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1.
Commun Biol ; 6(1): 26, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631659

RESUMO

F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the Fo motor that is transferred to the F1 motor to catalyze ATP production, with flexible F1/Fo coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the Fο motor. This highlights the importance of the central rotor for flexible coupling of the F1 and Fo motors and provides further insight into the regulatory mechanism mediated by subunit ε.


Assuntos
Trifosfato de Adenosina , Escherichia coli
2.
Proc Natl Acad Sci U S A ; 117(43): 26766-26772, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33051299

RESUMO

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.


Assuntos
Membrana Celular , Quimiotaxia/fisiologia , Haloferax volcanii , Modelos Biológicos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Flagelos/química , Flagelos/metabolismo , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Cinética , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo
3.
Nat Commun ; 11(1): 2615, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457314

RESUMO

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1-3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.


Assuntos
Proteínas de Escherichia coli/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Lipídeos/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rotação , Relação Estrutura-Atividade
4.
Methods Mol Biol ; 2073: 73-84, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31612437

RESUMO

ATP synthase is an essential enzyme found in all known forms of life, generating the majority of cellular energy via a rotary catalytic mechanism. Here, we describe the in-depth methods for expression, purification, and functional assessment of E. coli ATP synthase.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , ATPases Translocadoras de Prótons/química
5.
Elife ; 82019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30912741

RESUMO

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, ß and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate 'half-up' state to a condensed 'down' state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory 'up' state.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Microscopia Crioeletrônica , Conformação Proteica , Subunidades Proteicas/química
6.
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
7.
J Vis Exp ; (134)2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29683454

RESUMO

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to >10 microns, as well as ATP production by this chain.


Assuntos
Detergentes/uso terapêutico , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteolipídeos/metabolismo
8.
J Bioenerg Biomembr ; 49(2): 171-181, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28078625

RESUMO

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.


Assuntos
ATPases Bacterianas Próton-Translocadoras/genética , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Deleção de Sequência , Trifosfato de Adenosina/biossíntese , Sequência de Aminoácidos , ATPases Bacterianas Próton-Translocadoras/química , ATPases Bacterianas Próton-Translocadoras/metabolismo , Cisteína/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transporte de Íons , Mutagênese Sítio-Dirigida , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
9.
Elife ; 52016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28001127

RESUMO

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk's ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides.


Assuntos
ATPases Bacterianas Próton-Translocadoras/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/enzimologia
10.
Nat Commun ; 7: 13025, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27708275

RESUMO

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 µm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.


Assuntos
Trifosfato de Adenosina/química , Escherichia coli/enzimologia , Lipossomos/química , Proteolipídeos/química , ATPases Translocadoras de Prótons/química , Calibragem , Membrana Celular/enzimologia , Cobalto/química , Transporte de Elétrons , Fluoresceínas/química , Bicamadas Lipídicas/química , Fusão de Membrana , Proteínas de Membrana/química , Oxirredutases/química , Ligação Proteica , Bombas de Próton , Glycine max/química , Biologia Sintética
11.
Sci Rep ; 6: 20729, 2016 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-26853732

RESUMO

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.


Assuntos
Iluminação/métodos , Microscopia/métodos , Dispositivos Ópticos , Custos e Análise de Custo , Iluminação/economia , Microscopia/economia
12.
Proc Natl Acad Sci U S A ; 111(10): 3715-20, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24567403

RESUMO

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αß-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).


Assuntos
Aceleração , Escherichia coli/enzimologia , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Conformação Proteica , ATPases Translocadoras de Prótons/metabolismo , Rotação , Hidrólise , Imagem Molecular/métodos , ATPases Translocadoras de Prótons/isolamento & purificação
13.
FEBS Lett ; 587(7): 892-7, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23416299

RESUMO

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu(2+) catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2-3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.


Assuntos
Complexos de ATP Sintetase/metabolismo , ATPases Bacterianas Próton-Translocadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/genética , ATPases Bacterianas Próton-Translocadoras/química , ATPases Bacterianas Próton-Translocadoras/genética , Benzofenonas/química , Benzofenonas/farmacologia , Sítios de Ligação/genética , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/farmacologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Immunoblotting , Maleimidas/química , Maleimidas/farmacologia , Modelos Moleculares , Mutação , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Estrutura Terciária de Proteína
14.
Methods Mol Biol ; 778: 273-89, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21809213

RESUMO

Single-molecule measurements of rotation catalyzed by the F(1)-ATPase or the F(o)F(1) ATP synthase have provided new insights into the molecular mechanisms of the F(1) and F(o) molecular motors. We recently developed a method to record ATPase-driven rotation of F(1) or F(o)F(1) in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 µs at unprecedented signal-to-noise.


Assuntos
Proteínas Motores Moleculares/metabolismo , Nanotecnologia/métodos , Ouro/química , Microscopia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Nanotubos/química , ATPases Translocadoras de Prótons/metabolismo
15.
EMBO J ; 29(23): 3911-23, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21037553

RESUMO

Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F0F1-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F0 has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F0 subunits a and c by improving the time resolution to 10 µs at unprecedented S/N, and by using Escherichia coli F0F1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 µs to form and 175 µs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of ∼36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F0 motor where c-ring rotational diffusion is limited to 36°.


Assuntos
Escherichia coli/enzimologia , Bicamadas Lipídicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Difusão , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Nanotubos/química , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/genética , Prótons , Alinhamento de Sequência
16.
Biochemistry ; 48(33): 7979-85, 2009 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-19610671

RESUMO

Increases in the power stroke and dwell durations of single molecules of Escherichia coli F(1)-ATPase were measured in response to viscous loads applied to the motor and inhibition of ATP hydrolysis. The load was varied using different sizes of gold nanorods attached to the rotating gamma subunit and/or by increasing the viscosity of the medium using PEG-400, a noncompetitive inhibitor of ATPase activity. Conditions that increase the duration of the power stroke were found to cause 20-fold increases in the length of the dwell. These results suggest that the order of hydrolysis, product release, and substrate binding may change as the result of external load on the motor or inhibition of hydrolysis.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Motores Moleculares/química , Força Próton-Motriz , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Catálise/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/metabolismo , Hidrólise/efeitos dos fármacos , Proteínas Motores Moleculares/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Fatores de Tempo , Viscosidade
17.
Methods ; 2008 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-18565336

RESUMO

We report a novel method to detect angular conformational changes of a molecular motor in a manner sensitive enough to achieve acquisition rates with a time resolution of 2.5mus (equivalent to 400,000fps). We show that this method has sufficient sensitivity to resolve the velocity of the F(1)-ATPase gamma-subunit as it travels from one conformational state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-ATPase. Variations in scattered light intensity allow precise measurement of changes in angular position of the rod below the diffraction limit of light.

18.
Biochim Biophys Acta ; 1777(7-8): 579-82, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18471428

RESUMO

The torque generated by the power stroke of Escherichia coli F(1)-ATPase was determined as a function of the load from measurements of the velocity of the gamma-subunit obtained using a 0.25 micros time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the gamma-subunit was able to rotate with a 20x increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63+/-8 pN nm, independent of the load on the enzyme.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Escherichia coli/química , Cinética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/química , Torque
19.
Biochim Biophys Acta ; 1777(1): 32-8, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18068111

RESUMO

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N'-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F(1)-F(o) interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.


Assuntos
Complexos de ATP Sintetase/fisiologia , Trifosfato de Adenosina/biossíntese , Escherichia coli/enzimologia , Complexos de ATP Sintetase/química , Concentração de Íons de Hidrogênio , Subunidades Proteicas
20.
J Bioenerg Biomembr ; 39(5-6): 435-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18058004

RESUMO

The abundance of E. coli F1-ATPase molecules observed to rotate using gold nanorods attached to the gamma-subunit was quantitated. Individual F1 molecules were determined to be rotating based upon time dependent fluctuations of red and green light scattered from the nanorods when viewed through a polarizing filter. The average number of F1 molecules observed to rotate in the presence of GTP, ATP, and without nucleotide was approximately 50, approximately 25, and approximately 4% respectively. In some experiments, the fraction of molecules observed to rotate in the presence of GTP was as high as 65%. These data indicate that rotational measurements made using gold nanorods provide information of the F1-ATPase mechanism that is representative of the characteristics of the enzyme population as a whole.


Assuntos
Proteínas de Escherichia coli , Técnicas de Sonda Molecular , Sondas Moleculares , Nanotubos , ATPases Translocadoras de Prótons/metabolismo , Ouro , Nanopartículas Metálicas , Microscopia , Proteínas Motores Moleculares , Nanotecnologia/métodos , ATPases Translocadoras de Prótons/análise , Rotação
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