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G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs (1-5) have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the [β.sub.2] adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures., In the ligand-free basal state, GPCRs exist in an equilibrium of conformations (6). Ligand binding modulates receptor function by stabilizing different intramolecular interactions and establishing a new conformational equilibrium. Activating [...]

A synthetic process for the generation of large, diverse libraries of unnatural carbamate oligomers was designed utilizing the 'one bead, one peptide' technique. One acyclic and two cyclic libraries were generated and screened for binding to the integrin GPIIb/IIIa by employing a set of 27 structurally and functionally diverse monomers. Several types of oligocarbamate ligands for GPIIb/IIIa were found and two cyclic ligands have activities that are within a factor of 3 of kistrin, a snake venom protein that effectively prevents platelet aggregation.

We report NMR and kinetic studies of antibody AZ-28, which was generated against the diaryl-substituted cyclohexanol derivative 3 and catalyzes the oxy-Cope rearrangement of the corresponding hexadiene 1 to aldehyde 2 (Braisted, A. C., Schultz, P. G. J. Am. Chem. Soc. 1994, 116, 2211-2212). Conformational studies of free substrate and the antibody - substrate complex using transferred-NOE experiments demonstrate that the antibody binds the substrate in a cyclic conformation, consistent with the chair-like geometry of 3. In contrast, free substrate adopts an extended conformation in solution. In spite of the conformational restriction of the bound substrate revealed by NMR, the temperature dependence of the reaction indicates that the antibody functions primarily by lowering [Mathematical Expression Omitted], offsetting a decrease in [Mathematical Expression Omitted]; the values of [Mathematical Expression Omitted] and [Mathematical Expression Omitted] are 15.4 [+ or -] 2.4 kcal/mol and -23 [+ or -] 8 cal/mol K, respectively. A secondary kinetic isotope effect ([k.sub.cat]H/[k.sub.cat]D) of 0.61 [+ or -] 0.1 was measured for substrate 23 in which both allylic termini are dideuterated, indicating that the chemical rearrangement step is wholly or partially rate limiting under saturating conditions. The magnitude of this secondary isotope effect is consistent with a significant degree of bond formation between C1 and C6 of the substrate in the transition state. These results, together with the recently reported three-dimensional crystal structure for the antibody-hapten 3 complex (Ulrich, H.; Mundroff, E.; Santarsiero, B. D.; Driggers, E. M.; Stevens, R. C.; Schultz, P. G. Nature 1997, 389, 271-275), provide a detailed mechanistic model for this antibody - catalyzed reaction.

The bicyclic amide, hexahydroindol-4-one 3,6-diacid and some of its ester derivatives were attached to the peptide EALAKA-NH2. Their effect on the conformation was investigated in aqueous solution by circular dichroism and nuclear magnetic resonance. Amide alogs were observed to have a minimal effect on the appended peptide while, the ester-linked template in S,S-9-O induces a helical character at 20 degrees celsius and even at 70 degrees celsius.

Rapamycin is an important immunosuppressant, a possible anticancer therapeutic and widely used research tool. A study was conducted to investigate the affinities involved in the formation of the FKBP.rapamycin.FRB complex using fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. The result shows that the protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

Results indicate that the solution structure of the frenolicin acyl carrier protein exhibits three major alpha-helices and three short helices in intervening loops are packed in a bundle. Data suggest atomic rmsd values of 0.49 plus or minus 0.09 and 0.91 plus or minus 0.08 angstrom for backbone and hydrogen atoms, respectively.

Agriculturally-driven land transformation is increasing globally. Improving phosphorus (P) use efficiency to sustain optimum productivity in diverse ecosystems, based on knowledge of soil P dynamics, is also globally important in light of potential shortages of rock phosphate to manufacture P fertilizer. We investigated P chemical speciation and P cycling with solution 31P nuclear magnetic resonance, P K-edge X-ray absorption nearedge structure spectroscopy, phosphatase activity assays, and shotgun metagenomics in soil samples from long-term agricultural fields containing four different land-use types (native and tame grasslands, annual croplands, and roadside ditches). Across these land use types, native and tame grasslands showed high accumulation of organic P, principally orthophosphate monoesters, and high acid phosphomonoesterase activity but the lowest abundance of P cycling genes. The proportion of inositol hexaphosphates (IHP), especially the neo-IHP stereoisomer that likely originates from microbes rather than plants, was significantly increased in native grasslands than croplands. Annual croplands had the largest variances of soil P composition, and the highest potential capacity for P cycling processes based on the abundance of genes coding for P cycling processes. In contrast, roadside soils had the highest soil Olsen-P concentrations, lowest organic P, and highest tricalcium phosphate concentrations, which were likely facilitated by the neutral pH and high exchangeable Ca of these soils. Redundancy analysis demonstrated that IHP by NMR, potential phosphatase activity, Olsen-P, and pH were important P chemistry predictors of the P cycling bacterial community and functional gene composition. Combining chemical and metagenomics results provides important insights into soil P processes and dynamics in different land-use ecosystems. [ABSTRACT FROM AUTHOR]

Experiments were performed on 13D6.1, an antibody elicited against phosphonate diester 4 and catalyzes the transesterification of ester 2a to alcohol 1 which yielded an effective molarity of 27000M. Transferred nuclear Overhauser enhancement spectroscopy was performed and revealed the molecular conformation of the Michaelis complex of 13D6.1. The findings suggested the effectiveness of phosphonates and phosphonamidates in generating antibodies.