RESUMO
NMR chemical shift assignments are reported for backbone (15N, 1H) and partial side chain (13Cα and ß, side chain 1H) atoms of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of hydrolyzing phosphorus - fluorine bonds in a variety of toxic organophosphorus compounds. Analysis of residues lining the active site of DFPase highlight a number of residues whose chemical shifts can be used as a diagnostic of binding and detection of organophosphorus compounds.
Assuntos
Loligo , Hidrolases de Triester Fosfórico , Animais , Loligo/metabolismo , Ressonância Magnética Nuclear Biomolecular , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismoRESUMO
We report a critical advance in the generation and characterization of peptoid hetero-oligomers. A library of sub-monomers with amine and carboxylate side-chains are combined in different sequences using microwave-assisted synthesis. Their sequence-structure propensity is confirmed by circular dichroism, and conformer subtypes are enumerated by NMR. Biasing the ψ-angle backbone to trans (180°) in Monte Carlo modelling favors i to i + 3 naphthyl-naphthyl stacking, and matches experimental ensemble distributions. Taken together, high-yield synthesis of heterooligomers and NMR with structure prediction enables rapid determination of sequences that induce secondary structural propensities for predictive design of hydrophilic peptidomimetic foldamers and their future libraries.
RESUMO
Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.
Assuntos
Aminas/química , Elastina/química , Metais/química , Compostos Orgânicos/química , Polímeros/química , Piridinas/química , Solventes/química , Eletroforese em Gel de Poliacrilamida , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Multimerização Proteica , Espectroscopia de Prótons por Ressonância Magnética , Solubilidade , Espectrofotometria UltravioletaRESUMO
Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.
Assuntos
Produtos do Gene tat/química , Ensaios de Triagem em Larga Escala , Fosfotransferases/química , beta-Lactamases/química , Substituição de Aminoácidos , Aspergillus niger/química , Aspergillus niger/enzimologia , Sítios de Ligação , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Produtos do Gene tat/metabolismo , HIV/química , HIV/metabolismo , Modelos Moleculares , Mutação , Biblioteca de Peptídeos , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Solubilidade , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-Híbrido , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Two organometallic 1D infinite coordination polymers and two organometallic monometallic complexes of thorium diazide have been synthesized and characterized. Steric control of these self-assembled arrays, which are dense in thorium and nitrogen, has also been demonstrated: infinite chains can be circumvented by using steric bulk either at the metallocene or with a donor ligand in the wedge.
RESUMO
Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in Escherichia coli. Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one design incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. This technique can be extended to improve a wide variety of designed pathways.
Assuntos
Escherichia coli/metabolismo , Glucose/análogos & derivados , Biocatálise , Biomassa , Estabilidade Enzimática , Escherichia coli/enzimologia , Glucose/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Biologia SintéticaRESUMO
The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.
Assuntos
Celulose/química , Proteínas Fúngicas/química , Glucose-6-Fosfato/química , Glucose/análogos & derivados , Lipomyces/química , Fosfotransferases/química , Biocatálise , Biomassa , Celulose/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Glucose/química , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Cinética , Lipomyces/enzimologia , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Modelos Moleculares , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Human carbonic anhydrase II (HCA II) uses a Zn-bound OH(-)/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa of this residue, NMR spectroscopic measurements of [(13)C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.
Assuntos
Anidrases Carbônicas/química , Cristalografia por Raios X/métodos , Espectroscopia de Ressonância Magnética/métodos , Nêutrons , Tirosina/química , Catálise , Domínio Catalítico , Cátions , Enzimas/química , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Conformação Proteica , Prótons , Eletricidade Estática , Água/químicaRESUMO
(13)C-Labeled levoglucosan has been synthesized and purified in good yield, and on the gram scale in one step from commercially available (13)C glucose. This one-step protocol uses 2-chloro-1,3-dimethylimidazolinium chloride that serves to selectively activate the anomeric carbon toward substitution reactions. The labeled glucose is then smoothly converted to the anhydroglucose. Purification is efficiently achieved on large scale by chromatography on silica gel.
Assuntos
Glucose/análogos & derivados , Isótopos de Carbono/química , Técnicas de Química Sintética/métodos , Glucose/síntese química , Glucose/isolamento & purificaçãoRESUMO
Lewis bases react with borazine and polyborazylene, yielding borane adducts. In the case of NH3 (l), ammonia-borane (AB) is formed and quantified using NMR spectroscopy against an internal standard. Calculations indicate that the formation of B(NH2)3 may provide the driving force of this redistribution. Given the complexity and expense of currently known spent AB regeneration pathways, it is suggested that this redistribution chemistry be used to recover AB and improve regeneration methods.
