Fluorescent and colored trinitrophenylated analogs of ATP and GTP

Hiratsuka, Toshiaki

doi:10.1046/j.1432-1033.2003.03748.x

Cited by 58 publications

(89 citation statements)

References 38 publications

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“…2c). As noted before (27), and as observed for many other proteins (20), the affinity for MgTNP-ATP and MgTNP-ADP is Ϸ10-fold higher than for the natural nucleotides, MgATP and MgADP. (Fig.…”

Section: Subunitsupporting

confidence: 58%

“…As the trinitrophenyl group of 2Ј,3Ј-O- (2,4,6-trinitrophenyl)adenosine 5Ј-triphosphate (TNP-ATP) and 2Ј,3Ј-O-(2,4,6-trinitrophenyl)adenosine 5Ј-diphosphate (TNP-ADP) forms a Meisenheimer complex (a spiro complex with a negative charge on the trinitrophenyl moiety) with the 2Ј and 3Ј oxygen atoms of the ribose (20), the 2Ј oxygen was chosen as reference point. The ratio of both distances is plotted in Fig.…”

Section: Subunitmentioning

confidence: 99%

“…Thus, the identified N-and C-terminal regions of ␥ should be eminently suitable for the insertion of Trp residues for the intended energy transfer experiments. Binding of TNP-ATP or TNP-ADP to the spatially closer closed site should quench the fluorescence of the inserted Trp residue considerably due to energy transfer, whereas, because of the distance dependence of the transfer efficiency (20,23), binding to the more remote closed site (and binding to the half-closed site) should reduce the fluorescence by significantly less. The third asymmetric region of ␥ identified here, ␥176-␥179, appears to be less suitable, because even binding of the nucleotide analog to the closest catalytic site (␤ TP ) would result in little quenching of the fluorescence of an inserted Trp.…”

Section: Subunitmentioning

confidence: 99%

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Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

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

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ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each ␤ subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central ␥ subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into ␥ and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of ␥, but at considerable distance from its N terminus. Thus, the ␤TP site in the ATP synthase ͉ catalytic mechanism ͉ rotation F 1 F o -ATP synthase is responsible for the bulk of ATP synthesis from ADP and P i in most organisms. F 1 F o -ATP synthase consists of the membrane embedded F o subcomplex with, in Escherichia coli, a subunit composition of ab 2 c 10 , and the peripheral F 1 subcomplex, with a subunit composition of ␣ 3 ␤ 3 ␥␦ . The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, sodium ion) gradient. Proton flow, down the gradient, through F o is coupled to ATP synthesis on F 1 by a unique rotary mechanism. The protons flow through channels at the interface of a and c subunits, which drives rotation of the ring of c subunits. The c 10 ring, together with F 1 subunits ␥ and , forms the rotor. Rotation of ␥ leads to conformational changes in the catalytic nucleotide binding sites on the ␤ subunits, where ADP and P i are bound. The conformational changes result in formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in form of subunit rotation, and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see refs.

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Section: Subunitsupporting

confidence: 58%

Section: Subunitmentioning

confidence: 99%

Section: Subunitmentioning

confidence: 99%

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Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

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

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“…98 Going forward, such treatment strategies may also make use of energy transfer systems 116 to stabilize cellular metabolic capacity and to allow the stressed or dying cell to fully utilize Gibbs free energy during ATP hydrolysis. 117 Most crucially, the role of autophagy in delaying the onset of cell death by the modification of cellular metabolic performance deserves more study.…”

Section: Conclusion and Future Outlookmentioning

confidence: 99%

Cell death: A dynamic response concept

Loos

Engelbrecht²

2009

Autophagy

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Autophagy, apoptosis and necrosis have previously been described as distinct static processes that induce and execute cell death. Due to an increased use of novel techniques in mapping cellular death-techniques which allow for reporting of real-time data-the existence of "grey zones" between cell death modes and the existence of the "point of no return" within these have been revealed. This revelation demands the integration of new concepts in describing the cellular death process. Furthermore, since the contribution of autophagy in cell death or cell survival is still poorly understood, it is important to accurately describe its function within the dynamic framework of cell death.In this review cell death is viewed as a dynamic and integrative cellular response to ensure the highest likelihood of self-preservation. Suggestions are offered for conceptualizing cell death modes and their morphological features, both individually and in relation to one another. It addresses the need for distinguishing between dying cells and dead cells so as to better locate and control the onset of cell death. Most importantly, the fundamental role of autophagy, autophagic flux, and the effects of the intracellular metabolic environment on the kinetics of the cell death modes are stressed. It also contextualizes the kinetic dimension of cell death as a process and aims to contribute towards a better understanding of autophagy as a key mechanism within this process. Understanding the dynamic nature of the cell death process and autophagy's central role can reveal new insight for therapeutic intervention in preventing cell death.

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“…These distances allow fluorescence resonance energy transfer (FRET) (Lakowicz, 1999) from tryptophan (excitation wavelength, 280 nm; emission wavelength, 350 nm) to MANT (excitation wavelength, 350 nm; emission wavelength, 450 nm) Mou et al, 2005Mou et al, , 2006. Third, TNP nucleotides are environmentally sensitive fluorescence probes, too (Hiratsuka, 2003). Like MANT nucleotides, TNP nucleotides show a blue shift in emission in a hydrophobic environment (Hiratsuka, 2003).…”

mentioning

confidence: 99%

Molecular Analysis of the Interaction ofBordetella pertussisAdenylyl Cyclase with Fluorescent Nucleotides

Göttle¹,

Dove²,

Steindel³

et al. 2007

Mol Pharmacol

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The calmodulin (CaM)-dependent adenylyl cyclase (AC) toxin from Bordetella pertussis (CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2Ј,3Ј-Nmethylanthraniloyl (MANT)-, anthraniloyl-and trinitrophenyl (TNP)-substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5-to 170-fold increased potency of nucleotides. K i values of 3ЈMANT-2Јd-ATP and 2ЈMANT-3Јd-ATP in the AC activity assay using Mn 2ϩ were 220 and 340 nM, respectively. Natural nucleoside 5Ј-triphosphates, guanine-, hypoxanthine-and pyrimidine-MANTand TNP nucleotides and di-MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer (FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K d values of 300 nM for 3ЈMANT-2Јd-ATP and 400 nM for 2ЈMANT-3Јd-ATP. Fluorescence experiments and docking approaches indicate that the MANT-and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM-dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to lowaffinity MANT nucleotides, even low-affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design.

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Fluorescent and colored trinitrophenylated analogs of ATP and GTP

Cited by 58 publications

References 38 publications

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Cell death: A dynamic response concept

Molecular Analysis of the Interaction ofBordetella pertussisAdenylyl Cyclase with Fluorescent Nucleotides

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Fluorescent and colored trinitrophenylated analogs of ATP and GTP

Cited by 58 publications

References 38 publications

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Cell death: A dynamic response concept

Molecular Analysis of the Interaction ofBordetella pertussisAdenylyl Cyclase with Fluorescent Nucleotides

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Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site