Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

Evaluation of reproducible and transparent research practices in pulmonology.

BACKGROUND: Study reproducibility is valuable for validating or refuting results. Provision of reproducibility indicators, such as materials, protocols, and raw data in a study improve its potential for reproduction. Efforts to reproduce noteworthy studies in the biomedical sciences have resulted in an overwhelming majority of them being found to be unreplicable, causing concern for the integrity of research in other fields, including medical specialties. Here, we analyzed the reproducibility of studies in the field of pulmonology. METHODS: 500 pulmonology articles were randomly selected from an initial PubMed search for data extraction. Two authors scoured these articles for reproducibility indicators including materials, protocols, raw data, analysis scripts, inclusion in systematic reviews, and citations by replication studies as well as other factors of research transparency including open accessibility, funding source and competing interest disclosures, and study preregistration. FINDINGS: Few publications included statements regarding materials (10%), protocols (1%), data (15%), and analysis script (0%) availability. Less than 10% indicated preregistration. More than half of the publications analyzed failed to provide a funding statement. Conversely, 63% of the publications were open access and 73% included a conflict of interest statement. INTERPRETATION: Overall, our study indicates pulmonology research is currently lacking in efforts to increase replicability. Future studies should focus on providing sufficient information regarding materials, protocols, raw data, and analysis scripts, among other indicators, for the sake of clinical decisions that depend on replicable or refutable results from the primary literature.

Recent crustal uplift in yellowstone national park.

Comparison of precise leveling measurements made in 1923 with those made in 1975, 1976, and 1977 reveals that the 600,000-year-old Yellowstone caldera is being uplifted relative to its surroundings. Maximum relative uplift since 1923 is in excess of 700 millimeters-about 14 millimeters vertically per year. The most likely cause of this rapid and unusually large surface deformation is a recent influx of molten or partially molten materials to a location within the crust beneath Yellowstone National Park.

Phosphoaspartates in bacterial signal transduction.

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

Conformational studies of the 'RGD' containing peptide echistatin and close analogs by circular dichroism and fluorescence.

Echistatin, one of the smallest and most active natural disintegrins, and its [Trp13]echistatin, [Trp27]echistatin, [Phe13,Trp31]echistatin analogs have been investigated by far-UV circular dichroism spectroscopy and fluorescence spectroscopy. All analogs inhibited ADP-stimulated platelet aggregation with EC50 values between 30 and 50 nM. The analogs were related closely, both in the CD spectral properties, characteristic of turn conformations, and in the location of isodichroic points connected to conformational transitions upon temperature increase. The low fluorescence quantum yield for Trp13 of 0.018, which could be enhanced 2.7-fold by DTT reduction of the peptide, is ascribed to a close proximity of this Trp13 residue to a disulfide bond. Calculation of the efficiency of fluorescence resonance energy transfer (FRET) yielded distances of 11.5 +/- 0.8 A for Tyr31-Trp27 in [Trp27]echistatin, and more than 15 A for Tyr31-Trp13 in [Trp13]echistatin, in good agreement with the structure of echistatin deduced from earlier NMR-molecular modeling studies. Both Trp13 and Trp27 in the respective analogs were quenched effectively by acrylamide with bimolecular quenching constants of 3.36 x 10(9) M-1 s-1 and 3.72 x 10(9) M-1 s-1, respectively. Iodide anion had negligible quenching effect on Trp13, despite high exposure of this residue to water, but was only 2-fold less efficient than acrylamide in quenching Trp27 fluorescence. Steady-state fluorescence anisotropy data, together with mean fluorescence lifetimes of 1.25 ns for Trp13 and 3.84 ns for Trp27 derived from full fluorescence lifetime decay analyses, yielded long rotational relaxation times of 1.39 +/- 0.18 and 1.35 +/- 0.17 ns, respectively, for these residues comparable to the expected overall rotation time of the peptides. The 'RGD'-containing loop appears to be restricted in movement on the nanosecond timescale with respect to the compact core of the peptide.

Structural characterization of myo-inositol monophosphatase from bovine brain by secondary structure prediction, fluorescence, circular dichroism and Raman spectroscopy.

