{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/397464"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/397464","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Characterising the conformational dynamics of β₁-adrenergic receptor activation and G protein coupling","abstract":"The G protein-coupled receptor (GPCR) family of seven-transmembrane signalling proteins facilitates cellular responses to environmental stimuli. GPCRs are integral to numerous physiological signalling pathways and thus many therapeutics have been developed to modulate receptor activity. Stimulation by agonist binding into an orthosteric pocket induces conformational changes in a highly interconnected allosteric activation network which enables coupling to intracellular signalling partners, including selective coupling to specific G protein α-subtypes. Investigations in solution by nuclear magnetic resonance spectroscopy (NMR) have been utilised in this thesis to complement static studies of discrete agonist-bound and G protein-coupled conformations and demonstrate population of complex conformational equilibria. This research extends our understanding of the link between GPCR conformational dynamics and signalling properties, such as agonist-induced activation, G protein selectivity, and allosteric modulation, at the β₁-adrenergic receptor (β₁AR), a prototypical Class A GPCR. In Chapter 2, the structural, dynamic, and functional properties of conformational equilibria in a minimally thermostabilised avian β₁AR construct are characterised by selective labelling in combination with ¹⁹F and ¹³C NMR spectroscopy. These studies reveal agonist-stimulated β₁AR predominantly populates an active state, which in the receptor core and orthosteric pocket is comparable to conformations observed in static studies of β₁AR coupled to Gs protein. However, investigation of the receptor intracellular surface demonstrates the active state differs in conformation from known static structures, highlighting the importance of solution studies in structural investigations of GPCRs. Population of the active state is found to accelerate the association of G proteins with β₁AR in biolayer interferometry assays, relative to previously characterised pre-active and inactive states. These data therefore extend our understanding of the functional states populated in solution by the β₁AR. The impact of G protein coupling on the receptor conformations of the active state are assessed in Chapter 3. Further conformational rearrangements are observed upon binding partner coupling in NMR spectra. These changes are primarily localised to the intracellular surface of the receptor, supporting looser coupling of this region to orthosteric agonist efficacy information relative to the rest of the β₁AR. Extension of these studies to examine different G protein complexes at the β₁AR reveal only minor differences by solution NMR spectroscopy, whereas the kinetics of association to the receptor differ substantially between different G proteins. Importantly, these data suggest the agonist-bound solution active state acts as a kinetics-driven selectivity gate with differences in favourability of binding to different G proteins. The integrative investigation in Chapter 4 assesses how changes in the receptor conformational equilibria observed in the previous chapters could account for reductions in receptor activity. This work contrasts the influences on receptor signalling from the orthosteric agonist binding pocket or from other binding sites in the receptor, in order to characterise mechanisms of allosteric modulation at the β₁AR. NMR studies show that either orthosteric agonists with lower efficacies or mutations in a known allosteric pocket stabilise less active states in the receptor equilibria, which could account for reductions in G protein binding affinities and decreased downstream signalling. In contrast, binding of a small molecule modulator (AS408) into the same allosteric pocket decreases G protein coupling via a distinct mechanism. The AS408-bound receptor adopts an alternative conformation which differs from known states on the intracellular surface of β₁AR, whilst conformations in other regions of the receptor are influenced by both the orthosteric agonist and AS408. These investigations highlight the vital role of receptor plasticity in GPCR allostery and demonstrate the limitations of the conformational equilibrium model in understanding GPCR signalling. In summary, this thesis characterises the solution conformations of agonist-bound β₁AR and emphasises the impact of dynamics on GPCR signalling. The structural findings have important implications for investigations into the interaction between signalling partners and the GPCR active state, and the development of novel therapeutics.","abstract_html":"The G protein-coupled receptor (GPCR) family of seven-transmembrane signalling proteins facilitates cellular responses to environmental stimuli. GPCRs are integral to numerous physiological signalling pathways and thus many therapeutics have been developed to modulate receptor activity. Stimulation by agonist binding into an orthosteric pocket induces conformational changes in a highly interconnected allosteric activation network which enables coupling to intracellular signalling partners, including selective coupling to specific G protein α-subtypes. Investigations in solution by nuclear magnetic resonance spectroscopy (NMR) have been utilised in this thesis to complement static studies of discrete agonist-bound and G protein-coupled conformations and demonstrate population of complex conformational equilibria. This research extends our understanding of the link between GPCR conformational dynamics and signalling properties, such as agonist-induced activation, G protein selectivity, and allosteric modulation, at the β₁-adrenergic receptor (β₁AR), a prototypical Class A GPCR. In Chapter 2, the structural, dynamic, and functional properties of conformational equilibria in a minimally thermostabilised avian β₁AR construct are characterised by selective labelling in combination with ¹⁹F and ¹³C NMR spectroscopy. These studies reveal agonist-stimulated β₁AR predominantly populates an active state, which in the receptor core and orthosteric pocket is comparable to conformations observed in static studies of β₁AR coupled