Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.
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PMID:Synthesis of a membrane protein with two transmembrane regions. 1199 Dec 6

Subunit a of the Escherichia coli ATP synthase, a 30 kDa integral membrane protein, was purified to homogeneity by a novel procedure incorporating selective extraction into a monophasic mixture of chloroform, methanol and water, followed by Ni-NTA chromatography in the mixed solvent. Pure subunit a was reconstituted with subunits b and c and phospholipids to form a functional proton-translocating unit. Nuclear magnetic resonance (NMR) spectra of the pure subunit a in the mixed solvent show good chemical shift dispersion and demonstrate the potential of the solvent mixture for NMR studies of the large membrane proteins that are currently intractable in aqueous detergent solutions.
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PMID:Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent. 1470 21

The structure of the 30 KDa subunit a of the membrane component (F(0)) of E. coli ATP synthase is investigated in a mixture of chloroform, methanol and water, a solvent previously used for solving the structure of another integral membrane protein, subunit c. Near complete backbone chemical shift assignments were made from a set of TROSY experiments including HNCO, HNCA, HN(CA)CB, HN(CO)CACB and 4D HNCOCA and HNCACO. Secondary structure of subunit a was predicted from the backbone chemical shifts using TALOS program. The protein was found to consist of multiple elongated alpha-helical segments. This finding is generally consistent with previous predictions of multiple transmembrane alpha-helices in this polytopic protein.
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PMID:Backbone 1H, 15N and 13C assignments for the subunit a of the E. coli ATP synthase. 1521 58

Mitochondrial ATP synthase (F1Fo-ATPase) catalyzes the terminal step of oxidative phosphorylation. In this paper, we demonstrate the functional expression of the hexahistidine-tagged beta-subunit of yeast ATP synthase and the purification of the F1-ATPase from yeast cells. A gene encoding the beta-subunit from Saccharomyces cerevisiae was modified to encode a protein of which the original N-terminus import signal sequence was replaced by a sequence containing the import signal sequence of a mitochondrial ATPase inhibitor, its processing site, and six consecutive histidines. Expression of the modified gene generated a functional F1Fo complex in host yeast cells lacking a functional copy of the endogenous ATP2 gene, as judged by growth of rescued cells on lactate medium. F1 was extracted from the yeast mitochondria by chloroform treatment and purified by immobilized metal affinity chromatography and gel filtration chromatography. The specific activity of the purified F1 was comparable to that of the wild-type enzyme, and the F1 contained all of the 5 known subunits (alpha, beta, gamma, delta, and epsilon). Moreover, the activity of the F1 was completely inhibited by the specific ATPase inhibitor protein, IF1. These results indicate that F1 containing the tagged beta-subunit is fully assembled and active. The application of this novel procedure simplifies the number of steps required for the isolation of F1 used for studying the molecular mechanism of catalysis and regulation of the enzyme.
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PMID:Functional expression of hexahistidine-tagged beta-subunit of yeast F1-ATPase and isolation of the enzyme by immobilized metal affinity chromatography. 1529 86

The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.
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PMID:Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase. 1535 74

In F(o)F(1)-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F(o)-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF(o)c protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F(o) motor coupled with H(+)-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na(+)-ATPase.
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PMID:A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation. 1649 28

The properties of the soluble moiety (F(1)) of the mitochondrial H(+)-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F(1) preparation contained major polypeptides corresponding to alpha, beta, gamma, delta, and epsilon of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F(1)-ATPase, like the native enzyme, was inhibited by azide (I(50) = 10 micromolar), nitrate (I(50) = 7-10 millimolar), 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (I(50) = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I(50) = 3 micromolar). F(1)-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F(1)-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F(1). In contrast to the membrane-bound enzyme, the F(1)-ATPase was not inhibited by oligomycin or by N,N'-dicyclohexylcarbodiimide. The mitochondrial F(1)-ATPase from oat roots is analogous to other known F(1)F(0)-ATPases.
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PMID:Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria. 1666 52

An amiloride-resistant mutant with diminished Na+/H+ antiporter activity was isolated from Methanothermobacter thermoautotrophicus. To define the protein basis of amiloride resistance, the composition of membrane-associated proteins was partially characterized and compared with that of the wild type strain. An abundant 670-kDa membrane-associated protein that was present only in the mutant strain was analyzed by MALDI-TOF MS and identified as a coenzyme F420-reducing hydrogenase. The amiloride resistance was not accompanied by changes in protein size or changes in the level of subunits A or B of the A1A0-type ATP synthase; on the other hand, the SDS-PAGE patterns of the chloroform-methanol extract of membranes from both strains were different. Two bands with calculated molecular mass 16 and 11 kDa were identified as MtrD and AtpK, respectively. The observed over-expression of a 22.7-kDa protein in the mutant cells may represent the multimeric form of the MtrD subunit. These results show that the impairment of the Na+/H+ antiporter system in the amiloride-resistant mutant of Methanothermobacter thermoautotrophicus is accompanied by only small changes in a few membrane-associated proteins.
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PMID:Amiloride resistance in the methanoarcheon Methanothermobacter thermoautotrophicus: characterization of membrane-associated proteins. 1700 34

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
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PMID:Identification of two proteins associated with mammalian ATP synthase. 1757 25

Mixtures of organic solvents are often used as membrane mimetics in structure determination of transmembrane proteins by solution NMR; however, the mechanism through which these isotropic solvents mimic the anisotropic environment of cell membranes is not known. Here, we use molecular dynamics simulations to study the solvation thermodynamics of the c-subunit of Escherichia coli F1F0 ATP synthase in membrane mimetic mixtures of methanol, chloroform, and water with varying fractions of components as well as in lipid bilayers. We show that the protein induces a local phase separation of the solvent components into hydrophobic and hydrophilic layers, which provides the anisotropic solvation environment to stabilize the amphiphilic peptide. The extent of this effect varies with solvent composition and is most pronounced in the ternary methanol-chloroform-water mixtures. Analysis of the solvent structure, including the local mole fraction, density profiles, and pair distribution functions, reveals considerable variation among solvent mixtures in the solvation environment surrounding the hydrophobic transmembrane region of the protein. Hydrogen bond analysis indicates that this is primarily driven by the hydrogen-bonding propensity of the essential Asp(61) residue. The impact of the latter on the conformational stability of the solvated protein is discussed. Comparison with the simulations in explicit all-atom models of lipid bilayer indicates a higher flexibility and reduced structural integrity of the membrane mimetic solvated c-subunit. This was particularly true for the deprotonated form of the protein and found to be linked to solvent stabilization of the charged Asp(61).
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PMID:Solvation of transmembrane proteins by isotropic membrane mimetics: a molecular dynamics study. 1778 46


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