Structural aspects of myo-inositol monophosphatase were examined by spectroscopic techniques and empirical prediction methods. The enzyme belongs to the alpha/beta class of proteins, with approx. 33% alpha-helix and 29% beta-sheet, as shown by circular dichroism (CD), Raman spectroscopy and prediction based on the amino-acid sequence. The Raman spectrum also suggests that the three tryptophan residues in myo-inositol monophosphatase are not exposed to solvent. This was confirmed by a blue shift of 25 nm in the fluorescence emission spectrum, as compared to tryptophan in water, and by quenching studies with acrylamide. The enzyme shows a transition temperature of 87 degrees C for the CD signal at 222 nm. This remarkable heat stability is not due to the presence of disulfide bonds, since both the Raman spectrum and chemical modification studies clearly indicate that all six cysteine residues are in the reduced state.

Effects of [1-N alpha-trinitrophenylhistidine, 12-homoarginine]glucagon on cyclic AMP levels and free fatty acid release in isolated rat adipocytes.

[1-N alpha-Trinitrophenylhistidine, 12-homoarginine]glucagon (THG) stimulated, in a concentration-dependent fashion, lipolysis (2-fold) and cyclic AMP accumulation (50% over basal) in isolated rat adipocytes, but was much less effective than glucagon, which stimulated lipolysis 4-fold and cyclic AMP accumulation 10-15-fold. THG displaced to the right the concentration-response curves for glucagon and diminished in a concentration-dependent fashion the effects of a fixed concentration of glucagon. The data indicate that THG is a mixed agonist-antagonist (partial agonist) in isolated rat fat cells.

Metabolic effects and cyclic AMP levels produced by glucagon, (1-N alpha-Trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes.

[1-N alpha-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.

Endothelins--from receptors to medicine.

Since the discovery of endothelins, peptides with exceptional vasoconstrictor potency that were originally suggested to act by causing the opening of Ca2+ channels, it has emerged that these agents are important in intercellular communication in many tissues. They exert their effects through G protein-coupled receptors, of which two classes have been cloned. Robert Miller, John Pelton and John Huggins review the progress made towards a molecular understanding of ligand recognition by endothelin receptors. Receptor-selective agonists and antagonists have emerged from attempts to understand the three-dimensional structure of the endothelin pharmacophore, from structure-activity studies and from rapid-screening programmes. From the nature of the secretion and action of endothelins, it would seem that these peptides are involved in long-term changes rather than in acute responses to stimuli, and that they are likely to be important in a number of pathological states. Evidence suggests that receptor antagonists with appropriate affinity and selectivity may be useful in the treatment of conditions as diverse as hypertension, ulcerogenesis and ciclosporin toxicity.

The structure and specificity of endothelin receptors: their importance in physiology and medicine.

In addition to involvement in vascular endothelium-smooth muscle communication, the secretion of and receptors for, endothelins are widely distributed. Two cloned receptor subtypes are G-protein-coupled to several intracellular messengers, predominantly inositol phosphates. From a knowledge of structure-activity relationships and peptide conformations, details of receptor architecture and selective agents, including nonpeptides and antagonists, have been discovered. From the nature of the actions of endothelins, receptor distributions (including CNS) and plasma levels, it is concluded that they are paracrine factors normally involved in long-term cellular regulation, but which may be important in several pathologies, many of which are stress-related.

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

NMR structure of activated CheY.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

Refined solution structure and dynamics of the DNA-binding domain of the heat shock factor from Kluyveromyces lactis.