to Gs protein. However, investigation of the receptor intracellular surface demonstrates the active state differs in conformation from known static structures, highlighting the importance of solution studies in structural investigations of GPCRs. Population of the active state is found to accelerate the association of G proteins with β₁AR in biolayer interferometry assays, relative to previously characterised pre-active and inactive states. These data therefore extend our understanding of the functional states populated in solution by the β₁AR. The impact of G protein coupling on the receptor conformations of the active state are assessed in Chapter 3. Further conformational rearrangements are observed upon binding partner coupling in NMR spectra. These changes are primarily localised to the intracellular surface of the receptor, supporting looser coupling of this region to orthosteric agonist efficacy information relative to the rest of the β₁AR. Extension of these studies to examine different G protein complexes at the β₁AR reveal only minor differences by solution NMR spectroscopy, whereas the kinetics of association to the receptor differ substantially between different G proteins. Importantly, these data suggest the agonist-bound solution active state acts as a kinetics-driven selectivity gate with differences in favourability of binding to different G proteins. The integrative investigation in Chapter 4 assesses how changes in the receptor conformational equilibria observed in the previous chapters could account for reductions in receptor activity. This work contrasts the influences on receptor signalling from the orthosteric agonist binding pocket or from other binding sites in the receptor, in order to characterise mechanisms of allosteric modulation at the β₁AR. NMR studies show that either orthosteric agonists with lower efficacies or mutations in a known allosteric pocket stabilise less active states in the receptor equilibria, which could account for reductions in G protein binding affinities and decreased downstream signalling. In contrast, binding of a small molecule modulator (AS408) into the same allosteric pocket decreases G protein coupling via a distinct mechanism. The AS408-bound receptor adopts an alternative conformation which differs from known states on the intracellular surface of β₁AR, whilst conformations in other regions of the receptor are influenced by both the orthosteric agonist and AS408. These investigations highlight the vital role of receptor plasticity in GPCR allostery and demonstrate the limitations of the conformational equilibrium model in understanding GPCR signalling. In summary, this thesis characterises the solution conformations of agonist-bound β₁AR and emphasises the impact of dynamics on GPCR signalling. The structural findings have important implications for investigations into the interaction between signalling partners and the GPCR active state, and the development of novel therapeutics.","abstract_has_math":false,"creators":["Harman, Thomas"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Nietlispach, Daniel"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-09-30","date_published":"2025-09-30","updated_at":"2026-07-22T22:24:16Z","subjects":["Biochemistry","Protein Biology","Solution NMR","GPCR"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/0b3f7640-32e1-435c-805e-e4009b313fd6/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.126595","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Nietlispach, Daniel"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["AstraZeneca Studentship"]},{"key":"dc:creator","label":"Author","values":["Harman, Thomas"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-09-30"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/397464"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biochemistry","Protein Biology","Solution NMR","GPCR"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/0b3f7640-32e1-435c-805e-e4009b313fd6/download","http://purl.org/NET/rdflicense/allrightsreserved"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.126595"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/065f5e07-150a-450c-a5d8-1995ebc835c3/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The G protein-coupled receptor (GPCR) family of seven-transmembrane signalling proteins facilitates cellular responses to environmental stimuli. GPCRs are integral to numerous physiological signalling pathways and thus many therapeutics have been developed to modulate receptor activity. Stimulation by agonist binding into an orthosteric pocket induces conformational changes in a highly interconnected allosteric activation network which enables coupling to intracellular signalling partners, including selective coupling to specific G protein α-subtypes. Investigations in solution by nuclear magnetic resonance spectroscopy (NMR) have been utilised in this thesis to complement static studies of discrete agonist-bound and G protein-coupled conformations and demonstrate population of complex conformational equilibria. This research extends our understanding of the link between GPCR conformational dynamics and signalling properties, such as agonist-induced activation, G protein selectivity, and allosteric modulation, at the β₁-adrenergic receptor (β₁AR), a prototypical Class A GPCR. In Chapter 2, the structural, dynamic, and functional properties of conformational equilibria in a minimally thermostabilised avian β₁AR construct are characterised by selective labelling in combination with ¹⁹F and ¹³C NMR spectroscopy. These studies reveal agonist-stimulated β₁AR predominantly populates an active state, which in the receptor core and orthosteric pocket is comparable to conformations observed in static studies of β₁AR coupled to Gs protein. However, investigation of the receptor intracellular surface demonstrates the active state differs in conformation from known static structures, highlighting the importance of solution studies in structural investigations of GPCRs. Population of the active state is found to accelerate the association of G proteins with β₁AR in biolayer interferometry assays, relative to previously characterised pre-active and inactive states. These data therefore extend our understanding of the functional states populated in solution by the β₁AR. The impact of G protein