Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.823
Filtrar
1.
Proc Natl Acad Sci U S A ; 117(3): 1447-1456, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896579

RESUMO

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant ßE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.


Assuntos
Proteínas Mitocondriais/química , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Imagem Individual de Molécula
2.
Biochim Biophys Acta Bioenerg ; 1860(8): 679-687, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251901

RESUMO

Functioning as a nanomotor, ATP synthase plays a vital role in the cellular energy metabolism. Interactions at the rotor and stator interface are critical to the energy transmission in ATP synthase. From mutational studies, we found that the γC87K mutation impairs energy coupling between proton translocation and nucleotide synthesis/hydrolysis. An additional glutamine mutation at γR242 (γR242Q) can restore efficient energy coupling to the γC87K mutant. Arrhenius plots and molecular dynamics simulations suggest that an extra hydrogen bond could form between the side chains of γC87K and ßTPE381 in the γC87K mutant, thus impeding the free rotation of the rotor complex. In the enzyme with γC87K/γR242Q double mutations, the polar moiety of γR242Q side chain can form a hydrogen bond with γC87K, so that the amine group in the side chain of γC87K will not hydrogen-bond with ßE381. As a conclusion, the intra-subunit interaction between positions γC87 and γR242 modulates the energy transmission in ATP synthase. This study should provide more information of residue interactions at the rotor and stator interface in order to further elucidate the energetic mechanism of ATP synthase.


Assuntos
Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Aminoácidos/genética , Biocatálise , Metabolismo Energético , Proteínas de Escherichia coli , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/química
3.
Biochem J ; 476(12): 1771-1780, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31164401

RESUMO

The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/genética , Proteínas de Bactérias/genética , Cianobactérias/genética , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/genética
4.
Molecules ; 24(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857224

RESUMO

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.


Assuntos
Membrana Celular/enzimologia , Kluyveromyces/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Western Blotting , Cromatografia em Gel , Substâncias Macromoleculares/metabolismo
5.
Biochim Biophys Acta Bioenerg ; 1860(5): 361-368, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876890

RESUMO

The γ subunit located at the center of ATP synthase (FOF1) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H+ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting FOF1 assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α3ß3-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in FOF1.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/química , Substituição de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação de Sentido Incorreto , Domínios Proteicos , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/genética
6.
J Vis Exp ; (143)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30735175

RESUMO

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.


Assuntos
ATPases Translocadoras de Prótons/química , Trypanosoma brucei brucei/metabolismo , Animais
7.
Molecules ; 24(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704145

RESUMO

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αß)3δab2. Subunits c10 or (αß)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αß)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme's rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ouro , Cinética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Nanotubos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
8.
Artigo em Inglês | MEDLINE | ID: mdl-30196154

RESUMO

The mitochondrial F1FO-ATPase, the key enzyme in cell bioenergetics, apparently works in the same way in mollusks and in mammals. We previously pointed out a raft-like arrangement in mussel gill mitochondrial membranes, which apparently distinguishes bivalve mollusks from mammals. To explore the relationship between the microenvironmental features and the enzyme activity, the physico-chemical features of mitochondrial membranes and the F1FO-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum). Similarly to the mussel, clam gill mitochondrial membrane lipids exhibit a high sterol content (42 mg/g protein), mainly due to phytosterols (cholesterol only attains 42% of total sterols), and abundant polyunsaturated fatty acids (PUFA) (70% of total fatty acids), especially of the n-3 family. However, the F1FO-ATPase activation energies above and below the break in the Arrhenius plot (22.1 °C) are lower than in mussel and mammalian mitochondria. Laurdan fluorescence spectroscopy analyses carried out at 10 °C, 20 °C and 30 °C on mitochondrial membranes and on lipid vesicles obtained from total lipid extracts of mitochondria, indicate a physical state without coexisting domains. This mitochondrial membrane constitution, allowed by lipid-lipid and lipidprotein interactions and involving PUFA-rich phospholipids, phytosterols (much more diversified in clams than in mussels) and proteins, enables the maintenance of a homogeneous physical state in the range 10-30 °C. Consistently, this molecular interaction network would somehow extend the temperature range of the F1FO-ATPase activity and may contribute to clam resilience to temperature changes.


Assuntos
Bivalves/fisiologia , Mudança Climática , Metabolismo dos Lipídeos , Membranas Mitocondriais/metabolismo , Modelos Biológicos , ATPases Translocadoras de Prótons/metabolismo , Animais , Bivalves/enzimologia , Bivalves/crescimento & desenvolvimento , Ativação Enzimática , Estabilidade Enzimática , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-3/química , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/química , Feminino , Temperatura Alta/efeitos adversos , Itália , Bicamadas Lipídicas , Lipossomos , Masculino , Mar Mediterrâneo , Microdomínios da Membrana/química , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Membranas Mitocondriais/química , Fitosteróis/análise , Fitosteróis/metabolismo , ATPases Translocadoras de Prótons/química , Especificidade da Espécie , Esteróis/análise , Esteróis/metabolismo
9.
J Am Chem Soc ; 140(44): 14860-14869, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30339028

RESUMO

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3ß3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.


Assuntos
Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , ATPases Translocadoras de Prótons/metabolismo , Medição da Troca de Deutério , Escherichia coli/metabolismo , Espectrometria de Massas , ATPases Translocadoras de Prótons/química
10.
Biochem Biophys Res Commun ; 504(4): 709-714, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30213631

RESUMO

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.


Assuntos
Proteínas de Bactérias/química , Proteínas Motores Moleculares/química , ATPases Translocadoras de Prótons/química , Imagem Individual de Molécula/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Polarização de Fluorescência , Cinesina/química , Cinesina/metabolismo , Cinética , Microscopia de Fluorescência , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , Rotação
11.
J Biol Chem ; 293(44): 17095-17106, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217814

RESUMO

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a "head-to-tail" organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/genética , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Dimerização , Ligação Proteica , Domínios Proteicos , ATPases Translocadoras de Prótons/genética
12.
Acc Chem Res ; 51(9): 1911-1920, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30160941

RESUMO

Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors' active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature's nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme's diffusion due to active self-propulsion must be observed on top of the enzyme's passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme's substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme's diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes' catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.


Assuntos
Fosfatase Alcalina/química , ATPases Translocadoras de Prótons/química , Animais , Bovinos , Difusão , Fluorescência , Corantes Fluorescentes/química , Mucosa Intestinal/enzimologia , Conformação Proteica , Espectrometria de Fluorescência/métodos , Succinimidas/química
13.
Biochem J ; 475(18): 2925-2939, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30054433

RESUMO

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Šresolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a ß-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this ß-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3ß3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3ß3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the ß-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the ß-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Hidrólise , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/metabolismo
14.
Sci Rep ; 8(1): 11361, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30054535

RESUMO

ATP synthase is powered by the flow of protons through the molecular turbine composed of two α-helical integral membrane proteins, subunit a, which makes a stator, and a cylindrical rotor assembly made of multiple copies of subunit c. Transient protonation of a universally conserved carboxylate on subunit c (D61 in E. coli) gated by the electrostatic interaction with arginine on subunit a (R210 in E. coli) is believed to be a crucial step in proton transfer across the membrane. We used a fusion protein consisting of subunit a and the adjacent helices of subunit c to test by NMR spectroscopy if cD61 and aR210 are involved in an electrostatic interaction with each other, and found no evidence of such interaction. We have also determined that R140 does not form a salt bridge with either D44 or D124 as was suggested previously by mutation analysis. Our results demonstrate the potential of using arginines as NMR reporter groups for structural and functional studies of challenging membrane proteins.


Assuntos
Modelos Moleculares , Engenharia de Proteínas , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Sais/química , Arginina/química , Ácido Aspártico/química , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
15.
Int J Biol Macromol ; 116: 977-982, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29782980

RESUMO

The current study establishes the necessity of Glu-381, Glu-384, and Glu-385 in the ßDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. Inhibitory profiles of wild type and mutant E. coli membrane bound F1Fo ATP synthase were studied in the presence and absence of C-terminal NH2 bound melittin. Melittin-NH2 caused almost complete inhibition of wild type ATPase, while partial inhibition was observed for mutants where Glu was replaced with Ala, Arg, or Gln. Additionally, melittin-NH2 caused insignificant inhibition of a triple mutant where all three Glu residues were replaced with Ala residues, changing ßDELSEED-motif to ßDALSAAD-motif. Little or partial loss of oxidative phosphorylation in mutant strains corroborates their distinct location away from the catalytic site of ATP synthase. Moreover, abrogation of wild type E. coli cell growth and normal growth of mutant stains in the presence of melittin-NH2 further validated the necessity of Glu residues in the ßDELSEED-motif for peptide binding. Overall, while loss of one Glu residue at a time may allow partial peptide binding, loss of three Glu residues together-ßE381, ßE384, and ßE385-is detrimental for peptide binding and inhibition of ATP synthase.


Assuntos
Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Meliteno/química , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/química , Motivos de Aminoácidos , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(22): 5750-5755, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29760063

RESUMO

The angular velocity profile of the 120° F1-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a ΔµATP = -31.25 kBT at a time resolution of 10 µs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 kBT·rad-2). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative Ea values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 kBT·rad-2; equilibrium position, 90°) was also evident that mitigated the enthalpic cost of phase 2 rotation. The maximum ΔGǂ was 22.6 kBT, and maximum efficiency was 72%. An elastic coupling mechanism is proposed that uses the coiled-coil domain of the γ-subunit rotor as a torsion spring during phase 1, and then as a crankshaft driven by ATP-binding-dependent conformational changes during phase 2 to drive the power stroke.


Assuntos
Modelos Moleculares , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fenômenos Bioquímicos , Elasticidade , Termodinâmica
17.
PLoS One ; 13(3): e0193228, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29505581

RESUMO

Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. ATP13A1 complemented a deletion of the yeast P5A ATPase SPF1, while none of ATP13A2-5 could complement either the loss of SPF1 or that of the single P5B ATPase YPK9 in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.


Assuntos
Evolução Molecular , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Fosforilação , Filogenia , Conformação Proteica em alfa-Hélice , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , ATPases Translocadoras de Prótons/química
18.
Nat Commun ; 9(1): 501, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402931

RESUMO

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Membrana Celular/metabolismo , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Membrana Celular/ultraestrutura , Difusão , Recuperação de Fluorescência Após Fotodegradação , Cinética , Microdomínios da Membrana , Conformação Proteica , Transporte Proteico , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Biochim Biophys Acta Bioenerg ; 1859(5): 319-325, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29470949

RESUMO

F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3ß3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/genética , Domínios Proteicos , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Deleção de Sequência
20.
Phys Chem Chem Phys ; 20(3): 1872-1880, 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29292807

RESUMO

F1-ATPase (F1) is an efficient rotary protein motor, whose reactivity is modulated by the rotary angle to utilize thermal fluctuation. In order to elucidate how its kinetics are affected by the change in the fluctuation, we have extended the reaction-diffusion formalism [R. Watanabe et al., Biophys. J., 2013, 105, 2385] applicable to a wider range of temperatures based on experimental data analysis of F1 derived from thermophilic Bacillus under high ATP concentration conditions. Our simulation shows that the rotary angle distribution manifests a stronger non-equilibrium feature as the temperature increases, because ATP hydrolysis and Pi release are more accelerated compared with the timescale of rotary angle relaxation. This effect causes the rate coefficient obtained from dwell time fitting to deviate from the Arrhenius relation in Pi release, which has been assumed in the previous activation thermodynamic quantities estimation using linear Arrhenius fitting. Larger negative correlation is also found between hydrolysis and Pi release waiting time in a catalytic dwell with the increase in temperature. This loss of independence between the two successive reactions at the catalytic dwell sheds doubt on the conventional dwell time fitting to obtain rate coefficients with a double exponential function at temperatures higher than 65 °C, which is close to the physiological temperature of the thermophilic Bacillus.


Assuntos
Proteínas de Bactérias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/química , Biocatálise , Hidrólise , Cinética , ATPases Translocadoras de Prótons/química , Temperatura , Termodinâmica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA