Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

Hiratsuka, Toshiaki

doi:10.1016/0304-4165(82)90240-9

Cited by 65 publications

(46 citation statements)

References 28 publications

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“…5). The fluorescence increase was coupled with a blue shift of the fluorescence maximum from 542 to 528 nm, suggesting that the TNP-nucleotides bind to a hydrophobic region of the protein (15). Competition experiments confirmed that nucleotide binding was specific, since addition of an unlabeled nucleotide to TNP-nucleotide-protein complexes considerably reduced the fluorescence ( Fig.…”

Section: Resultsmentioning

confidence: 75%

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Schröder

Lanka

2003

J Bacteriol

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TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We charac- Type IV secretion is an energy-driven mechanism for delivery of effector molecules from bacterial donors to recipient cells. It mediates the transfer of toxic components into eukaryotic hosts by many pathogenic bacteria such as Helicobacter pylori, Legionella pneumophila, Brucella spp., and Bartonella henselae and enables DNA transfer in bacterial conjugation systems such as that encoded by plasmid RP4.

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Section: Resultsmentioning

confidence: 75%

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Schröder

Lanka

2003

J Bacteriol

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“…E. coli strain JM109 was used for preparation of plasmids, and CJ236 (36) was used for generating uracilcontaining single-stranded plasmids for site-directed mutagenesis. TNP-ATP was prepared according to Hiratsuka and Uchida (45,46). Protein concentration was determined by the method of Bradford (47) using bovine serum albumin as a standard or by the UV absorbance using the factor 0.45 at 280 nm as 1 mg/ml (48).…”

Section: Measurement Of Tnp-atp Binding Monitored By Fluorescentmentioning

confidence: 99%

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

Kato

Matsui²,

Tanaka

et al. 1997

Journal of Biological Chemistry

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of thermophilic F 1 -ATPase were prepared, and their catalytic properties were compared to know the role of ␦ and ⑀ subunits in catalysis. The presence of ␦ subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of ⑀ subunit was more profound. The (؊⑀) complexes, ␣ 3 ␤ 3 ␥ and ␣ 3 ␤ 3 ␥␦, initiated ATP hydrolysis without a lag. In contrast, the (؉⑀) complexes, ␣ 3 ␤ 3 ␥⑀ and ␣ 3 ␤ 3 ␥␦⑀, started hydrolysis of ATP (<700 M) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (؉⑀) complexes became shorter, and it was not observed above 1 mM ATP. Analysis of binding and hydrolysis of the ATP analog, 2 ,3 -O-(2,4,6-trinitrophenyl)-ATP, suggested that the (؉⑀) complexes bound substrate only slowly. Differing from Escherichia coli F 1 -ATPase, the activation of the (؉⑀) complexes from the lag phase was not due to dissociation of ⑀ subunit since the re-isolated activated complex retained ⑀ subunit. This indicates that there are two alternative forms of the (؉⑀) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

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“…It is a preferred ATP analogue for photochemical crosslinking with azide derivatives (5). These applications utilize the enhancement of fluorescence or absorption of visible light of the TNP group upon binding to a protein (6). Because of its sensitivity, competition with ATP/ADP has been a valuable means for examining mutational effects on nucleotide affinity (7).…”

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confidence: 99%

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺

Toyoshima

Yonekura

Tsueda

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca 2þ -ATPase, a representative P-type ATPase, in the absence of Ca 2þ with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca 2þ -ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca 2þ -ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.crystallography | ion pump | nucleotide derivatives T rinitrophenyl (TNP)-nucleotides (1) are often used for probing the structure of ATP-binding sites and conformational changes arising from nucleotide binding (2, 3), and for measuring the affinity of ATP by competition experiments (2, 4). It is a preferred ATP analogue for photochemical crosslinking with azide derivatives (5). These applications utilize the enhancement of fluorescence or absorption of visible light of the TNP group upon binding to a protein (6). Because of its sensitivity, competition with ATP/ADP has been a valuable means for examining mutational effects on nucleotide affinity (7). Thus, TNP nucleotides have been widely used with F1 (8), myosin (1), and P-type ATPases (2-5, 7, 9, 10), among others.Nonetheless, whether TNP derivatives are good mimics of authentic adenine nucleotides (AxPs) may be questionable. In several proteins TNP nucleotides have much higher affinities than the genuine AxPs. For instance, TNP-ATP is a high affinity (nM) antagonist of P2X receptors, which have IC 50 for ATP (or AMPPCP) in the μM range (11). The affinity is at least one order of magnitude higher in the E2 states of Ca 2þ -ATPase (3, 12) and Na þ , K þ -ATPase (4), representative P-type ATPases. Furthermore, TNP-AMP binds to Ca 2þ -ATPase similarly to or even more strongly than TNP-ATP (12), in marked contrast to AxPs. Thus, a substantially different binding mode of TNP derivatives is suggested. Although more than 20 entries are registered in the Protein Data Bank (PDB) for Ca 2þ -ATPase (reviewed in ref. 13), no structure with a bound TNP nucleotide exists. In fact, only three crystal structures have been published with bound TNP nucleotides. They are a bacterial h...

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Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

Cited by 65 publications

References 28 publications

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺

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Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

Cited by 65 publications

References 28 publications

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca 2+ -ATPase in the absence of Ca 2+

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Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