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[PMID]:28455448
[Au] Autor:Goodrich AC; Meyers DJ; Frueh DP
[Ad] Endereço:From the Department of Biophysics and Biophysical Chemistry and.
[Ti] Título:Molecular impact of covalent modifications on nonribosomal peptide synthetase carrier protein communication.
[So] Source:J Biol Chem;292(24):10002-10013, 2017 06 16.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. Delineating the mechanisms involved in orchestrating these various processes will deepen our understanding of domain-domain communication in nonribosomal peptide synthetases (NRPSs) and lay the groundwork for the rational reengineering of NRPSs by swapping domains handling different substrates to generate novel natural products. Although many structural and biochemical studies of NRPSs exist, few studies have focused on the energetics and dynamics governing the interactions in these systems. Here, we present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. Results from fluorescence anisotropy, isothermal titration calorimetry, and NMR titrations indicated that covalent modifications to a carrier protein modulate domain communication, suggesting that chemical modifications to carrier proteins during NRPS synthesis may impart directionality to sequential NRPS domain interactions. Comparison of the structure and dynamics of an apo-aryl carrier protein with those of its modified forms revealed structural fluctuations induced by post-translational modifications and mediated by modulations of protein dynamics. The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Proteínas de Transporte/metabolismo
Coenzima A Ligases/metabolismo
Modelos Moleculares
Peptídeo Sintases/metabolismo
Modificação Traducional de Proteínas
Processamento de Proteína Pós-Traducional
Yersinia pestis/metabolismo
[Mh] Termos MeSH secundário: Substituição de Aminoácidos
Apoenzimas/química
Apoenzimas/genética
Apoenzimas/metabolismo
Apoproteínas/química
Apoproteínas/genética
Apoproteínas/metabolismo
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Calorimetria
Isótopos de Carbono
Proteínas de Transporte/química
Proteínas de Transporte/genética
Coenzima A Ligases/química
Coenzima A Ligases/genética
Polarização de Fluorescência
Holoenzimas/química
Holoenzimas/genética
Holoenzimas/metabolismo
Cinética
Mutação
Isótopos de Nitrogênio
Ressonância Magnética Nuclear Biomolecular
Peptídeo Sintases/química
Peptídeo Sintases/genética
Conformação Proteica
Domínios e Motivos de Interação entre Proteínas
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Titulometria
Yersinia pestis/enzimologia
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Apoenzymes); 0 (Apoproteins); 0 (Bacterial Proteins); 0 (Carbon Isotopes); 0 (Carrier Proteins); 0 (Holoenzymes); 0 (Nitrogen Isotopes); 0 (Recombinant Proteins); EC 6.2.1.- (Coenzyme A Ligases); EC 6.2.1.- (YbtE protein, Yersinia pestis); EC 6.3.2.- (Peptide Synthases); EC 6.3.2.- (non-ribosomal peptide synthase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171228
[Lr] Data última revisão:
171228
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170430
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M116.766220


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[PMID]:28837132
[Au] Autor:Baccouche A; Okumura S; Sieskind R; Henry E; Aubert-Kato N; Bredeche N; Bartolo JF; Taly V; Rondelez Y; Fujii T; Genot AJ
[Ad] Endereço:LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.
[Ti] Título:Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.
[So] Source:Nat Protoc;12(9):1912-1932, 2017 Sep.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 µl of reaction mix.
[Mh] Termos MeSH primário: Bioensaio/instrumentação
Bioensaio/métodos
Técnicas Analíticas Microfluídicas/instrumentação
Técnicas Analíticas Microfluídicas/métodos
Titulometria/instrumentação
Titulometria/métodos
[Mh] Termos MeSH secundário: Desenho de Equipamento
Corantes Fluorescentes/análise
Corantes Fluorescentes/química
Processamento de Imagem Assistida por Computador/métodos
Tensoativos/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fluorescent Dyes); 0 (Surface-Active Agents)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170906
[Lr] Data última revisão:
170906
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170825
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.092


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[PMID]:28832133
[Au] Autor:Arnott ZLP; Nozaki S; Monteiro DCF; Morgan HE; Pearson AR; Niki H; Webb ME
[Ad] Endereço:Astbury Centre for Structural Molecular Biology and School of Chemistry, University of Leeds , Leeds LS2 9JT, U.K.
[Ti] Título:The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).
[So] Source:Biochemistry;56(37):4931-4939, 2017 Sep 19.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.
[Mh] Termos MeSH primário: Acetilcoenzima A/metabolismo
Carboxiliases/metabolismo
Escherichia coli K12/metabolismo
Proteínas de Escherichia coli/metabolismo
Glutamato Descarboxilase/metabolismo
Modelos Moleculares
[Mh] Termos MeSH secundário: Acetilcoenzima A/análogos & derivados
Acetilcoenzima A/química
Substituição de Aminoácidos
Antibacterianos/farmacologia
Antimetabólitos/farmacologia
Sítios de Ligação
Calorimetria
Carboxiliases/química
Carboxiliases/genética
Coenzima A/síntese química
Coenzima A/química
Coenzima A/metabolismo
Cristalografia por Raios X
Ativação Enzimática/efeitos dos fármacos
Escherichia coli K12/efeitos dos fármacos
Escherichia coli K12/crescimento & desenvolvimento
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Deleção de Genes
Glutamato Descarboxilase/antagonistas & inibidores
Glutamato Descarboxilase/química
Glutamato Descarboxilase/genética
Cinética
Mutação
Ácido Pantotênico/análogos & derivados
Ácido Pantotênico/farmacologia
Conformação Proteica
Multimerização Proteica
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Titulometria
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (Antimetabolites); 0 (Escherichia coli Proteins); 0 (N-pentylpantothenamide); 0 (PanZ protein, E coli); 0 (Recombinant Proteins); 19F5HK2737 (Pantothenic Acid); 72-89-9 (Acetyl Coenzyme A); EC 4.1.1.- (Carboxy-Lyases); EC 4.1.1.- (PanD protein, E coli); EC 4.1.1.11 (aspartate-alpha-decarboxylase); EC 4.1.1.15 (Glutamate Decarboxylase); SAA04E81UX (Coenzyme A)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171003
[Lr] Data última revisão:
171003
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170824
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00509


  4 / 3181 MEDLINE  
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[PMID]:28654569
[Au] Autor:Lebet R; Hayakawa J; Chamblee TB; Tala JA; Singh N; Wypij D; Curley MAQ
[Ad] Endereço:Ruth Lebet, MSN, CCNS-P, is Lecturer, School of Nursing, University of Pennsylvania, Philadelphia. Jennifer Hayakawa, DNP, PCNS-BC, CNRN, CCRN, is Clinical Nurse Specialist, Pediatric Intensive Care Unit, CHOC Children's Hospital, Orange, California, and Clinical Faculty, Western University of Health Sciences, Pomona, California. Tracy B. Chamblee, PhD, APRN, PCNS-BC, is Clinical Nurse Specialist, Pediatric Intensive Care Unit, Children's Medical Center Dallas, Texas. Joana A. Tala, MD, is Research Coordinator, Pediatric Intensive Care Unit, Yale New Haven Hospital/Yale University, Connecticut. Nakul Singh, ScM, is Biostatistician, Department of Cardiology, Boston Children's Hospital, Massachusetts. David Wypij, PhD, is Senior Biostatistician, Department of Cardiology, Boston Children's Hospital; Associate Professor, Department of Pediatrics, Harvard Medical School; and Senior Lecturer, Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, Massachusetts. Martha A. Q. Curley, RN, PhD, FAAN, is Ellen and Robert Kapito Professor in Nursing Science, School of Nursing, University of Pennsylvania, Philadelphia, and Nurse Scientist, Boston Children's Hospital, Massachusetts.
[Ti] Título:Maintaining Interrater Agreement of Core Assessment Instruments in a Multisite Randomized Controlled Clinical Trial: The Randomized Evaluation of Sedation Titration for Respiratory Failure (RESTORE) Trial.
[So] Source:Nurs Res;66(4):323-329, 2017 Jul/Aug.
[Is] ISSN:1538-9847
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: RESTORE (Randomized Evaluation of Sedation Titration for Respiratory Failure) was a cluster randomized clinical trial evaluating a sedation strategy in children 2 weeks to <18 years of age with acute respiratory failure supported on mechanical ventilation. A total of 31 U.S. pediatric intensive care units (PICUs) participated in the trial. Staff nurse rater agreement on measures used to assess a critical component of treatment fidelity was essential throughout the 4-year data collection period. OBJECTIVE: The purpose of the study is to describe the method of establishing and maintaining interrater agreement (IRA) of two core clinical assessment instruments over the course of the clinical trial. METHODS: IRA cycles were carried out at all control and intervention sites and included a minimum of five measurements of the State Behavioral Scale (SBS) and Withdrawal Assessment Tool-Version 1 (WAT-1). Glasgow Coma Scale scores were also obtained. PICUs demonstrating <80% agreement repeated their IRA cycle. Fleiss's kappa coefficient was used to assess IRA. RESULTS: Repeated IRA cycles were required for 8% of 226 SBS cycles and 2% of 222 WAT-1 cycles. Fleiss's kappa coefficients from more than 1,350 paired assessments were .86 for SBS and .92 for WAT-1, demonstrating strong agreement and similar to .91 for the Glasgow Coma Scale. There was no difference in Fleiss's kappa for any of the instruments based on unit size or timing of assessment (earlier or later in the study). For SBS scores, Fleiss's kappa was significantly different in larger and smaller PICUs (.82 vs. .92, p = .003); however, Fleiss's kappa for both groups indicated excellent agreement. CONCLUSION: Monitoring measurement reliability is an essential step in ensuring treatment fidelity and, thus, the validity of study results. Standardization on the use of these core assessment instruments among participating sites was achieved and maintained throughout the trial.
[Mh] Termos MeSH primário: Sedação Consciente/normas
Hipnóticos e Sedativos/normas
Unidades de Terapia Intensiva Pediátrica/normas
Monitorização Fisiológica/normas
Respiração Artificial/normas
Insuficiência Respiratória/terapia
Titulometria/normas
[Mh] Termos MeSH secundário: Adolescente
Criança
Pré-Escolar
Feminino
Seres Humanos
Lactente
Recém-Nascido
Masculino
Estudos Prospectivos
Reprodutibilidade dos Testes
Estados Unidos
[Pt] Tipo de publicação:JOURNAL ARTICLE; MULTICENTER STUDY; RANDOMIZED CONTROLLED TRIAL
[Nm] Nome de substância:
0 (Hypnotics and Sedatives)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170731
[Lr] Data última revisão:
170731
[Sb] Subgrupo de revista:AIM; IM; N
[Da] Data de entrada para processamento:170628
[St] Status:MEDLINE
[do] DOI:10.1097/NNR.0000000000000224


  5 / 3181 MEDLINE  
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[PMID]:28615444
[Au] Autor:Qian Y; Johnson KA
[Ad] Endereço:From the Institute for Cellular and Molecular Biology and Department of Molecular Biosciences, University of Texas, Austin, Texas 78712.
[Ti] Título:The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange.
[So] Source:J Biol Chem;292(31):13068-13084, 2017 Aug 04.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) and (SSB) , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) and (SSB) complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 10 m s ) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein.
[Mh] Termos MeSH primário: DNA Mitocondrial/metabolismo
DNA de Cadeia Simples/metabolismo
Proteínas de Ligação a DNA/metabolismo
Proteínas de Escherichia coli/metabolismo
Proteínas Mitocondriais/metabolismo
Modelos Moleculares
[Mh] Termos MeSH secundário: Substituição de Aminoácidos
Sítios de Ligação
Calorimetria
DNA Mitocondrial/química
DNA de Cadeia Simples/química
Proteínas de Ligação a DNA/química
Proteínas de Ligação a DNA/genética
Ensaio de Desvio de Mobilidade Eletroforética
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Transferência Ressonante de Energia de Fluorescência
Corantes Fluorescentes/química
Seres Humanos
Cinética
Proteínas Mitocondriais/química
Proteínas Mitocondriais/genética
Mutação
Poli T/química
Poli T/metabolismo
Domínios e Motivos de Interação entre Proteínas
Multimerização Proteica
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Termodinâmica
Titulometria
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Mitochondrial); 0 (DNA, Single-Stranded); 0 (DNA-Binding Proteins); 0 (Escherichia coli Proteins); 0 (Fluorescent Dyes); 0 (Mitochondrial Proteins); 0 (Recombinant Proteins); 0 (SSB protein, E coli); 0 (SSBP1 protein, human); 25086-81-1 (Poly T)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170825
[Lr] Data última revisão:
170825
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170616
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M117.791392


