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Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases. [ABSTRACT FROM AUTHOR]

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the [Pah.sup.enu2/enu2] PKU mouse model in short-and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically effective PAl. (Av, Anabaena variabilis) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of effective protein therapeutics. A PEG-Av-p.C503S/p.C565S-PAL effectively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a dose-and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG-Av-p.C503S/p.C565S-PAL as a treatment for PKU are underway. enzyme substitution therapy | hyperphenylalaninemia | long-term efficacy | PKU mouse model | injectable nonmammalian protein

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-[Angstrom] crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs. anandamide | crystal structure | endocannabinoid | fatty acid amides | hydrolase

Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85- [Angstrom] crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site,and the presence of an intriguing, ubiquitin-like N-terminal domain. To gain insight into the active- site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin--aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs. membrane-associated protease | ubiquitin-like domain

In the Joint Center for Structural Genomics, one-dimensional (1D) [sup.1]H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D [sup.1]H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1D [sup.1]H NMR spectra, the proteins are classified into four groups, 'A' to ''D.' A-type proteins are candidates for structure determination by NMR or crystallography; 'B'-type are earmarked for crystallography; 'C' indicates folded globular proteins with broadened line shapes; and 'D' are nonglobular, 'unfolded' polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D [sup.1]H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C- and D- type proteins. crystallographic resolution | NMR structure | x-ray structure | unfolded proteins

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (B[H.sub.4])]. Several hypotheses have been put forward to explain B[H.sub.4] responsiveness, but the molecular basis for the corrective effect(s) of B[H.sub.4] has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with B[H.sub.4]-responsive mutations (F39L, 165T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the B[H.sub.4] responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased [K.sub.m] (B[H.sub.4]) and reduced cooperativity of substrate binding, but no decoupling of cofactor (B[H.sub.4]) oxidation. For some, B[H.sub.4] seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo. Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that B[H.sub.4] appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen/high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins, When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-[Angstrom] resolution. The antibody-bound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.

Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-[Angstrom] and 1.9-[Angstrom] resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.

Comprehensive modeling of a whole cell requires an integration of vast amounts of information on various aspects of the cell and its parts. To divide and conquer this task, we introduce Bayesian metamodeling, a general approach to modeling complex systems by integrating a collection of heterogeneous input models. Each input model can in principle be based on any type of data and can describe a different aspect of the modeled system using any mathematical representation, scale, and level of granularity. These input models are 1) converted to a standardized statistical representation relying on probabilistic graphical models, 2) coupled by modeling their mutual relations with the physical world, and 3) finally harmonized with respect to each other. To illustrate Bayesian metamodeling, we provide a proof-of-principle metamodel of glucose-stimulated insulin secretion by human pancreatic ß-cells. The input models include a coarse-grained spatiotemporal simulation of insulin vesicle trafficking, docking, and exocytosis; a molecular network model of glucose-stimulated insulin secretion signaling; a network model of insulin metabolism; a structural model of glucagon-like peptide-1 receptor activation; a linear model of a pancreatic cell population; and ordinary differential equations for systemic postprandial insulin response. Metamodeling benefits from decentralized computing, while often producing a more accurate, precise, and complete model that contextualizes input models as well as resolves conflicting information. We anticipate Bayesian metamodeling will facilitate collaborative science by providing a framework for sharing expertise, resources, data, and models, as exemplified by the Pancreatic ß-Cell Consortium. [ABSTRACT FROM AUTHOR]

X-ray and neutron diffraction studies of the absolute configuration of (+)-neopentyl-1-d alcohol reveal that the neopentyl phthalate molecules have an S configuration. The yeast system transports hydrogen to the re face of the carbonyl group to mediate aldehyde reduction. The agreement factors at 100 K and at room temperature for 960 and 2768 reflections are 0. 144 and 0. 073, respectively.

Aspartate transcarbamoyltransferase (ATCase) is an allosteric enzyme which is inhibited by pyrimidine derivatives, and activated by purine derivatives. A model for the heterotropic regulation of ATCase at near atomic resolution is presented, based on recent structural and biochemical data. The features of the model explain the pattern of action of CTP and ATP on the tensed or relaxed states of the enzyme. This model is called the nucleotide perturbation mechanism.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A2A adenosine receptor (A2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19F-NMR experiments with probes at the A2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A2AAR and a constitutively active mutant established relations between the 19F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant. [ABSTRACT FROM AUTHOR]

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17 [degrees] C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 [Angstrom], 1.6 [Angstrom], and 2.0 [Angstrom], respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the [Alpha]/[Beta] hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxy-tryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femto-second crystallography with an X-ray free-electron laser. The anti-body binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCRtargeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. [ABSTRACT FROM AUTHOR]

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216- AcrF mutation leads to conformational changes in key active site residues--both in the free enzyme and upon formation of the acylenzyme intermediate--that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities. [ABSTRACT FROM AUTHOR]

In the Joint Center for Structural Genomics, one-dimensional (1 D) 1H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D 1H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1ID 1H NMR spectra, the proteins are classified into four groups, "A" to "D" A-type proteins are candidates for structure determination by NMR or crystallography; "B"-type are earmarked for crystallography; "C" indicates folded globular proteins with broadened line shapes; and "D" are nonglobular, "unfolded" polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D 1H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C-and D- type proteins. [ABSTRACT FROM AUTHOR]

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-Å resolution. The antibodybound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration, The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction. [ABSTRACT FROM AUTHOR]

The recently developed double-click reaction sequence [G. Meng et al. ,  Nature 574 , 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

Characterizing relationships between Zn 2+ , insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn 2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn 2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn 2+ and Ca 2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn 2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn 2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A 2A adenosine receptor (A 2A AR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19 F-NMR experiments with probes at the A 2A AR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A 2A AR and one inactive substate that is not seen in complexes with agonists, suggesting that A 2A AR activation includes both induced fit and conformational selection mechanisms. Comparison of A 2A AR and a constitutively active mutant established relations between the 19 F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant., Competing Interests: The authors declare no conflict of interest.

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

The structure of the dynorphin (1-13) peptide (dynorphin) bound to the human kappa opioid receptor (KOR) has been determined by liquid-state NMR spectroscopy. (1)H and (15)N chemical shift variations indicated that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM KOR. Radioligand binding indicated an intermediate-affinity interaction, with a Kd of ∼200 nM. Transferred nuclear Overhauser enhancement spectroscopy was used to determine the structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly disordered, the central part of the peptide from L5 to R9 to form a helical turn, and the C-terminal segment from P10 to K13 to be flexibly disordered in this intermediate-affinity bound state. Combining molecular modeling with NMR provided an initial framework for understanding multistep activation of a G protein-coupled receptor by its cognate peptide ligand.

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein-detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.