Freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques reveal the distribution of states and their interconversion rates along the reaction pathway of a ...bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD). The crystal structure of apo-FAcD exhibits asymmetry around the dimer interface and cap domain, priming one protomer for substrate binding. This asymmetry is dynamically averaged through conformational exchange on a millisecond time scale. During catalysis, the protomer conformational exchange rate becomes enhanced, the empty protomer exhibits increased local disorder, and water egresses. Computational studies identify allosteric pathways between protomers. Water release and enhanced dynamics associated with catalysis compensate for entropic losses from substrate binding while facilitating sampling of the transition state. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.
Abstract Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19 F NMR is used to delineate the effects of cations on ...functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.
G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ...ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.
Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define ...these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A
receptor (A
R), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a "gatekeeper" for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.
► A new fucosylated polysaccharide sulfate was purified from a special sea cucumber, Acaudina molpadioidea. ► Two derivatives were prepared from native polysaccharide and their anticoagulant ...activities were investigated. ► Sulfated derivative showed a stronger anticoagulant ability than native polysaccharide and carboxymethylated derivative.
A fucosylated polysaccharide sulfate, AMP-2, was purified by DEAE-Sepharose Fast Flow and Sephadex G-100 columns in successive steps from a special sea cucumber in southeastern China. HPLC and cellulose acetate membrane electrophoresis experiments confirmed AMP-2 was a homogenous carbohydrate with a relative molecular weight of ca. 2.4×104Da, and methylation analysis indicated that polysaccharide was composed of 1-substituted-Galp, 1,4-disubstituted-GalNp, 1,2-disubstituted-FucSp, 1,4,6-trisubstituted-Glcp in a molar ratio of ca. 0.5:2.0:1.0:3.0, together with a small amount of different substituted Manp. Sulfated derivative and carboxymethylated derivative were prepared using dry pyridine and chlorosulfonic acid, and chloroacetic acid, respectively. Anticoagulant activities in vitro investigation showed that sulfated derivative showed a stronger ability than native polysaccharide and carboxymethylated derivative, which might be caused by their different percentages and types of functional groups in their structures.
•Two derivatives, TAPA1-ac and TAPA1-deac, were prepared by chemical modification.•Chemical modifications were proved to occur by spectra and calculational results.•Immunostimulating activity of ...TAPA1, TAPA1-deac and TAPA1-ac, were determined.•The relationship between the group O-Ac and activities were preliminarily investigated.
TAPA1 was previously isolated from Tremella aurantialba fruiting bodies. In this paper, an acetylated derivative (TAPA1-ac) and a deactylated derivative (TAPA1-deac) of TAPA1 were prepared, and their characterization and immunostimulating activities were reported. Acetylation and deacetylation were found to occur actually by FT-IR and NMR spectra, together with calculational results. The degree of substitution (DS) of acetyl groups in TAPA1-ac was 0.23 and the content was 5.82%, which was higher than those of TAPA1 (0.03% and 0.70%, respectively) and TAPA1-deac (all were zero). Compared with TAPA1, TAPA1-ac showed significant immunostimulation effects on mouse spleen lymphocytes (MSLs) proliferation and nitric oxide (NO) production by macrophages RAW264.7, whereas TAPA1-deac showed markedly lower effects. These findings seemed to suggest that immunostimulating activities, including MSLs stimulation activity and NO production potency, might relate to the DS and content of acetyl groups, indicating that acetylation of TAPA1 was an effective way of enhancing immuno-stimulating activities.
► First report on oxalate successfully limited postharvest deterioration of mangoes. ► Oxalate increased activity of POD and PPO, and total phenolic content in mangoes. ► These effects of oxalate ...might be involved in induced resistance of mango fruit.
Effects of oxalate on the incidence of decay and ripening in mango fruit, and its physiological effects on the peel and flesh of mango were investigated after mango fruit (
Mangifera indica L.) were dipped in different oxalate solutions for 10
min and then stored at 25
°C. Oxalate application decreased the incidence of decay and delayed the ripening process in mango fruit during storage. Potassium oxalate treatment resulted in increased activities of peroxidase (POD) in both the peel and the flesh and polyphenol oxidase (PPO) in the peel, without activation of phenylalanine ammonia-lyase activity, and elevated total phenolic content in the peel. The physiological effects of oxalate in increasing activities of POD and PPO and elevating total phenolic level could be involved in induced resistance of mango fruit against postharvest disease. Oxalate application could be a promising method to suppress postharvest deterioration and extend the useful shelf-life of mangoes.
In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in ...insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded.
expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can't compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of
derived GPCR along with Gα and Gβγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.
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