- Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs
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Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.
- Norahan, Mohamad Javad,Horvath, Raphael,Woitzik, Nathalie,Jouy, Pierre,Eigenmann, Florian,Gerwert, Klaus,K?tting, Carsten
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p. 6779 - 6783
(2021/05/31)
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- Synthetic method of nucleoside tetraphosphate
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The invention discloses a synthetic method of nucleoside tetraphosphate. The synthetic method comprises the steps of carrying out selective phosphorylation reaction by virtue of nucleoside and a cyclic phosphorylation reagent, and carrying out oxidation and hydrolysis loop opening, so as to obtain nucleoside tetraphosphate. The structure of the cyclic phosphorylation reagent is represented by a formula I (shown in the description). According to the synthetic method, 5'-nucleoside tetraphosphate is selectively generated from nucleoside under the effect of the high-selectivity phosphorylation reagent, and 3'-OH (and 2'-OH) does not need to be protected in the process, namely that the generaiton of 3'(and 2'-)tetraphosphate can be effectively inhibited. Nucleoside tetraphosphate synthesized by virtue of the method has wide use ranges in the biology fields of DNA sequencing, labeling, extension and the like; currently, the selling prices is expensive, a synthetic method is complex, the reaction selectivity is poor; and the synthetic method provided by the invention is good in selectivity and easy in separation and purification, required experimental conditions are simple, and the synthetic processes are all conventional chemical reactions, so that the synthetic method is applicable to large-scale popularization and use.
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- Photo-electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode
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A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD+. This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.
- Sabir, Nadeem,Khan, Nazimuddin,V?lkner, Johannes,Widdascheck, Felix,Del Pino, Pablo,Witte, Gregor,Riedel, Marc,Lisdat, Fred,Konrad, Manfred,Parak, Wolfgang J.
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p. 5844 - 5850
(2016/01/25)
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- Dual-color control of nucleotide polymerization sensed by a fluorescence actuator
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Spatial and temporal control of molecular mechanisms can be achieved using photolabile bonds that connect biomolecules to protective caging groups, which can be cleaved upon irradiation of a specific wavelength, releasing the biomolecule ready-to-use. Here we apply and improve a previously reported strategy to tightly control in vitro transcription reactions. The strategy involves two caging molecules that block both ATP and GTP nucleotides. Additionally, we designed a molecular beacon complementary to the synthesized mRNA to infer its presence through a light signal. Upon release of both nucleotides through a specific monochromatic light (390 and 325 nm) we attain a light signal indicative of a successful in vitro transcription reaction. Similarly, in the absence of irradiation, no intense fluorescence signal was obtained. We believe this strategy could further be applied to DNA synthesis or the development of logic gates. This journal is
- Reimao-Pinto, Madalena M.,Cordeiro, Ana,Almeida, Carina,Pinheiro, Andre V.,Moro, Artur,Lima, Joao C.,Baptista, Pedro V.
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p. 751 - 756
(2014/05/06)
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- Enzymatic and molecular characterization of arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26
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Not only in bacteria but also in plant cells, guanosine- 3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrola
- Ito, Daisuke,Kato, Takahiro,Maruta, Takanori,Tamoi, Masahiro,Yoshimura, Kazuya,Shigeoka, Shigeru
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p. 2236 - 2241
(2013/02/25)
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- Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: Implications for the design of better anti-HIV analogues
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The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the d-/l-configurations of the nucleotides, the l-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the l-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an l-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.
- Varga, Andrea,Chaloin, Laurent,Sagi, Gyula,Sendula, Robert,Graczer, Eva,Liliom, Karoly,Zavodszky, Peter,Lionne, Corinne,Vas, Maria
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experimental part
p. 1863 - 1873
(2012/04/17)
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- An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
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Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
- Gillerman, Irina,Fischer, Bilha
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p. 245 - 256
(2011/08/06)
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- Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-32P]d/rNTP
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Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k m and k cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α- 32P]rNTP and [α-32P]dNTP.