coupling on the receptor conformations of the active state are assessed in Chapter 3. Further conformational rearrangements are observed upon binding partner coupling in NMR spectra. These changes are primarily localised to the intracellular surface of the receptor, supporting looser coupling of this region to orthosteric agonist efficacy information relative to the rest of the β₁AR. Extension of these studies to examine different G protein complexes at the β₁AR reveal only minor differences by solution NMR spectroscopy, whereas the kinetics of association to the receptor differ substantially between different G proteins. Importantly, these data suggest the agonist-bound solution active state acts as a kinetics-driven selectivity gate with differences in favourability of binding to different G proteins. The integrative investigation in Chapter 4 assesses how changes in the receptor conformational equilibria observed in the previous chapters could account for reductions in receptor activity. This work contrasts the influences on receptor signalling from the orthosteric agonist binding pocket or from other binding sites in the receptor, in order to characterise mechanisms of allosteric modulation at the β₁AR. NMR studies show that either orthosteric agonists with lower efficacies or mutations in a known allosteric pocket stabilise less active states in the receptor equilibria, which could account for reductions in G protein binding affinities and decreased downstream signalling. In contrast, binding of a small molecule modulator (AS408) into the same allosteric pocket decreases G protein coupling via a distinct mechanism. The AS408-bound receptor adopts an alternative conformation which differs from known states on the intracellular surface of β₁AR, whilst conformations in other regions of the receptor are influenced by both the orthosteric agonist and AS408. These investigations highlight the vital role of receptor plasticity in GPCR allostery and demonstrate the limitations of the conformational equilibrium model in understanding GPCR signalling. In summary, this thesis characterises the solution conformations of agonist-bound β₁AR and emphasises the impact of dynamics on GPCR signalling. 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Stimulation by agonist binding into an orthosteric pocket induces conformational changes in a highly interconnected allosteric activation network which enables coupling to intracellular signalling partners, including selective coupling to specific G protein α-subtypes. Investigations in solution by nuclear magnetic resonance spectroscopy (NMR) have been utilised in this thesis to complement static studies of discrete agonist-bound and G protein-coupled conformations and demonstrate population of complex conformational equilibria. This research extends our understanding of the link between GPCR conformational dynamics and signalling properties, such as agonist-induced activation, G protein selectivity, and allosteric modulation, at the β₁-adrenergic receptor (β₁AR), a prototypical Class A GPCR. In Chapter 2, the structural, dynamic, and functional properties of conformational equilibria in a minimally thermostabilised avian β₁AR construct are characterised by selective labelling in combination with ¹⁹F and ¹³C NMR spectroscopy. These studies reveal agonist-stimulated β₁AR predominantly populates an active state, which in the receptor core and orthosteric pocket is comparable to conformations observed in static studies of β₁AR coupled to Gs protein. However, investigation of the receptor intracellular surface demonstrates the active state differs in conformation from known static structures, highlighting the importance of solution studies in structural investigations of GPCRs. Population of the active state is found to accelerate the association of G proteins with β₁AR in biolayer interferometry assays, relative to previously characterised pre-active and inactive states. These data therefore extend our understanding of the functional states populated in solution by the β₁AR. The impact of G protein coupling on the receptor conformations of the active state are assessed in Chapter 3. Further conformational rearrangements are observed upon binding partner coupling in NMR spectra. These changes are primarily localised to the intracellular surface of the receptor, supporting looser coupling of this region to orthosteric agonist efficacy information relative to the rest of the β₁AR. Extension of these studies to examine different G protein complexes at the β₁AR reveal only minor differences by solution NMR spectroscopy, whereas the kinetics of association to the receptor differ substantially between different G proteins. Importantly, these data suggest the agonist-bound solution active state acts as a kinetics-driven selectivity gate with differences in favourability of binding to different G proteins. The integrative investigation in Chapter 4 assesses how changes in the receptor conformational equilibria observed in the previous chapters could account for reductions in receptor activity. This work contrasts the influences on receptor signalling from the orthosteric agonist binding pocket or from other binding sites in the receptor, in order to characterise mechanisms of allosteric modulation at the β₁AR. NMR studies show that either orthosteric agonists with lower efficacies or mutations in a known allosteric pocket stabilise less active states in the receptor equilibria, which could account for reductions in G protein binding affinities and decreased downstream signalling. In contrast, binding of a small molecule modulator (AS408) into the same allosteric pocket decreases G protein coupling via a distinct mechanism. The AS408-bound receptor adopts an alternative conformation which differs from known states on the intracellular surface of β₁AR, whilst conformations in other regions of the receptor are influenced by both the orthosteric agonist and AS408. These investigations highlight the vital role of receptor plasticity in GPCR allostery and demonstrate the limitations of the conformational equilibrium model in understanding GPCR signalling. In summary, this thesis characterises the solution conformations of agonist-bound β₁AR and emphasises the impact of dynamics on GPCR signalling. 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