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[PMID]:28570045
[Au] Autor:Reed B; Yakovleva L; Shuman S; Ghose R
[Ad] Endereço:Department of Chemistry and Biochemistry, The City College of New York , New York, New York 10031, United States.
[Ti] Título:Characterization of DNA Binding by the Isolated N-Terminal Domain of Vaccinia Virus DNA Topoisomerase IB.
[So] Source:Biochemistry;56(26):3307-3317, 2017 Jul 05.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5'-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3'-phosphotyrosyl )-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with a cleavage site affinity lower than that of the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in the DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ∼2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution nuclear magnetic resonance methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB is underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.
[Mh] Termos MeSH primário: DNA Topoisomerases Tipo I/metabolismo
DNA/metabolismo
Modelos Moleculares
Vírus Vaccinia/enzimologia
Proteínas Virais/metabolismo
[Mh] Termos MeSH secundário: Substituição de Aminoácidos
Calorimetria
DNA/química
DNA Topoisomerases Tipo I/química
DNA Topoisomerases Tipo I/genética
Hidrodinâmica
Isoenzimas/química
Isoenzimas/genética
Isoenzimas/metabolismo
Cinética
Mutação
Ressonância Magnética Nuclear Biomolecular
Motivos de Nucleotídeos
Concentração Osmolar
Fragmentos de Peptídeos/química
Fragmentos de Peptídeos/genética
Fragmentos de Peptídeos/metabolismo
Domínios e Motivos de Interação entre Proteínas
Proteínas Recombinantes de Fusão/química
Proteínas Recombinantes de Fusão/metabolismo
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Titulometria
Proteínas Virais/química
Proteínas Virais/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Isoenzymes); 0 (Peptide Fragments); 0 (Recombinant Fusion Proteins); 0 (Recombinant Proteins); 0 (Viral Proteins); 9007-49-2 (DNA); EC 5.99.1.2 (DNA Topoisomerases, Type I)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170726
[Lr] Data última revisão:
170726
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170602
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00042


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[PMID]:28562023
[Au] Autor:Pedroso MM; Ely F; Carpenter MC; Mitic N; Gahan LR; Ollis DL; Wilcox DE; Schenk G
[Ad] Endereço:School of Chemistry and Molecular BioSciences, The University of Queensland , St Lucia, QLD 4072, Australia.
[Ti] Título:Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.
[So] Source:Biochemistry;56(26):3328-3336, 2017 Jul 05.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Cobalto/metabolismo
Enterobacter aerogenes/enzimologia
Manganês/metabolismo
Diester Fosfórico Hidrolases/metabolismo
[Mh] Termos MeSH secundário: Substituição de Aminoácidos
Asparagina/química
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Sítios de Ligação
Calorimetria
Domínio Catalítico
Ativação Enzimática
Ligações de Hidrogênio
Cinética
Mutação
Diester Fosfórico Hidrolases/química
Diester Fosfórico Hidrolases/genética
Fósforo/química
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Serina/química
Termodinâmica
Titulometria
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Recombinant Proteins); 27YLU75U4W (Phosphorus); 3G0H8C9362 (Cobalt); 42Z2K6ZL8P (Manganese); 452VLY9402 (Serine); 7006-34-0 (Asparagine); EC 3.1.4.- (Phosphoric Diester Hydrolases); EC 3.1.4.46 (glycerophosphodiester phosphodiesterase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170726
[Lr] Data última revisão:
170726
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170601
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b01200


  8 / 3181 MEDLINE  
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[PMID]:28533433
[Au] Autor:Stevens CM; Rayani K; Singh G; Lotfalisalmasi B; Tieleman DP; Tibbits GF
[Ad] Endereço:From the Cardiovascular Sciences, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia V5Z 4H4, Canada.
[Ti] Título:Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca -sensitizing mutations.
[So] Source:J Biol Chem;292(28):11915-11926, 2017 Jul 14.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca TnC binding Ca initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnI ). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca and TnI Surprisingly the Ca affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca -binding site of cTnC.
[Mh] Termos MeSH primário: Sinalização do Cálcio
Cardiomiopatia Hipertrófica Familiar/genética
Cardiomiopatia Hipertrófica/genética
Modelos Moleculares
Mutação
Troponina C/metabolismo
Troponina I/metabolismo
[Mh] Termos MeSH secundário: Substituição de Aminoácidos
Sítios de Ligação
Calorimetria
Cardiomiopatia Hipertrófica/metabolismo
Cardiomiopatia Hipertrófica Familiar/metabolismo
Transferência de Energia
Seres Humanos
Cinética
Simulação de Dinâmica Molecular
Fragmentos de Peptídeos/química
Fragmentos de Peptídeos/genética
Fragmentos de Peptídeos/metabolismo
Conformação Proteica
Domínios e Motivos de Interação entre Proteínas
Redobramento de Proteína
Estabilidade Proteica
Desdobramento de Proteína
Proteínas Recombinantes/metabolismo
Titulometria
Troponina C/antagonistas & inibidores
Troponina C/química
Troponina C/genética
Troponina I/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Peptide Fragments); 0 (Recombinant Proteins); 0 (Troponin C); 0 (Troponin I)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170803
[Lr] Data última revisão:
170803
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170524
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M116.770776


  9 / 3181 MEDLINE  
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[PMID]:28437073
[Au] Autor:Kiran U; Regur P; Kreutz MR; Sharma Y; Chakraborty A
[Ad] Endereço:CSIR-Centre for Cellular and Molecular Biology (CCMB) , Uppal Road, Hyderabad 500007, India.
[Ti] Título:Intermotif Communication Induces Hierarchical Ca Filling of Caldendrin.
[So] Source:Biochemistry;56(19):2467-2476, 2017 May 16.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A crucial event in calcium signaling is the transition of a calcium sensor from the apo (Ca free) to the holo (Ca -saturated) state. Caldendrin (CDD) is a neuronal Ca -binding protein with two functional (EF3 and EF4) and two atypical (EF1 and EF2), non-Ca -binding EF-hand motifs. During the transition from the apo to the holo state, guided by the stepwise filling of Ca , the protein passes through distinct states and acquires a stable conformational state when only EF3 is occupied by Ca . This state is characterized by a Ca -derived structural gain in EF3 with destabilization of the EF4 motif. At higher Ca levels, when Ca fills in EF4, the motif regains stability. EF3 controls initial Ca binding and dictates structural destabilization of EF4. It is likely that this unexpected intermotif communication will have an impact on Ca -dependent target interactions.
[Mh] Termos MeSH primário: Sinalização do Cálcio
Proteínas de Ligação ao Cálcio/metabolismo
Modelos Moleculares
Proteínas do Tecido Nervoso/metabolismo
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Substituição de Aminoácidos
Animais
Apoproteínas/química
Apoproteínas/genética
Apoproteínas/metabolismo
Sítios de Ligação
Proteínas de Ligação ao Cálcio/química
Proteínas de Ligação ao Cálcio/genética
Calorimetria
Dicroísmo Circular
Mutagênese Sítio-Dirigida
Mutação
Proteínas do Tecido Nervoso/química
Proteínas do Tecido Nervoso/genética
Estabilidade Proteica
Desdobramento de Proteína
Ratos
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Espectrometria de Fluorescência
Termodinâmica
Titulometria
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Apoproteins); 0 (Calcium-Binding Proteins); 0 (Nerve Tissue Proteins); 0 (Recombinant Proteins); EC 5.3.4.- (Ca2+-binding protein-1)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170623
[Lr] Data última revisão:
170623
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170425
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00132


  10 / 3181 MEDLINE  
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[PMID]:28410941
[Au] Autor:Cardiano P; Crea F; Foti C; Giuffrè O; Sammartano S
[Ad] Endereço:Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy.
[Ti] Título:Potentiometric, UV and H NMR study on the interaction of Cu with ampicillin and amoxicillin in aqueous solution.
[So] Source:Biophys Chem;224:59-66, 2017 May.
[Is] ISSN:1873-4200
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:A potentiometric, UV and H NMR study on Cu -ampicillin [(2S,5R,6R)-6-([(2R)-2-amino-2-phenylacetyl]amino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid] and -amoxicillin [(2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-24-carboxylic acid] systems in NaCl aqueous solution at I=0.15molL and t=25°C is reported. On the basis of potentiometric results two speciation models were proposed for each system. It was found that spectrophotometric and H NMR measurements are essential for selecting the most reliable speciation models. They included ML, MLOH and ML(OH) species in both systems and, only for Cu -ampicillin, also MLH species. The stability constants obtained by UV and H NMR titrations were comparable to the ones calculated by potentiometry. The sequestering ability of the ligands under study towards Cu by pL empiric parameter (ligand concentration required to sequester 50% of the metal cation present in traces) at several pH values was calculated as well. For ampicillin and amoxicillin, pL =7.19 and 6.67, respectively, at physiological pH, I=0.15molL and t=25°C were obtained.
[Mh] Termos MeSH primário: Amoxicilina/química
Ampicilina/química
Cobre/química
[Mh] Termos MeSH secundário: Potenciometria
Espectroscopia de Prótons por Ressonância Magnética
Soluções
Espectrofotometria Ultravioleta
Titulometria
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Solutions); 789U1901C5 (Copper); 7C782967RD (Ampicillin); 804826J2HU (Amoxicillin)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170613
[Lr] Data última revisão:
170613
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170416
[St] Status:MEDLINE



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