Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor.

A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and β-arrestin. Using 13C methyl methionine NMR for the β1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level

Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19 F NMR of the β 1 -adrenergic receptor

Abstract: G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed

Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19 F NMR of the β 1 -adrenergic receptor

Abstract: G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed

Recent progress with hot carrier solar cells

Hot carrier solar cells offer one of the most promising options for high performance “third generation” photovoltaic devices. For successful operation, these need to be thin, strongly absorbing, radioactively efficient devices in a simple 2-terminal configuration. Nonetheless, they offer potential performance close to the maximum possible for solar conversion, equivalent to a multi-cell stack of six or more tandem cells possibly without some of the limitations, such as spectral sensitivity. However, hot carrier cells offer some quite fundamental challenges in implementation that our team is addressing in an internationally collaborative effort

Production de chitine deacetylase hétérologue chez la levure Pichia pastoris en vue de transformer la chitine en chitosan par un bioprocédé

La chitine est produite en quantité importante dans la biosphère. Elle existe dans les squelettes externes des Arthropodes (crustacés et insectes) et dans la paroi des champignons. La forme deacétylée, appelée chitosan, est rare dans la nature. Cependant, il s'agit d'un produit très interessant pour la médicine, l'agriculture et pour plusieurs branches de l'industrie (pharmacie, cosmétique, traitement des eaux, microélectronique). La production mondiale actuelle de chitosan est très faible et peu rentable sur le plan commercial. Le chitosan du commerce est traditionellement produit par déacétylation chimique de la chitine des crustacés à haute température. Cette technologie produit du chitosan de mauvaise qualité. L'utilisation de grandes quantités d'alcalis concentrés provoque une dégradation du produit final et génère des déchets dangereux pour l'environnement. Un procédé alternatif, basé sur la déacétylation enzymatique de la chitine, pourrait être une alternative attrayante et éviterait les inconvénients de la méthode chimique. De nombreuses recherches sont menées dans ce sens au niveau international. Le clonage d'un gène codant une chitine-déacétylase dans un hôte bien adapté pourrait aboutir à une production rentable de chitine-déacétylase et donc de chitosan.Le gène codant la chitine déacétylase de Colletotrichum lindemuthianum UPS9 a été identifié, cloné et surexprimé chez Escherichia coli, Corynebacterium glutamicum et Pichia pastoris. Seule P. pastoris a permis la sécrétion d'une chitine déacétylase active. La production a été realisée en fermenteur. L'enzyme a été purifiée et caractérisée au niveau enzymatique grâce à la mise au poin d'une nouvelle méthodeChitin is produced in enormous quantities in the biosphere. It exists in the outer skeleton of arthropods (crustaceans and insects) and in the cell wall of many fungi. The deacetylated form of chitin, chitosan, is rarely found in nature. Chitosan is an interesting fine chemical which has wide applications in various branches of industry (pharmaceutical, cosmetics, food, textile, water purification and microelectronics). Chitosan is commercially produced by thermochemical deacetylation of crustacean chitin waste using strong alkaline condition at high temperature. Chitosan obtained from this method is usually damaged by chemical degradation. Moreover release of large quantities of alkaline waste water are produced. An alternative method of deacetylation by an enzymatic method might overcome the drawbacks of the thermochemical method. Research on the enzymatic deacetylation carried out so far by other institutes has not resulted in a satisfactory process. The cloning of gene encoding chitin deacetylase, the expression in a suitable host and further engineering of enzyme may help in high level production of functional chitin deacetylase.The gene encoding the chitin deacetylase of Colletotrichum lindemuthianum has been identified and cloned in Escherichia coli, Corynebacterium glutamicum and Pichia pastoris. P. pastoris was found to be capable of expressing active chitin deacetylase in the extracellular medium. The production of chitin deacetylase was carried out in a fermenter. The enzyme was purified and characterized with the help of a new method of enzymatic assay developed in this study.ORSAY-PARIS 11-BU Sciences (914712101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.

The increasing demand for production and characterization of diverse groups of recombinant proteins necessitates the analysis of several constructs and fusion tags in a variety of expression systems. The challenge is to screen multiple clones quickly for the desired properties. When using a eukaryotic system, such as baculovirus-mediated expression in insect cells, the total time required and the volume of culture needed to obtain reasonable results are limiting factors. This chapter focuses on addressing these issues by describing rapid small-scale expression as a mode of screening. The method allows the rapid identification of the best clone before scaling-up and the production of heterologous protein

Development of a biochemical and biophysical suite for integral membrane protein targets: A review

The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches

Transfection of insect cell in suspension for efficient baculovirus generation

Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation. This significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination. The method is economical and doesn’t require any special adjustment in existing labs

Insect cell culture in reagent bottles

Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way

Uniform isotope labeling of proteins expressed in insect cells using a custom medium

Production of proteins uniformly labeled with 2H, 13C and 15N isotopes is essential for advanced NMR studies. Here we present an affordable strategy for uniform isotope labeling in the insect cell system. Compared to commercial media the cost of the newly developed media are about 10-fold lower at comparable isotope incorporation. Additionally, there is no restriction on the labeling pattern (only 15N and 13C,15N are available commercially), but 2H, 13C and 15N in every combination can be labeled. The method was evaluated by the NMR studies on expressed proteins with four different uniform labeling patterns