Assuntos
Compostos de Boro/química , Bases de Lewis/química , Amônia/química , Boranos/química , Boro/química , Hidrogênio/química , Espectroscopia de Ressonância Magnética , TermodinâmicaRESUMO
The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D (1)H, (13)C and (15)N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 microM to 2.0 microM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation.
Assuntos
Rhodococcus/metabolismo , Sideróforos/química , Ágar/química , Cromatografia Líquida de Alta Pressão/métodos , Epinefrina/análogos & derivados , Epinefrina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ferro/química , Ferro/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metais/química , Modelos Químicos , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria/métodos , Espectrofotometria Ultravioleta/métodos , Fatores de TempoRESUMO
The sarcin-ricin domain is a universal element of the RNA from the large ribosomal subunit. The domain is part of the binding site for elongation factors and is specifically cleaved by the toxins alpha-sarcin and ricin. In this work, we have mapped the energetics and dynamics of individual structural motifs in a 29-mer RNA oligomer containing the sarcin-ricin domain. The stability of individual base pairs in the structure was characterized from measurements of the exchange rates of imino protons using nuclear magnetic resonance spectroscopy at 10 degrees C. The measurements also provided the rates of opening and closing for selected base pairs. The results reveal that the structural stabilization free energies in the sarcin-ricin domain are broadly distributed between 2.9 and 10.6 kcal/mol. One of the least stable sites in the structure is the noncanonical G-A base pair located next to the phosphodiester bond that is cleaved by alpha-sarcin. The low stability of this base pair supports the proposal that cleavage by alpha-sarcin occurs by a base flipping mechanism. The opening dynamics of other base pairs is affected by elements of the structure such as the bulged-G motif and its cross-strand stacking. Participation in these motifs increases the lifetimes of the bases in an open, solvent-accessible conformation.
Assuntos
Pareamento de Bases/efeitos dos fármacos , RNA Ribossômico 28S/química , Animais , Sequência de Bases , Endorribonucleases/química , Proteínas Fúngicas/química , Guanina/química , Iminas/química , Ressonância Magnética Nuclear Biomolecular , Ratos , Ricina/química , Uracila/químicaRESUMO
The iron complexation of a fluorescent green pyoverdin siderophore produced by the environmental bacterium Pseudomonas putida was characterized by solution thermodynamic methods. Pyoverdin binds iron through three bidentate chelate groups, a catecholate, a hydroxamate, and an alpha-hydroxycarboxylic acid. The deprotonation constants of the free pyoverdin and Fe(III)-pyoverdin complex were determined through a series of potentiometric and spectrophotometric experiments. The ferric complex of pyoverdin forms at very low pH (pH < 2), but full iron coordination does not occur until neutral pH. The calculated pM value of 25.13 is slightly lower than that for pyoverdin PaA (pM = 27), which coordinates iron by a catecholate and two hydroxamate groups. The redox potential of Fe-pyoverdin was found to be very pH sensitive. At high pH (approximately pH 9-11) where pyoverdin coordinates Fe in a hexadentate mode the redox potential is -0.480 V (NHE); however, at neutral pH where full Fe coordination is incomplete, the redox potential is more positive (E(1/2) = -0.395 V). The positive shift in the redox potential and the partial dissociation of the Fe-pyoverdin complex with pH decrease provides a path toward in vivo iron release.
Assuntos
Compostos Férricos/química , Oligopeptídeos/química , Compostos Organometálicos/química , Pseudomonas putida/química , Sideróforos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/biossíntese , Oligopeptídeos/isolamento & purificação , Potenciometria , Pseudomonas putida/metabolismo , Sideróforos/biossíntese , Sideróforos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria UltravioletaRESUMO
We have conducted an extensive comparison of cellular biochemical composition obtained from infrared and Raman spectra of intact cells with measurements using standard extraction and chemical analysis (including NMR), and flow cytometric assay on fixed cells. Measurements were conducted on a rat fibroblast carcinogenesis model consisting of normal and tumorigenic cells assayed as exponentially growing and plateau-phase cultures. Estimates of protein, DNA, RNA, lipids, and glycogen amounts were obtained from a previous publication in which vibrational spectra were fit to a set of basis spectra representing protein, DNA, RNA, lipids, and glycogen. The Raman spectral estimates of absolute cellular composition were quite similar to the independent biochemical and flow cytometric assays. The infrared spectra gave similar results for protein, lipid, and glycogen but underestimated the DNA content while overestimating the RNA level. When ratios of biochemical concentrations in exponential and plateau-phase cultures were examined, the Raman spectroscopic results were the same, within errors, as the independent methods, in all cases. Several changes in relative biochemical composition due to tumorigenic and proliferative status previously reported using vibrational spectroscopy were confirmed by the independent methods. These results demonstrate that vibrational spectroscopy can provide reliable estimates of the biochemical composition of mammalian cells.
