10 research outputs found
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Investigating the molecular signatures of β1-adrenergic receptor activation
In this thesis I have investigated the molecular signatures of receptor activation, using the β1-adrenergic receptor (β1AR) as a prototypical class A G-protein coupled receptor (GPCR). I have used a minimally thermostabilised turkey β1AR and expressed it functionally in insect cells using a baculovirus system. The work described here established the labelling, expression, purification and sample preparation of the receptor in LMNG detergent micelles for use in nuclear magnetic resonance (NMR) spectroscopy. GPCRs are highly dynamic molecular systems, and the use of solution NMR is highly suited to the study of fine structural changes that take place within the receptor as a consequence of receptor activation. To this end the receptor was selectively labelled with 13C at the methyl position of methionine residues. The labelling was carried out during insect cell expression, by supplementing methionine deficient media with the labelled amino acid. In this way the methionines throughout the receptor served as reporters. The radio frequency signals emitted by the nuclei of these labelled residues were monitored. NMR experiments were recorded on the receptor in the presence of ligands of various efficacies together with and without G-protein mimetic nanobodies, and changes in the signal were recorded. This allowed for a pattern of molecular signatures to be established, reporting on the effect ligands and G-protein mimetics have on the receptor. This identified two conformational equilibria, between an inactive and a ligand bound-pre-activated state and between a more and a less active ternary state when bound to a G-protein mimetic. Furthermore, it was also observed that ligand binding to the G-protein mimetic saturated basal active state elicits further changes on the receptor cytoplasmic side, demonstrating that ligand efficacy modulates the nature of receptor interaction with G-proteins, which may underpin partial agonism. It was also observed that ligand binding affects the dynamics and rigidity of the receptor, with a full agonist bound receptor exhibiting extensive µs to ms timescale dynamics, compared to a more rigid nanobody bound state. The increased dynamics suggest that full agonist binding primes the receptor for interaction with various downstream signalling partners. Once this coupling takes place, ligand efficacy determines the quality of interaction in this rigidified system.
In addition to activation by ligands, certain proteins, such as antibodies can cause receptor agonism in the absence of a small molecule agonist. An example of this takes place in chronic Chagas’ heart disease, where anti-Trypanosoma cruzi antibodies inappropriately cross-react to β1AR, leading to chronic cardiac overstimulation and heart failure. In this thesis, the production of a published monoclonal antibody fragment was explored, in order to generate a tool for the study of this activation mechanism.MRC CASE, NIBR, Wolfson College, Biochemical Society, Philosophical Societ
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The role of NMR spectroscopy in mapping the conformational landscape of GPCRs.
Over recent years, nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful mechanistic tool for the investigation of G protein-coupled receptors (GPCRs). NMR provides insights which underpin the dynamic nature of these important receptors and reveals experimental evidence for a complex conformational energy landscape that is explored during receptor activation resulting in signalling. NMR studies have highlighted both the dynamic properties of different receptor states as well as the exchange pathways and intermediates formed during activation, extending the static view of GPCRs obtained from other techniques. NMR studies can be undertaken in realistic membrane-like phospholipid environments and an ever-increasing choice of labelling strategies provides comprehensive, receptor-wide information. Combined with other structural methods, NMR is contributing to our understanding of allosteric signal propagation and the interaction of GPCRs with intracellular binding partners (IBP), crucial to explaining cellular signalling.BBSRC BB/K01983 X/
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
An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins.
Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy
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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
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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
Insight into partial agonism by observing ligand-modulated conformational equilibria of a Gs-mimetic nanobody-bound B1-adrenergic receptor
Signal transduction of extracellular stimuli via G-protein-coupled receptors (GPCRs) involves formation of agonist-bound active receptor complexes with intracellular cytoplasmic binding partners such as G-proteins and β-arrestins. Current mechanistic understanding of activation of these highly dynamic signalling receptors relies primarily on crystallographic information but many questions remain. Using 13C-methyl-methionine NMR we show that following cytoplasmic coupling of Gs-mimetic nanobodies to the β1-adrenergic receptor, the receptor is found in a dynamic ligand-efficacy dependent equilibrium between an active ternary complex when bound to full-agonist and a less-active conformation distinctive of basal activity. Structural differences between the conformations of these ternary complexes concentrate on the cytoplasmic side of the receptor indicating ligand-induced variations in G-protein mimetic interaction as the likely cause, providing a mechanistic framework for partial agonism for the Gs pathway. We compare differences in structure and dynamics for receptors bound to different orthosteric ligands, including observation of states representative of constitutive activity
An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins
Evolthon: A community endeavor to evolve lab evolution.
In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation