The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Matthew I. Gibson - One of the best experts on this subject based on the ideXlab platform.
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antifreeze Protein Mimetic metallohelices with potent ice recrystallization inhibition activity
Journal of the American Chemical Society, 2017Co-Authors: Daniel E Mitchell, Guy J Clarkson, Rebecca Ann Vipond, Peter Scott, Matthew I. GibsonAbstract:Antifreeze Proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure–property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze Protein Mimetics and provides insight into the origins of ice-growth inhibition.
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influence of block copolymerization on the antifreeze Protein Mimetic ice recrystallization inhibition activity of poly vinyl alcohol
Biomacromolecules, 2016Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:Antifreeze (glyco) Proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These Proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications—from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze Protein Mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain ...
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latent ice recrystallization inhibition activity in nonantifreeze Proteins ca2 activated plant lectins and cation activated antimicrobial peptides
Biomacromolecules, 2015Co-Authors: Daniel E Mitchell, Matthew I. GibsonAbstract:Organisms living in polar regions have evolved a series of antifreeze (glyco) Proteins (AFGPs) to enable them to survive by modulating the structure of ice. These Proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for bioMimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these Proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) Proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze Protein Mimetic macromolecules and may help in the development of synthetic mimics of AFPs.
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quantitative study on the antifreeze Protein Mimetic ice growth inhibition properties of poly ampholytes derived from vinyl based polymers
Biomaterials Science, 2014Co-Authors: Daniel E Mitchell, Mary Lilliman, Sebastian G Spain, Matthew I. GibsonAbstract:Antifreeze (glyco) Proteins (AF(G)Ps) from the blood of polar fish species are extremely potent ice recrystallization inhibitors (IRI), but are difficult to synthesise or extract from natural sources. Despite this challenge, materials which display IRI are appealing due to their ability to enhance cellular cryopreservation, for applications including regenerative and transplantation medicine. Here, poly(ampholytes), which contain a mixture of cationic and anionic side chains are quantitatively evaluated for their IRI activity. Poly(aminoethyl methacrylate), obtained by RAFT polymerization, is functionalised with succinic anhydride to generate the poly(ampholytes). The charge balance of the side chains is shown to be crucial, with only 50 : 50 mixtures having strong IRI activity, which also scales with molecular weight. This is the first example of a non-hydroxylated synthetic polymer with quantifiable IRI activity and raises questions about the mechanism of IRI, as the polymers have no obvious ice-binding motif. The ampholytic structure is shown to be transferable to carbohydrate-centred polymers with activity retained, but poly(betaines) are shown to be inactive.
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antifreeze glyco Protein Mimetic behavior of poly vinyl alcohol detailed structure ice recrystallization inhibition activity study
Biomacromolecules, 2013Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a bioMimetic surrogate for antifreeze(glyco)Proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze Proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure–activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is “switched on” when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains...
Thomas Congdon - One of the best experts on this subject based on the ideXlab platform.
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influence of block copolymerization on the antifreeze Protein Mimetic ice recrystallization inhibition activity of poly vinyl alcohol
Biomacromolecules, 2016Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:Antifreeze (glyco) Proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These Proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications—from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze Protein Mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain ...
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antifreeze glyco Protein Mimetic behavior of poly vinyl alcohol detailed structure ice recrystallization inhibition activity study
Biomacromolecules, 2013Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a bioMimetic surrogate for antifreeze(glyco)Proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze Proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure–activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is “switched on” when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains...
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Antifreeze (Glyco)Protein Mimetic behavior of poly(vinyl alcohol): Detailed structure ice recrystallization inhibition activity study
Biomacromolecules, 2013Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a bioMimetic surrogate for antifreeze(glyco)Proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze Proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure-activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important. © 2013 American Chemical Society.
Rebecca Notman - One of the best experts on this subject based on the ideXlab platform.
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influence of block copolymerization on the antifreeze Protein Mimetic ice recrystallization inhibition activity of poly vinyl alcohol
Biomacromolecules, 2016Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:Antifreeze (glyco) Proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These Proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications—from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze Protein Mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain ...
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antifreeze glyco Protein Mimetic behavior of poly vinyl alcohol detailed structure ice recrystallization inhibition activity study
Biomacromolecules, 2013Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a bioMimetic surrogate for antifreeze(glyco)Proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze Proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure–activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is “switched on” when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains...
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Antifreeze (Glyco)Protein Mimetic behavior of poly(vinyl alcohol): Detailed structure ice recrystallization inhibition activity study
Biomacromolecules, 2013Co-Authors: Thomas Congdon, Rebecca Notman, Matthew I. GibsonAbstract:This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a bioMimetic surrogate for antifreeze(glyco)Proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze Proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure-activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important. © 2013 American Chemical Society.
Daniel E Mitchell - One of the best experts on this subject based on the ideXlab platform.
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antifreeze Protein Mimetic metallohelices with potent ice recrystallization inhibition activity
Journal of the American Chemical Society, 2017Co-Authors: Daniel E Mitchell, Guy J Clarkson, Rebecca Ann Vipond, Peter Scott, Matthew I. GibsonAbstract:Antifreeze Proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure–property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze Protein Mimetics and provides insight into the origins of ice-growth inhibition.
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latent ice recrystallization inhibition activity in nonantifreeze Proteins ca2 activated plant lectins and cation activated antimicrobial peptides
Biomacromolecules, 2015Co-Authors: Daniel E Mitchell, Matthew I. GibsonAbstract:Organisms living in polar regions have evolved a series of antifreeze (glyco) Proteins (AFGPs) to enable them to survive by modulating the structure of ice. These Proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for bioMimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these Proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) Proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze Protein Mimetic macromolecules and may help in the development of synthetic mimics of AFPs.
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quantitative study on the antifreeze Protein Mimetic ice growth inhibition properties of poly ampholytes derived from vinyl based polymers
Biomaterials Science, 2014Co-Authors: Daniel E Mitchell, Mary Lilliman, Sebastian G Spain, Matthew I. GibsonAbstract:Antifreeze (glyco) Proteins (AF(G)Ps) from the blood of polar fish species are extremely potent ice recrystallization inhibitors (IRI), but are difficult to synthesise or extract from natural sources. Despite this challenge, materials which display IRI are appealing due to their ability to enhance cellular cryopreservation, for applications including regenerative and transplantation medicine. Here, poly(ampholytes), which contain a mixture of cationic and anionic side chains are quantitatively evaluated for their IRI activity. Poly(aminoethyl methacrylate), obtained by RAFT polymerization, is functionalised with succinic anhydride to generate the poly(ampholytes). The charge balance of the side chains is shown to be crucial, with only 50 : 50 mixtures having strong IRI activity, which also scales with molecular weight. This is the first example of a non-hydroxylated synthetic polymer with quantifiable IRI activity and raises questions about the mechanism of IRI, as the polymers have no obvious ice-binding motif. The ampholytic structure is shown to be transferable to carbohydrate-centred polymers with activity retained, but poly(betaines) are shown to be inactive.
Aashish Manglik - One of the best experts on this subject based on the ideXlab platform.
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structural insights into µ opioid receptor activation
Nature, 2015Co-Authors: Weijiao Huang, Aashish Manglik, A J Venkatakrishnan, Toon Laeremans, Evan N Feinberg, Adrian L Sanborn, Hideaki E Kato, Kathryn E Livingston, Thor S. Thorsen, Ralf C KlingAbstract:Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 A X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G Protein Mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-Protein-coupled receptors. X-ray crystallography and molecular dynamics simulations of the μ-opioid receptor reveal the conformational changes in the extracellular and intracellular domains of this G-Protein-coupled receptor that are associated with its activation. The μ-opioid receptor is a G-Protein-coupled receptor (GPCR) activated by various analgesics, endogenous endorphins and drugs of abuse such as heroin and opium. Our understanding of the mechanism by which agonist binding leads to recognition, coupling, and activation of a particular G Protein subtype is incomplete. In two papers in this issue of Nature, the authors used X-ray crystallography, molecular dynamics simulations, and NMR spectroscopy to probe the structural basis for receptor activation. As well as revealing the conformational changes in the extracellular and intracellular domains of this GPCR associated with receptor activation, these studies help explain why the allosteric coupling between the agonist-binding pocket and the cytoplasmic G-Protein-coupling interface of this receptor is relatively weak.
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Propagation of conformational changes during μ-opioid receptor activation
Nature, 2015Co-Authors: Remy Sounier, Aashish Manglik, Weijiao Huang, Toon Laeremans, Jan Steyaert, Brian Kobilka, Héléne Démèné, Sebastien GranierAbstract:NMR spectroscopy reveals the conformational changes of the μ-opioid receptor that are associated with receptor activation, helping to explain why the allosteric coupling between the agonist-binding pocket and the cytoplasmic G-Protein-coupling interface of this receptor is relatively weak. The μ-opioid receptor is a G-Protein-coupled receptor (GPCR) activated by various analgesics, endogenous endorphins and drugs of abuse such as heroin and opium. Our understanding of the mechanism by which agonist binding leads to recognition, coupling, and activation of a particular G Protein subtype is incomplete. In two papers in this issue of Nature , the authors used X-ray crystallography, molecular dynamics simulations, and NMR spectroscopy to probe the structural basis for receptor activation. As well as revealing the conformational changes in the extracellular and intracellular domains of this GPCR associated with receptor activation, these studies help explain why the allosteric coupling between the agonist-binding pocket and the cytoplasmic G-Protein-coupling interface of this receptor is relatively weak. µ-Opioid receptors (µORs) are G-Protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive^ 1 and agonist-induced active states (Huang et al. , ref. 2 ) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G Protein Mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G Protein, are almost completely dependent on the presence of both the agonist and the G Protein Mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-Protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor^ 3 . Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G Protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G Protein and intracellular loop 1 and/or helix 8 may be involved in G-Protein coupling specificity, as has been suggested for other family A G-Protein-coupled receptors.
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Propagation of conformational changes during μ-opioid receptor activation
Nature, 2015Co-Authors: Remy Sounier, Aashish Manglik, Weijiao Huang, Toon Laeremans, Camille Mas, Jan Steyaert, Brian Kobilka, Héléne Démèné, Sebastien GranierAbstract:µ-Opioid receptors (µORs) are G-Protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G Protein Mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G Protein, are almost completely dependent on the presence of both the agonist and the G Protein Mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-Protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G Protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G Protein and intracellular loop 1 and/or helix 8 may be involved in G-Protein coupling specificity, as has been suggested for other family A G-Protein-coupled receptors.
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structural insights into the dynamic process of β2 adrenergic receptor signaling
Cell, 2015Co-Authors: Aashish Manglik, Matthieu Masureel, Christian Altenbach, Zhongyu Yang, Daniel Hilger, Michael T Lerch, Tong Sun Kobilka, Foon Sun Thian, Wayne L Hubbell, Scott R ProsserAbstract:G-Protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular Proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G Proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G Protein or an intracellular G Protein Mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-Protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.
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Activation and allosteric modulation of a muscarinic acetylcholine receptor
Nature, 2013Co-Authors: Andrew C. Kruse, Aashish Manglik, Aaron M. Ring, Jianxin Hu, Kelly Hu, Katrin Eitel, Harald Hübner, Els Pardon, Celine Valant, Patrick M. SextonAbstract:The structures of many G-Protein-coupled receptors (GPCRs), including members of the class B and class F families, are now available but little is known about the transitions from the inactive to active states. In this study the authors solve the X-ray crystal structures of the human M2 muscarinic acetylcholine receptor in the active state bound to the agonist iperoxo alone and in combination with LY2119620, a positive allosteric modulator. The structures reveal that the activated M2 receptor has an extremely small orthosteric binding site, with LY2119620 'sitting' right on top of the agonist. The authors also note that the region that makes up the allosteric site in the inactive conformation of the M2 receptor is too large to bind to LY2119620; this means that the extracellular region needs to contract (by binding to the high-affinity agonist) before LY2119620 can bind to the allosteric site. This GPCR is essential for the physiological control of cardiovascular function, cognition, and pain perception, and since allosteric sites are less conserved in sequence and structure than the orthosteric binding site, the hope is that ligands that bind to allosteric sites could be turned into drugs that selectively interact with only one of the five muscarinic receptor subtypes. Despite recent advances in crystallography and the availability of G-Protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β_2 adrenergic receptor (β_2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-Protein Mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β_2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors. Very little is known about how a G-Protein-coupled receptor (GPCR) transitions from an inactive to an active state, but this study has solved the X-ray crystal structures of the human M2 muscarinic acetylcholine receptor bound to a high-affinity agonist in an active state and to a high-affinity agonist and a small-molecule allosteric modulator in an active state; the structures provide insights into the activation mechanism and allosteric modulation of muscarinic receptors.