The solution structure of the 92 residue (11 kDa) winged helix-turn-helix DNA-binding domain from the kluyveromyces lactis heat shock factor was refined using a total of 932 NOE, 35 phi, 25 chi 1, 5 chi 2 and 44 hydrogen bond restraints. The overall root-mean-square deviation for structured regions was 0.75(+/- 0.15) A. The three-helix bundle and four-stranded beta-sheet are well defined with rmsd of 0.53(+/- 0.10) A and 0.60(+/- 0.17) A, respectively. Helix H2 is underwound and bent near Pro45. The angle between helix H2 and the proposed recognition helix H3 is 96(+/- 6) degrees. Detailed comparisons are made with the X-ray structure of this protein as well as other structural studies on HSF. Overall, the results are consistent with the earlier studies. Differences are related to protein-protein interactions in the crystal and dynamics in solution. Backbone dynamics was investigated via 15N relaxation. The average R1, R2 and NOE values for residues in segments of secondary structure were 1.9(+/- 0.9) s-1, 7.8(+/- 0.9) s-1 and 0.81(+/- 0.05), respectively. The correlation time based on these data was 5.6(+/- 0.4) ns. Motional order parameters were calculated by fitting the relaxation data to one of three models. Low-order parameters were found for residues that comprise the turn between helices H2 and H3 (residues Lys49 to Phe53), and most strikingly, the 16 residue wing (residues Val68 to Arg83). These data are consistent with the lack of long-range NOEs identified in these regions. The data provide a basis for comparison with results of the protein-DNA complex. The relationship between structure and function is discussed.

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.

Solution structure of the DNA-binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The solution structure of the 92-residue DNA-binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three-dimensional (3D) triple resonance, 1H-13C-13C-1H total correlation spectroscopy, and 15N-separated total correlation spectroscopy-heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side-chain 1H, 15N, and 13C resonances. Five-hundred eighty-three NOE constraints identified in 3D 13C- and 15N-separated NOE spectroscopy (NOESY)-heteronuclear multiple quantum correlation spectra and a 4-dimensional 13C/13C-edited NOESY spectrum, along with 35 phi, 9 chi 1, and 30 hydrogen bond constraints, were used to calculate 30 structures by hybrid distance geometry/stimulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3-helix bundle packs against a small 4-stranded antiparallel beta-sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 +/- 0.19 A with respect to the average structure. The topology is analogous to that of the C-terminal domain of the catabolite gene activator protein and appears to be in the helix-turn-helix family of DNA-binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X-ray structures of 1.77 +/- 0.20 A. Several differences were identified some of which may be due to protein-protein interactions in the crystal.

Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy.

The structure and dynamics of the N-terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA-binding domain as a structural reference, we show that the protein backbone of the N-terminal activation domain undergoes rapid, large-amplitude motions and is therefore unstructured. Difference CD data also show that the N-terminal activation domain remains random-coil, even in the presence of DNA. Implications for a "polypeptide lasso" model of transcriptional activation are discussed.

Conformational study of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], an endothelin-A receptor-selective antagonist.

The conformation of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], (BQ123), an endothelin-A receptor-selective antagonist, has been studied in 20% acetonitrile in water by CD and NMR spectroscopy. CD studies showed the peptide adopted a similar, constrained conformation in both water alone and 20% acetonitrile in water. NMR spectra showed the proline residue to be in the trans conformation and 2 of the NH protons to exchange slowly with the solvent, indicating hydrogen bonding. Structural constraints derived from the NMR spectra were used to define the conformation in molecular dynamics simulations. A single backbone conformation is observed for the cycle, comprising a beta type II turn and a gamma' turn.

1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure.

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.

Complete 1H and 13C assignment of Lys and Leu sidechains of staphylococcal nuclease using HCCH-COSY and HCCH-TOCSY 3D NMR spectroscopy.

Complete proton and carbon sidechain assignments are reported for 22 lysine and 11 leucine residues in staphylococcal nuclease, an enzyme with 149 residues. These assignments are readily obtained in a direct manner from the correlations observed in the 3D HCCH-COSY and HCCH-TOCSY spectra and the known protein backbone assignments. These assignments open the way to detailed studies of the sidechain structure and dynamics at the active site, in the hydrophobic core and on the surface of the protein.

In vitro processing of rANF7-28-NH2 by rat kidney homogenates.

The major rat kidney in vitro degradation products of the rat atrial natriuretic factor analog, H-Cys7-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly -Cys-Asn-Ser-Phe-Arg-Tyr28-NH2 (with a Cys27-Cys23 disulfide bridge), have been identified. The degradation products are the C-terminal modified compounds rANF7-27, rANF7-26, and rANF7-25.