expressionsmodi. Die Qualität des Rezeptors war nach Produktion im Präzipitat erzeugenden P CF Modus mit anschließender post-translationeller Solubilisierung deutlich besser im Vergleich zur Synthese mit direkter ko-translationeller Solubilisierung im D CF Modus nach Zugabe von Detergenzien in den Reaktionsansatz. Dieses zunächst überraschende Ergebnis wurde durch Gelfiltrations¬experimente bestätigt. In Übereinstimmung offenbarten Festkörper-Kernmagnetische Resonanz Analysen zudem alpha-helikale Sekundärstrukturelemente in den P-CF generierten ETA Präzipitaten. Als weiteres effektives Mittel zur Modulation der mizellaren Umgebung von P CF synthetisiertem ETA wurde der Detergenzaustausch nach Immobilisierung durch Metallchelat-Affinitätschromatographie verwendet. Als äußerst geeignet wurde Brij 35 identifiziert. ETA zeigte hier in analytischer Größenausschlusschromatographie schlanke Elutionsprofile, in deren Fraktionen das Protein einen hohen Grad an Homogenität im Verbund mit Stabilität aufwies. Negativfärbung belegte die Monodispersität der Proben, die sich außerdem durch eine hohe thermische Sekundärstrukturstabilität auszeichneten. Analysen durch native und denaturierende Gelelektrophorese deuteten auf die potentielle Bildung SDS-resistenter Dimere/Oligomere des ETA Rezeptors hin. Diese Annahme wurde durch Massenspektrometrie unterstützt. Vielwinkel-Lichtstreuung zeigte außerdem, dass Gleichgewichte zwischen monomerem und dimerem ETA Rezeptor offenbar durch das umgebende Mizellenmillieu gesteuert werden können. Zudem wurden erste Hinweise auf das Oligomerisierungs-Potential von zellfrei exprimiertem ETA und ETB durch Ko-Expression und anschließenden Komplexanalysen unter Einbezug hergestellter, fragmentierter Derivate erlangt. Die Ligandenbindungs-Kompetenz des solubilisierten Rezeptors wurde durch Affinitätschromatographie und Fluoreszenz-Anisotropie untersucht. Dieser Ansatz führte zu einem effizienten Protokoll für die Reinigung Liganden-gebundener Rezeptorfraktionen für z.B. Kristallisationsanalysen. Nach Synthese in den identifizierten optimalen Bedingungen im P-CF Modus und Detergenzaustausch in Brij 35 wurden Bindungseffizienzen von bis zu 50 % erzielt, die unter optimalen Bedingungen eine Ausbeute an funktionellem Rezeptor von 0.5 mg/mL zellfreiem Reaktionsansatz ergeben. Übereinstimmende Nachweise der funktionellen Faltung des synthetisierten ETA Rezeptors in Abhängigkeit der Expressionsbedingungen wurden erhalten durch Fluoreszenz-Anisotropie, elektronenmikroskopische Gefrierbruchanalysen von rekonstituierten Proben sowie durch Radioliganden-Experimente zur Bestimmung der Gleichgewichtsdissoziations-konstanten (KD) und der inhibitorischen Konzentrationen (IC50) des natürlichen Liganden Endothelin 1. Die Arbeit bietet eine grundlegende Richtlinie zur Qualitätsoptimierung zellfrei synthetisierter Membranproteine und sie stellt zudem ein Orientierungshilfe zur Prozessentwicklung individueller Expressionsprotokolle dar. Die präparative Produktion hochwertiger Proben an ETA Rezeptor resultierte in der Durchführung umfassender Kristallisationsanalysen als Basis für nachfolgende Arbeiten zur Strukturbestimmung

Structural basis of ligand recognition at the human MT1 melatonin receptor

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors