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1.
Chemistry ; 25(27): 6728-6740, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30801798

RESUMO

The m7 G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7 G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7 GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.


Assuntos
Endorribonucleases/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Corantes Fluorescentes/química , Animais , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Hidrólise , Concentração Inibidora 50 , Cinética , Ligantes , Camundongos , Ligação Proteica , Pirenos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência
2.
Chem Commun (Camb) ; 53(88): 11992-11995, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-28984884

RESUMO

The 10DM24 deoxyribozyme can site-specifically label RNAs with fluorophore-GTP conjugates; however, the 2',5'-branched RNA linkage is readily cleaved by debranchase. To prevent loss of labels upon cleavage, we synthesized phosphorothioate-modified, fluorescent GTP derivatives and elaborated conditions for their incorporation by 10DM24. RNAs labeled with fluorescent derivatives of Sp-GTPS were found to be resistant to debranchase.


Assuntos
DNA Catalítico/química , Corantes Fluorescentes/química , Guanosina Trifosfato/química , RNA/análise , RNA/química , Coloração e Rotulagem/métodos , DNA Catalítico/metabolismo , Corantes Fluorescentes/síntese química , Guanosina Trifosfato/síntese química , Compostos de Sulfidrila/química
3.
J Biochem ; 159(1): 41-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26184075

RESUMO

A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Corantes Fluorescentes/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Cinética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/metabolismo
4.
Nucleic Acids Res ; 44(6): e52, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26657632

RESUMO

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.


Assuntos
Trifosfato de Adenosina/síntese química , Guanosina Trifosfato/síntese química , Marcação por Isótopo/métodos , Nucleotídeos/síntese química , Bacillus anthracis/química , Bacillus anthracis/genética , Isótopos de Carbono , Coronavirus Humano 229E/química , Coronavirus Humano 229E/genética , Creatina Quinase/química , Creatina Quinase/genética , Espectroscopia de Ressonância Magnética , Pentosiltransferases/química , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Elementos de Resposta , Ribose/química , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/genética , Riboswitch , Transcrição Gênica
5.
Bioconjug Chem ; 24(3): 301-4, 2013 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-23425139

RESUMO

Uridine, adenosine, guanosine, and cytidine that carry a propargyl group attached to the 2'-oxygen were converted efficiently to the corresponding nucleoside triphosphates (pNTPs). Primer extension experiments revealed that pUTP, pATP, and pGTP can be successfully incorporated in oligonucleotides in the so-called 9°N and Therminator DNA polymerases. Most importantly, the ethynyl group as single 2'-modification of the enzymatically prepared oligonucleotides can be applied for postsynthetic labeling. This was representatively shown by PAGE analysis after the "click"-type cycloaddition with the fluorescent nile red azide. These results show that the 2'-position as one of the most important modification sites in oligonucleotides is now accessible not only for synthetic, but also for enzymatic oligonucleotide preparation.


Assuntos
Trifosfato de Adenosina/síntese química , Química Click/métodos , DNA/síntese química , Guanosina Trifosfato/síntese química , Oxazinas/síntese química , Uridina Trifosfato/síntese química , Trifosfato de Adenosina/genética , DNA/genética , Corantes Fluorescentes/síntese química , Guanosina Trifosfato/genética , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Uridina Trifosfato/genética
6.
Bioorg Med Chem ; 20(7): 2416-8, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22364745

RESUMO

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiguanina/síntese química , Guanosina Trifosfato/análogos & derivados , Selênio/química , Trifosfato de Adenosina/síntese química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiguanina/química , Guanosina Trifosfato/síntese química , Mutação , Compostos Organosselênicos , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
Bioorg Med Chem Lett ; 19(7): 1921-5, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19269171

RESUMO

We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5'-(1-thiotriphosphate). These new analogs of mRNA 5'cap (referred to as m(7)GTPalphaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m(7)GTPalphaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate approximately 8-fold and approximately 15-fold more efficiently than m(7)GTP and m(7)GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.


Assuntos
Guanosina Trifosfato/análogos & derivados , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , Análogos de Capuz de RNA/química , Capuzes de RNA/antagonistas & inibidores , Animais , Endorribonucleases/metabolismo , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Guanosina Trifosfato/farmacologia , Humanos , Camundongos , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/síntese química , Análogos de Capuz de RNA/síntese química , Análogos de Capuz de RNA/farmacologia , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Estereoisomerismo , Tionucleotídeos/síntese química , Tionucleotídeos/química , Tionucleotídeos/farmacologia
8.
Nucleic Acids Res ; 37(5): 1638-49, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19153138

RESUMO

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M(2+))-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 +/- 0.026) min(-1). A pH rate profile analysis revealed pK(a)'s of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M(2+)-free conditions at 37 degrees C.


Assuntos
Aminas/química , DNA Catalítico/química , Guanidinas/química , Imidazóis/química , Sequência de Bases , Cátions Bivalentes/química , DNA/metabolismo , DNA Catalítico/metabolismo , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxiuracil/química , Guanidinas/síntese química , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Concentração Osmolar , RNA/metabolismo , Temperatura
10.
ACS Chem Biol ; 3(8): 499-511, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18707057

RESUMO

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.


Assuntos
Enzimas/química , Nucleotídeos de Purina/síntese química , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Isótopos de Carbono , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Enzimas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Estrutura Molecular , Isótopos de Nitrogênio , Plasmídeos , Engenharia de Proteínas , Nucleotídeos de Purina/química , RNA/química , Especificidade por Substrato
11.
Bioorg Med Chem ; 15(3): 1330-40, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17157508

RESUMO

As a model system for designing new inhibitors of bacterial cell division, we studied the essential and highly conserved FtsZ GTPase from Pseudomonas aeruginosa. A collection of GTP analogues were prepared using the solid-phase parallel synthesis approach. The synthesized GTP analogues inhibited the GTPase activity of FtsZ with IC(50) values between 450microM and 2.6mM, and 5 compounds inhibited Staphylococcus aureus growth in a biological assay. The FtsZ spectrophotometric assay developed for screening of synthesized compounds is the first step in identification of antibacterials targeting the bacterial cell division essential proteins.


Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/antagonistas & inibidores , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/farmacologia , Testes de Sensibilidade Microbiana , Staphylococcus aureus/crescimento & desenvolvimento
12.
Biol Chem ; 386(11): 1105-14, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16307476

RESUMO

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by guanine nucleotide-binding proteins (GNBPs) is a fundamental enzyme reaction in living cells that acts as an important timer in a variety of biological processes. This reaction is intrinsically slow but can be stimulated by GTPase-activating proteins (GAPs) by several orders of magnitude. In the present study, we synthesized and characterized a new fluorescent nucleotide, 2'(3')-O-(N-ethylcarbamoyl-(5''-carboxytetramethylrhodamine) amide)-GTP, or tamraGTP, which is sensitive towards conformational changes of certain GNBPs induced by GTP hydrolysis. Unlike other fluorescent nucleotides, tamra-GTP allows real-time monitoring of the kinetics of the intrinsic and GAP-catalyzed GTP hydrolysis reactions of small GNBPs from the Rho family.


Assuntos
Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/análogos & derivados , Sítios de Ligação , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Hidrólise , Cinética , Modelos Moleculares , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
13.
Biochemistry ; 44(21): 7879-84, 2005 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-15910002

RESUMO

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.


Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/síntese química , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/ultraestrutura , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Luz , Espalhamento de Radiação
14.
Chembiochem ; 5(10): 1448-53, 2004 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-15457534

RESUMO

A practical and convenient method for the synthesis of acid- and base-sensitive GTP analogues carrying a further substituent at the terminal phosphate has been developed. Key to the successful synthesis of these potential ligands of the Ras protein is the use of Pd0-sensitive allyl protecting groups in a one-pot synthesis that avoids evaporation steps. Initial biochemical analysis of a representative compound revealed that such GTP analogues can bind to Ras and might open up the possibility of developing small molecules that can act as deactivators of oncogenic Ras.


Assuntos
Guanosina Trifosfato/química , Guanosina Trifosfato/síntese química , Proteínas ras/química , Ligação Competitiva , Guanosina Difosfato/química , Guanosina Trifosfato/análogos & derivados , Ligantes , Conformação Molecular
15.
Mol Med ; 8(7): 382-92, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12393936

RESUMO

BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.


Assuntos
Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas Musculares , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Chá/química , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Calpaína/análise , Carcinoma/patologia , Proteínas de Transporte/metabolismo , Caspases/análise , Linhagem Celular , Cisteína Endopeptidases/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Masculino , Proteínas dos Microfilamentos/metabolismo , Complexos Multienzimáticos/efeitos dos fármacos , Fenóis/síntese química , Fenóis/isolamento & purificação , Polímeros/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas/metabolismo , Estereoisomerismo , Fatores de Elongação da Transcrição , Proteína X Associada a bcl-2
16.
Eur J Biochem ; 269(13): 3270-8, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12084068

RESUMO

Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPgammaS are widely used to stabilize rapidly hydrolyzing protein-nucleotide complexes and to investigate biochemical reaction pathways. Here we describe the chemical synthesis of guanosine 5'-O-(gamma-amidotriphosphate) (GTPgammaNH(2)) and a new synthesis of guanosine 5'-O-(gamma-fluorotriphosphate) (GTPgammaF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on (31)P, (19)F and (1)H NMR resonances are tabulated. For GTPgammaNH(2) the enthalpy of magnesium coordination is DeltaH degrees = 3.9 kcal.mol(-1) and the association constant K(a) is 0.82 mm(-1). The activation energy for GTPgammaNH(2).Mg2+ complex formation is DeltaH++ = 7.8 +/- 0.15 kcal.mol(-1), similar to that for the natural substrate GTP. For GTPgammaF we obtained a similar enthalpy of DeltaH degrees = 3.9 kcal.mol(-1) while the magnesium association constant is only K(a) = 0.2 mm(-1). The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated. The rate of hydrolysis of GTPgammaNH(2) bound to Ras protein lay between the rates found for Ras-bound GTPgammaS and GppNHp, while Ras-catalysed hydrolysis of GTPgammaF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.


Assuntos
Bioquímica/métodos , Guanosina Trifosfato/química , Calorimetria/métodos , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/metabolismo , Hidrólise , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Metais/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo
17.
Protein Sci ; 8(11): 2524-8, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10595559

RESUMO

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2'-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2'-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and RabS. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of approximately 100 (p21ras) or approximately 10(5) (Rab5), and the resulting equilibrium dissociation constants are in the nm or microM range, respectively. The data demonstrate that 2'halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the microM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Cristalografia por Raios X/métodos , Proteínas de Escherichia coli , Guanosina Trifosfato/análogos & derivados , Proteínas/química , Trifosfato de Adenosina/síntese química , Guanosina Trifosfato/síntese química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Halogênios , Indicadores e Reagentes , Cinética , Núcleosídeo-Fosfato Quinase , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/metabolismo
18.
Arch Biochem Biophys ; 372(1): 205-13, 1999 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10562435

RESUMO

A new affinity label, 8-(4-bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate (8-BDB-TGTP), has been synthesized by initial reaction of GTP to form 8-Br-GTP, followed by its conversion to 8-thio-GTP, and finally coupling with 1,4-dibromobutanedione to produce 8-BDB-TGTP. 8-BDB-TGTP and its synthetic intermediates were characterized by thin-layer chromatography, UV, (31)P NMR spectroscopy, as well as by bromide and phosphorus analysis. Escherichia coli adenylosuccinate synthetase is inactivated by 8-BDB-TGTP at pH 7.0 at 25 degrees C. Pretreatment of the enzyme with N-ethylmaleimide (NEM) blocks the exposed Cys(291) and leads to simple pseudo-first-order kinetics of inactivation. The inactivation exhibits a nonlinear relationship of initial inactivation rate versus 8-BDB-TGTP concentration, indicating the reversible association of 8-BDB-TGTP with the enzyme prior to the formation of a covalent bond. The inactivation kinetics exhibit an apparent K(I) of 115 microM and a k(max) of 0.0262 min(-1). Reaction of the NEM-treated adenylosuccinate synthetase with 8-BDB-[(3)H]TGTP results in 1 mol of reagent incorporated/mol of enzyme subunit. Adenylosuccinate or IMP plus GTP completely protects the enzyme against 8-BDB-TGTP inactivation, whereas IMP or GTP alone provide partial protection against inactivation. AMP is much less effective in protection. The results of ligand protection studies suggest that E. coli adenylosuccinate synthetase may accommodate 8-BDB-TGTP as a GTP analog. The new affinity label may be useful for identifying catalytic amino acid residues of protein proximal to the guanosine ring.


Assuntos
Marcadores de Afinidade , Guanosina Trifosfato/análogos & derivados , Proteínas/química , Nucleotídeos de Purina/química , Adenilossuccinato Sintase/antagonistas & inibidores , Adenilossuccinato Sintase/metabolismo , Marcadores de Afinidade/síntese química , Sítios de Ligação , Domínio Catalítico , Escherichia coli/enzimologia , Etilmaleimida/farmacologia , Guanosina Trifosfato/síntese química , Cinética , Ligantes , Espectroscopia de Ressonância Magnética
19.
J Biochem Biophys Methods ; 35(1): 61-6, 1997 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9310869

RESUMO

8-N3 GMP was synthesised from 8-BrGMP by the addition of LiN3. 8-N3GMP was then phosphorylated to N3-GDP and N3-GTP by controlled enzymatic reaction. 8-N3GDP can be converted to N3-ppGpp with crude Rel A, which phosphorylates the 3'-OH of GDP.


Assuntos
Guanosina Trifosfato/síntese química , Cromatografia em Camada Fina , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/isolamento & purificação
20.
RNA ; 3(5): 464-75, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9149228

RESUMO

Two new photoreactive nucleotide derivatives have been applied in site-directed crosslinking studies with mRNA analogues. 6-Thioguanosine triphosphate or 5-methyleneaminouridine triphosphate was incorporated into mRNA analogues by T7 transcription; after transcription, the 5-methyleneaminouridine residues were converted to a diazirine derivative. mRNA analogues carrying either 6-thioguanosine or the diazirine derivative were bound to Escherichia coli ribosomes in the presence of tRNA(f)(Met), and photo-crosslinking was induced by irradiation at 350 nm. With 6-thioguanosine, specific crosslinks were observed from downstream positions +8 or +9 of the mRNA to nt 1196 in helix 34 of the 16S rRNA, and from position +12 to nt 530 in helix 18. With the diazirine derivative, a crosslink from position +2 (within the AUG codon) to nt 926 in helix 28 was found. Taken together with previous data obtained from downstream sites in mRNA analogues carrying 4-thiouridine residues, specific crosslinks have now been identified from downstream mRNA positions +2, +4, +6, +7, +8, +9, +11, and +12. The data confirm that the three 16S rRNA regions involved-helices 18, 28, and 34-are in the direct neighborhood of the decoding area of the 30S subunit.


Assuntos
Escherichia coli/metabolismo , Guanosina Trifosfato/análogos & derivados , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/biossíntese , Ribossomos/metabolismo , Tionucleotídeos/metabolismo , Uridina Trifosfato/análogos & derivados , Bacteriófago T7/metabolismo , Sequência de Bases , Códon , Reagentes de Ligações Cruzadas , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/biossíntese , RNA Bacteriano/química , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Ribossômico 16S/química , RNA de Transferência de Metionina/metabolismo , Tionucleotídeos/síntese química , Transcrição Gênica , Uridina Trifosfato/síntese química , Uridina Trifosfato/metabolismo
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