Assuntos
Bioquímica/métodos , Fatores Biológicos/análise , Contagem de Células/métodos , Fibroblastos/química , Citometria de Fluxo/métodos , Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodos , Animais , Linhagem Celular , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
15N spin relaxation parameters provide a powerful tool for probing the internal dynamics and thermodynamics of proteins. The biological insight provided by these experiments often involves interpretation of small changes in relaxation parameters. This, in turn, requires careful data analysis, especially in the identification and treatment of systematic error. While progress continues on reduction of experiment-specific errors associated with pulse sequences, system-specific sources of error have received far less attention. The impact of these errors varies between facilities, spectrometers, and biological samples. We demonstrate that performing a series of control experiments along with relaxation measurements can help identify, quantify, and isolate sources of system-specific error, and, in some cases, correct for systematic changes. We further demonstrate that control experiments can be performed without significant loss of spectrometer time, and lead to more accurate relaxation parameter values.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ubiquitina/química , Processamento de Sinais Assistido por Computador , TemperaturaRESUMO
MOTIVATION: High-throughput NMR structure determination is a goal that will require progress on many fronts, one of which is rapid resonance assignment. An important rate-limiting step in the resonance assignment process is accurate identification of resonance peaks in the NMR spectra. Peak-picking schemes range from incomplete (which lose essential assignment connectivities) to noisy (which obscure true connectivities with many false ones). We introduce an automated preassignment process that removes false peaks from noisy peak lists by requiring consensus between multiple NMR experiments and exploiting a priori information about NMR spectra. This process is designed to accept multiple input formats and generate multiple output formats, in an effort to be compatible with a variety of user preferences. RESULTS: Automated preprocessing with APART rapidly identifies and removes false peaks from initial peak lists, reduces the burden of manual data entry, and documents and standardizes the peak filtering process. Successful preprocessing is demonstrated by the increased number of correct assignments obtained when data are submitted to an automated assignment program. AVAILABILITY: APART is available from http://sir.lanl.gov/NMR/APART.htm CONTACT: npawley@lanl.gov; rmichalczyk@lanl.gov SUPPLEMENTARY INFORMATION: Manual pages with installation instructions, procedures and screen shots can also be found at http://sir.lanl.gov/NMR/APART_Manual1.pdf.
Assuntos
Algoritmos , Espectroscopia de Ressonância Magnética/métodos , Reconhecimento Automatizado de Padrão/métodos , Proteínas/análise , Proteínas/química , Software , Modelos Químicos , Modelos Estatísticos , Conformação Proteica , Processamento de Sinais Assistido por Computador , Processos EstocásticosRESUMO
In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.
Assuntos
DNA/química , Ressonância Magnética Nuclear Biomolecular , Oligodesoxirribonucleotídeos/química , Maleabilidade , Poli A/química , Poli T/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , DNA/metabolismo , Óxido de Deutério/química , Eletroforese em Gel de Poliacrilamida , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Soluções , Análise Espectral Raman , Eletricidade Estática , Água/químicaRESUMO
We report CW-EPR, ESEEM, and structural NMR results, as well as DFT calculations, on model compounds relevant to the unusual cross-linked Tyr-His (YH) moiety at the active site of the heme-copper oxidases. CW-EPR spectra of an (15)N isotopically labeled 4-methyl-2-(4-methyl-imidazole-1-yl)-phenol radical are nearly identical to those of the natural abundance (14)N compound. We obtain good simulations of these EPR spectra without including hyperfine couplings to the nitrogen nuclei. This implies that the electron distribution of the radical is largely localized on the phenol ring with only a small amount of spin delocalized onto the nitrogens of the imidazole. Using three-pulse ESEEM spectroscopy, we have successfully detected the two imidazole ring nitrogens, one near the "exact cancellation" ESEEM condition and the other more weakly coupled. We assign these to the imino and amino nitrogens, respectively, based on DFT calculations performed on this radical species. The experimental results and the supporting density functional calculations clearly show that the imidazole substituent has only a minor effect on the electronic structure of the substituted phenol radical.
Assuntos
Heme/química , Histidina/química , Oxirredutases/química , Tirosina/química , Simulação por Computador , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Análise de Fourier , Modelos Químicos , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
Our group has used Ti-promoted aldol additions with an oxazolidineselone as the chiral auxiliary with much success. In these reactions, the Se atom in the auxiliary both promotes stereospecific addition as well as reports on, through the use of 77Se NMR spectroscopy, the ratio of diastereomers produced and the geometry of intermediates as the reaction proceeds. Through stable isotope labeling and NMR spectroscopy, we are able to experimentally observe a Ti enolate in solution and gain insight into its structure and reactivity. Results from molecular modeling calculations are also presented for comparison with NMR data.