Fluorescent analogs of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel

Pringle, Michael J.; Taber, Magdalena

doi:10.1021/bi00346a051

Cited by 21 publications

(14 citation statements)

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“…A conserved localization of 1.3 ± 2.4 Å and 1.8 ± 2.8 Å from the center of the bilayer was found for the ATP synthases of I. tartaricus and E. coli, respectively. These data correspond very well with a distance of 18 Å from the membrane surface in case of the mitochondrial enzyme [23]. Data supporting membrane localization were also found for the chloroplast enzyme [49,50].…”

Section: Discussionsupporting

confidence: 86%

“…To validate and extend this new finding we investigated the localization of the binding site both for the Na + ‐translocating ATP synthase of I. tartaricus and for the H + ‐translocating ATP synthase of E. coli by parallax analysis of fluorescence quenching. The method was originally described by Chattopadhyay and London [22] and has been applied successfully for the localization of the DCCD binding residues in bovine F 1 F 0 ATP synthase [23], vacuolar H + ‐ATPase [24] and other proteins [25,26]. We show here a conserved localization of the binding site in Na + ‐ or H + ‐translocating ATP synthases.…”

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confidence: 79%

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Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Ballmoos

Meier

Dimroth

2002

European Journal of Biochemistry

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Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na + -translocating F 1 F 0 ATP synthase of Ilyobacter tartaricus within the hydrophobic part of the bilayer. Similarly, a membrane embedded H + -binding site is accepted for the H + -translocating F 1 F 0 ATP synthase of Escherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from both I. tartaricus and E. coli. Both ATP synthases were specifically labelled at their c subunit sites with N-cyclohexyl-N¢-(1-pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined for I. tartaricus and E. coli to be 1.3 ± 2.4 Å and 1.8 ± 2.8 Å from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na + ions to the binding sites in the reconstituted I. tartaricus c 11 oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane.Keywords: coupling ion binding site; parallax analysis; membrane localization; c subunit; ATP synthase.Structurally similar F 1 F 0 ATP synthases are present in mitochondria, chloroplasts or eubacteria, where they catalyze ATP formation with the energy stored in a transmembrane electrochemical gradient of protons or Na + ions (reviewed in [1]). The enzyme is composed of an extrinsic membrane domain, F 1 , which harbors the catalytic sites for ATP synthesis. The subunit composition of F 1 is a 3 b 3 cde [2,3]. Alternating a and b subunits form a cylinder around a central a-helical stalk of the c subunit [4][5][6]. Rotation of the c subunit with respect to the a 3 b 3 subcomplex has been directly observed [7]. There is strong evidence to support a mechanism in which the central stalk of the soluble F 1 domain, together with the oligomeric c-ring in the membrane domain, rotates as an assembly coupling ion movement with ATP synthesis or hydrolysis [8][9][10]. The F 0 membrane domain consists of three different subunits in the stoichiometry ab 2 c n (n ¼ 10-14) (reviewed in [11]. The single a subunit and the two b subunits are supposed to contact the c-ring laterally [12][13][14][15]. The number of c subunits forming the ring varies among species, being 10 for yeast mitochondria [5], 14 for spinach chloroplasts [16] and 11 for the Na + translocating F 1 F 0 ATP synthase from Ilyobacter tartaricus [17]. Each monomeric unit folds as a helical hairpin. The N-terminal helices form a tightly packed inner ring and the C-terminal helices form a more loosely packed outer ring [5,18]. Cavities between neighbouring outer helices and the inner ring were suggested to act ...

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Section: Discussionsupporting

confidence: 86%

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confidence: 79%

Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Ballmoos

Meier

Dimroth

2002

European Journal of Biochemistry

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“…Doxyl-stearic acids have been shown to be effective quenchers the lipid/protein molar ratio is about 80, the M r of the ATPase is 110 000, and the avarage M r of the phospholipids is 750. In of NCD-4 fluorescence in cytochrome c oxidase [15], F0F1-ATPase [16] and in cytochrome b 6 [17]. We have used n-Doxyl-these experiments quenching of all the NCD-4 fluorescence was considered.…”

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confidence: 99%

Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca²⁺‐transporting ATPase

Velasco-Guillén¹,

Corbalán-Garcı́a²,

Gómez‐Fernández³

et al. 1998

European Journal of Biochemistry

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The Ca 2ϩ -transporting ATPase has been labeled with N-cyclohexyl-N′-(4-dimethyl-amino-A-naphthyl)-carbodiimide (NCD-4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD-4 labels a transmembrane portion of the protein at the high-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the fluorescence by nitroxide-substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16-nitroxide derivatives). We have found that all the calcium-sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n-(4,4-dimethyl-3-oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD-4 label which is involved in the high-affinity calcium-binding site is located near the water/lipid interface.The fluorescence of the NCD-4 bound to that site can be quenched by acrylamide and Cu 2ϩ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD-4 located at this site. The hydrophobic location of NCD-4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6-tetramethyl-1-piperidine-Noxyl but not by 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-N-oxyl which is much less hydrophobic.Keywords : Ca 2ϩ -transporting ATPase; sarcoplasmic reticulum; fluorescent carbodiimide; fluorescence quenching.Calcium transport through sarcoplasmic reticulum memIt has been also described that the derivatization of sarcoplasmic reticulum ATPase with NCD-4 resulted in inhibition of branes is initiated by cooperative binding of calcium to the calcium-dependent ATP utilization but not of calcium-indepencalcium pump. Two high-affinity calcium-binding sites in the dent partial reactions such as enzyme phosphorylation by inorcalcium-transporting ATPase have been demonstrated: a molar ganic phosphate [6]. stoichiometry of calcium bound/phosphorylation site of 2 :1 was Another approach to locate the high-specific-binding site of found [1]; and one molecule of ATP is hydrolyzed to transport calcium has been site-directed mutagenesis, which showed that two calcium ions [2, 3]. However, little is known concerning the the mutations of any of six amino acids within the transmemprecise localization of these calcium-binding sites in the molebrane region of the ATPase interfere with enzyme activation by cule of Ca 2ϩ -ATPase, and their relationship with the phosphorycalcium [7]. lation site, which is an essential question to understand the moTherefore both types of approaches agree on the membrane lecular mechanism of calcium transport. location of this site, although both of them, and specially the It has been shown that N,N′-dicyclohexylcarbodiimide, a hysite-directed mutagenesis one, are substantially based on the asdrophobic reagent for acidic amino acid residues, inhibits the sumptions made by predictions on domain structure after hydroca...

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“…Fig. 2 (16), and the acetylcholine receptor (17,18). Assumptions were made in our estimates of the distance between the fluorophores.…”

Section: Resultsmentioning

confidence: 99%

Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer.

Peerce¹,

Wright²

1986

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

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Covalent fluorescent probes were used to label the rabbit intestinal brush border Na+-glucose cotrapsporter at the putative glucose and Na' binding sites, and a steady-state fluorescence energy transfer technique was used to measure the distance between the two binding sites. In, both intact brush border membrane vesicles and partially purified soluble protein, the distance (R2/3) between the Na+ and glucose sites was -35 A. This distance was the same with four different donor/acceptor pairs with different transfer efficiencies, by donor quantum yield measurements, or sensitized acceptor fluorescence, The fact that the Na' site and glucose site probes bind to a 75-kDa polypeptide, copurify with the same isoelectric point (pI 5.3) and retain function, and exhibit energy transfer indicates that the sites are on the same 75-kDa polypeptide. The large distance between the Na' and glucose site probes raises questions about simple models of frictional interactions between the two substrates during the transport cycle.Uphill glucose transport across the intestinal brush border membrane occurs by Na'-glucose cotransport (1). We have identified this cotransporter as a 75-kDa polypeptide using fluorescent group-specific reagents: fluorescein isothiocyanates (FITCs) react with lysine residues at or near the glucose binding site, whereas fluorescent N-acetylimidazoles react with tyrosine residues at or near the Na' binding sites (2)(3)(4). In the present study, we have measured the distance between the sodium and glucose binding sites of the glucose cotransporter using the technique of fluorescence energy transfer (5, 6). In both native brush border membrane vesicles and partially purified, soluble proteins, our results demonstrate that the sodium and glucose sites are on the same polypeptide (75 kDa; pI 5.3) and that the Na' site is 35 A away from the glucose site. METHODSRabbit intestinal brush border membrane vesicles were prepared by a Ca2' precipitation procedure and treated With KSCN (3, 4). The vesicles were then resuspended in 300 mM mannitol and 10 mM Hepes/Tris HCl, pH 7.5, and stored at -800C until needed.Chromatofocusing of vesicles was performed as described by Lin et al. (7), except we have substituted the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for octyl glucoside. Chromatofocusing was performed in a 35 cm x 1.5 cm column at a flow rate of 30 ml/hr and over the pH range of 7.2-4.0. Fractions were precipitated with (NH4)2SO4 and resuspended in buffer and the (NH4)2SO4 was removed by passage through a Sephadex G-50 column equilibrated with buffer. Following removal of (NH4)2SO4, the protein was lyophilized and stored at -800C.FITC and other isothiocyanate derivative labeling of the cotransporter at the glucose site and N-acetylimidazole derivative labeling of the cotransporter at the Na' site were carried out as reported (2-4). The conditions were optimal for specific, complete labeling of the sites on the transport protein.NaDodSO4/pQlyacrylamide electro...

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Fluorescent analogs of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel

Cited by 21 publications

References 20 publications

Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca²⁺‐transporting ATPase

Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer.

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Fluorescent analogs of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel

Cited by 21 publications

References 20 publications

Membrane embedded location of Na+or H+binding sites on the rotor ring of F1F0ATP synthases

Membrane embedded location of Na+or H+binding sites on the rotor ring of F1F0ATP synthases

Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca2+‐transporting ATPase

Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer.

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Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Membrane embedded location of Na⁺or H⁺binding sites on the rotor ring of F₁F₀ATP synthases

Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca²⁺‐transporting ATPase