Assuntos
ATPases Translocadoras de Prótons , Trifosfato de Adenosina/biossíntese , Animais , Transporte Biológico , Escherichia coli/enzimologia , Bombas de Próton/química , Bombas de Próton/fisiologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/fisiologia , Prótons , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/fisiologiaRESUMO
Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm. A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported. The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala. These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.
Assuntos
ATPases Translocadoras de Prótons/metabolismo , Serina/química , Actinas/química , Trifosfato de Adenosina/metabolismo , Alanina/química , Escherichia coli/química , Escherichia coli/enzimologia , Glicina/química , Hidrólise , Isoleucina/química , Modelos Moleculares , Mutação , Fenilalanina/química , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.
Assuntos
Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrólise , Plasmídeos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/química , Venturicidinas/farmacologiaRESUMO
Proton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (gammaepsilonc(10-12)) which is formed from peripheral and intrinsic membrane subunits.
Assuntos
Proteínas de Membrana/química , ATPases Translocadoras de Prótons/química , Animais , Catálise , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Motores Moleculares , Estrutura Molecular , Mutação , ATPases Translocadoras de Prótons/genética , Prótons , RotaçãoRESUMO
Acidic pH is known to cause pain sensation through nociceptive neurons as well as taste transduction in mammals. Caenorhabditis elegans avoids an acidic environment (pH lower than approximately 4.0) formed by organic or inorganic acids. This avoidance behavior was dependent on multiple amphid chemosensory neurons, and inhibited by a mutation of capsaicin receptor homologue, and by the addition of amiloride and ruthenium red (inhibitors of proton-gated Na+ channels and capsaicin receptors, respectively). These results indicate that C. elegans recognizes protons as a nociceptive stimulus, through multiple neurons, which elicits avoidance behavior. It is of special interest that a system similar to that of mammalian signal transduction is responsible for this nematode's acid avoidance.
Assuntos
Aprendizagem da Esquiva/fisiologia , Neurônios Aferentes/fisiologia , Dor , Amilorida/farmacologia , Animais , Caenorhabditis elegans , Células Quimiorreceptoras/fisiologia , Concentração de Íons de Hidrogênio , Receptores de Superfície Celular , Receptores de Droga/genética , Receptores de Droga/fisiologia , Rutênio Vermelho/farmacologia , Bloqueadores dos Canais de Sódio , Canais de Sódio/fisiologiaRESUMO
Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c(552) through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c(552), there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Grupo dos Citocromos c/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Estabilidade Enzimática , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Desnaturação Proteica , TemperaturaRESUMO
Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.
Assuntos
ATPases Translocadoras de Prótons/química , Bactérias/enzimologia , Sítios de Ligação , Cloroplastos/enzimologia , Cinética , Modelos Moleculares , Proteínas Motores Moleculares , Mutação , Nucleotídeos/metabolismo , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , PrótonsRESUMO
Proton-translocating ATP synthase (F(o)F(1)) synthesizes ATP from ADP and phosphate, coupled with an electrochemical proton gradient across the biological membrane. It has been established that the rotation of a subunit assembly is an essential feature of the enzyme mechanism and that F(o)F(1) can be regarded as a molecular motor. Thus, experimentally, in the reverse direction (ATP hydrolysis), the chemical reaction drives the rotation of a gammaepsilonc(10-14) subunit assembly followed by proton translocation. We discuss our very recent results regarding subunit rotation in Escherichia coli F(o)F(1) with a combined biophysical and mutational approach.
Assuntos
ATPases Bacterianas Próton-Translocadoras/química , ATPases Bacterianas Próton-Translocadoras/metabolismo , Substituição de Aminoácidos , ATPases Bacterianas Próton-Translocadoras/genética , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Subunidades Proteicas , Força Próton-Motriz , TermodinâmicaRESUMO
A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Pseudomonas aeruginosa/enzimologia , Substituição de Aminoácidos , Grupo dos Citocromos c/genética , Estabilidade Enzimática , Guanidina , Espectroscopia de Ressonância Magnética , Mutação , Conformação Proteica , Desnaturação ProteicaRESUMO
F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Biotinilação , Transferência de Energia , Enzimas Imobilizadas , Escherichia coli/enzimologia , Hidrólise , Força Próton-Motriz , Desacopladores/metabolismo , Desacopladores/farmacologia , Venturicidinas/farmacologia , Gravação em VídeoRESUMO
The rotation of the gamma-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (FOF1). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the gamma-subunit of the F1 sector rotated on addition of ATP. This progress enabled us to analyze the gammaM23K (the gamma-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the gammaM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.
Assuntos
Actinas/química , Escherichia coli/enzimologia , Escherichia coli/genética , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Substituição de Aminoácidos , Cinética , Substâncias Macromoleculares , Mutagênese Insercional , Óperon , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotação , TorqueRESUMO
We developed a quantitative assay for Caenorhabditis elegans avoidance behavior. This was then used to demonstrate that the worm moved away from toxic concentrations of Cd2+ and Cu2+, but not Ni2+, all ions that prevented development from larval to adult stages. Mutants that have structural defects in ciliated neurons (che-2 and osm-3) as well as worms with three laser-operated neurons (ADL, ASE, and ASH), showed no avoidance behavior from Cd2+ and Cu2+. These results suggest that the avoidance from Cd2+ and Cu2+ are mediated through multiple neural pathways including ADL, ASE, and ASH neurons. We hypothesize that the three sensing neurons provide increased accuracy of the sensory response and a survival advantage in the natural environment of the worm.
Assuntos
Cádmio/farmacologia , Caenorhabditis elegans/fisiologia , Células Quimiorreceptoras/fisiologia , Cobre/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/fisiologia , Cílios/fisiologia , Larva , Terapia a Laser , Mutação , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios Aferentes/fisiologia , Níquel/farmacologiaRESUMO
A cDNA encoding a putative copper chaperone protein, CUC-1, was cloned from Caenorhabditis elegans. CUC-1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively. Expression of CUC-1 cDNA complemented a null atx1 mutant, the yeast copper chaperone gene, thus demonstrating that CUC-1 is a functional copper chaperone. Studies with transgenic worms indicated that cuc-1 and cua-1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae. cua-1 was also expressed in pharyngeal muscle but cuc-1 was not. These results suggest that CUC-1 and CUA-1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA-1 may have a different function in pharyngeal muscle.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas de Transporte de Cátions , Clonagem Molecular , Cobre/metabolismo , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Sequência Conservada , Proteínas de Transporte de Cobre , Proteínas Fúngicas/genética , Expressão Gênica , Genes Reporter , Teste de Complementação Genética , Humanos , Intestinos , Larva , Metalochaperonas , Chaperonas Moleculares/química , Dados de Sequência Molecular , Especificidade de Órgãos , Músculos Faríngeos , Saccharomyces cerevisiae/genéticaRESUMO
Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast delta ccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast delta ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.
Assuntos
Adenosina Trifosfatases/química , Caenorhabditis elegans/enzimologia , Cobre/farmacocinética , Oligopeptídeos/análise , Sequência de Aminoácidos , Animais , Transporte Biológico , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver. The ATP7B gene, which encodes a copper transporting P-type ATPase, is defective in patients with Wilson disease. To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (delta ccc2), the yeast homologue of ATP7B. Wild type and the H1069Q mutant could rescue delta ccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease. In addition, the mutant containing only the sixth copper binding domain could rescue delta ccc2, indicating its functional importance.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Saccharomyces cerevisiae/crescimento & desenvolvimento , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , Cobre/metabolismo , ATPases Transportadoras de Cobre , DNA Complementar , Degeneração Hepatolenticular/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
The solution structure of a thermostable cytochrome c-552 from a thermophilic hydrogen oxidizing bacterium Hydrogenobacter thermophilus was determined by proton nuclear magnetic resonance spectroscopy. Twenty structures were calculated by the X-PLOR program on the basis of 902 interproton distances, 21 hydrogen bonds, and 13 torsion angle constraints. The pairwise average root-mean-square deviation for the main chain heavy atoms was 0.91 +/- 0.11 A. The main chain folding of the cytochrome c-552 was almost the same as that of Pseudomonas aeruginosa cytochrome c-551 that has 59% sequence identity to the cytochrome c-552 but is less thermostable. We found several differences in local structures between the cytochromes c-552 and c-551. In the cytochrome c-552, aromatic-amino interactions were uniquely formed between Arg 35 and Tyr 32 and/or Tyr 41, the latter also having hydrophobic contacts with the side chains of Tyr 32, Ala 38, and Leu 42. Small hydrophobic cores were more tightly packed in the cytochrome c-552 because of the occupancies of Ala 5, Met 11, and Ile 76, each substituted by Phe 7, Val 13, and Val 78, respectively, in the cytochrome c-551. Some of these structural differences may contribute to the higher thermostability of the cytochrome c-552.
Assuntos
Bactérias Aeróbias/enzimologia , Proteínas de Bactérias , Grupo dos Citocromos c/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Arginina/química , Cristalografia por Raios X , Heme/química , Ligação de Hidrogênio , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Prótons , Pseudomonas aeruginosa/enzimologia , Homologia de Sequência de Aminoácidos , Soluções , Termodinâmica , Tirosina/químicaRESUMO
The biogenesis of bacterial c-type cytochromes generally involves many gene products--some of which may also have roles in other processes--and their interaction with the disulphide-bond-forming system of the bacterial periplasm. However, in some bacteria a simpler process appears to operate that might be related to the formation of c-type cytochromes in thylakoids of photosynthetic cells. The corresponding process in fungal mitochondria is distinct.
Assuntos
Bactérias/metabolismo , Cloroplastos/metabolismo , Grupo dos Citocromos c/biossíntese , Mitocôndrias/metabolismo , Bactérias/genética , Proteínas de Bactérias/biossíntese , Citoplasma/metabolismo , Dissulfetos , HemeRESUMO
The full-length cDNA coding for a putative copper transporting P-type ATPase (Cu2+-ATPase) was cloned from Caenorhabditis elegans. The putative Cu2+-ATPase is a 1,238-amino acid protein, and highly homologous to the Menkes and Wilson disease gene products mutations of which are responsible for human defects of copper metabolism. The Saccharomyces cerevisiae mutant with a disrupted CCC2 gene (yeast Menkes/Wilson disease gene homologue) was rescued by the cDNA for the C. elegans Cu2+-ATPase but not by the cDNA with an Asp-786 (an invariant phosphorylation site) to Asn mutation, suggesting that the C. elegans Cu2+-ATPase functions as a copper transporter in yeast. The expressed C. elegans protein was detected in yeast vacuolar membranes by immunofluorescence microscopy. The yeast expression system may facilitate further studies on copper transporting P-type ATPases.
Assuntos
Caenorhabditis elegans/genética , Deleção de Genes , Genes Fúngicos , Degeneração Hepatolenticular/genética , Síndrome dos Cabelos Torcidos/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Mutação , Homologia de Sequência do Ácido NucleicoRESUMO
Absence through mutation of two proteins involved in periplasmic disulphide bond formation, DsbA and DsbB, results in failure of anaerobically grown Escherichia coli to synthesise the holo forms of either its endogenous c-type cytochrome nitrite reductase or exogenous cytochrome c550 from Paracoccus denitrificans. The synthesis of both cytochromes can be restored to the mutants by inclusion in the growth media of compounds containing disulphide bonds, e.g., the oxidised form of glutathione. The results suggest that the attachment of haem to the CXXCH motif of a periplasmic c-type cytochrome may be preceeded by the formation of one or more intra- or intermolecular disulphide bonds involving the cysteine residues of this motif.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/biossíntese , Escherichia coli/metabolismo , Glutationa/farmacologia , Isomerases/metabolismo , Proteínas de Membrana/metabolismo , Fosfatase Alcalina/biossíntese , Proteínas de Bactérias/genética , Meios de Cultura , Escherichia coli/enzimologia , Escherichia coli/genética , Isomerases/genética , Proteínas de Membrana/genética , Mutação , Oxirredução , Isomerases de Dissulfetos de ProteínasRESUMO
The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.