- Skoblov,Mikoulinskaia,Taran,Miroshnikov,Feofanov,Skoblov
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experimental part
p. 734 - 738
(2010/08/07)
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- A kinetic study of the rat liver adenosine kinase reverse reaction
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Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH3. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels. Copyright Taylor & Francis Group, LLC.
- Vannoni,Giglioni,Santoro,Aceto,Marinello,Leoncini
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p. 872 - 875
(2008/12/21)
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- Borate-nucleotide complex formation depends on charge and phosphorylation state
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Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 μM nucleotide and 500 μM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3
- Kim, Danny H.,Faull, Kym F.,Norris, Andrew J.,Eckhert, Curtis D.
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p. 743 - 751
(2007/10/03)
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- Derivatives of L-adenosine and L-guanosine as substrates for human deoxycytidine kinase
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A series of analogues of L-adenosine and of L-guanosine, including β- L-dA, β-L-Ado, β-L-araA, and β-L-dG, have been shown to be substrates of human deoxycytidine kinase thus demonstrating the complete lack of enantioselectivity of this enzyme.
- Gaubert,Gosselin,Imbach,Eriksson,Maury
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p. 857 - 860
(2007/10/03)
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- Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis
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The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.
- Noguchi, Toshitada,Shiba, Toshikazu
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p. 1594 - 1596
(2007/10/03)
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- Hydrolytic Susceptibilities of Modified 5'-mRNA Cap Analogues to the Yellow Lupin Ap4A Hydrolases
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Several new dinucleoside tri- and tetraphosphates, 5'-mRNA cap analogues, have been tested as potential substrates for two enzymes: highly specific homogeneous Ap3A and (asymmetrical) Ap4A hydrolase from yellow lupin seeds.The Ap3A hydrolase cleaves all examined dinucleotide triphosphates yielding in most cases GMP or m7GMP and modified diphosphates as the main hydrolysis products (75-100percent).Only m7Gp3G has been hydrolyzed randomly.Compounds with unmodified guanosine have been degraded at comparable rate as natural substrate Ap3A.For the analogues modified in guanosine or sugar moiety significant differences have been observed in the rate of hydrolysis.Ap4A hydrolase seems to be more specific enzyme than Ap3A hydrolase.Dinucleoside tetraphosphates containing guanosine or 7-methylguanosine have been degraded more slowly than Ap4A.In contrast, di- and trimethylated compounds have been hydrolyzed with the rate 2-fold higher in comparison with Ap4A.For all methylated dinucleoside tetraphosphates, GTP and methylated nucleoside monophosphates have been found as the main hydrolysis products.
- Bojarska, Elzbieta,Stepinski, Janusz,Guranowski, Andrzej,Starzynska, Elzbieta,Chlebicka, Lidia,et al.
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p. S192 - S196
(2007/10/03)
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- Chemo-enzymic Synthesis of Guanosine 5'-Diphosphomannose (GDP-Mannose) and Selected Analogues
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Guanosine 5'-diphosphomannose (GDP-mannose, 1) has been prepared via two routes from α-D-mannose 1-phosphate 5.The non-enzymic route involves coupling of the phosphate 5 to guanosine 5'-monophosphate dibutyl phosphinothioic anhydride (GMP-anhydride 11) to give compound 1 in 40percent yield.The enzymic routes require coupling of compound 5 with guanosine 5'-triphosphate (GTP) catalysed by GDP-mannose pyrophosphorylase (37percent).
- Pallanca, Jane E.,Turner, Nicolas J.
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p. 3017 - 3022
(2007/10/02)
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- Mg(II) ION-MEDIATED CONVERSION OF MONO- AND OLIGONUCLEOTIDES TO 5'-POLYPHOSPHATES IN AQUEOUS SOLUTION
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5'-Polyphosphates of mono- and oligonucleotides were prepared from the corresponding 5'-monophosphates with phosphorotriimidazolide or phosphorotribenzimidazolide mediated by Mg(II) or Mn(II) ion in aqueous solution.
- Sawai, Hiroaki,Inaba, Yoshiko,Hirano, Atsushi,Wakai, Hiromichi,Shimazu, Masamitsu
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p. 4801 - 4804
(2007/10/02)
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