Biomedical subjects

Eiro Muneyuki

Publications and source records attributed to Eiro Muneyuki.

13 recordsLinked to original sources

PubMed2006

Single molecule energetics of F1-ATPase motor.

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P(i) in its individual actions at a single molecule level. The F(1) portion of ATP synthase, also called F(1)-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of DeltaG(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

Adenosine Triphosphate↗

PubMed2006

AFM and TEM observations of alpha-helix to beta-sheet conformational change occurring on carbon nanotubes.

Bacteriorhodopsin (BR), which is rich in alpha-helical structure, was spread onto water with single-wall carbon nanotubes (SCNTs). After a Langmuir trough was used to apply compressive surface pressure to maintain the alpha-helices monolayer of denatured BR, the composite films comprising alpha-helices and SCNTs were transferred horizontally onto substrates. Atomic force microscopy (AFM) and fluorescence microscopy observation suggested that alpha-helices in contact with SCNTs changed into beta-sheets. High-resolution transmission electron microscopy (HR-TEM) showed 0.54 nm periodicity characteristic of the turn of alpha-helical structure in the SCNTs-free alpha-helix monolayer region and showed the 0.70 nm periodicity of beta-sheet pleated structure in the region where SCNTs were covered with unfolded BR. Unique features of carbon nanotubes that trigger conformational changes of a protein were revealed.

Bacteriorhodopsins↗

PubMed2006

Kinetic mechanism of quinol oxidation by cytochrome bd studied with ubiquinone-2 analogs.

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S(n) on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.

Cytochrome b Group↗

PubMed2005

An alternative reaction pathway of F1-ATPase suggested by rotation without 80 degrees/40 degrees substeps of a sluggish mutant at low ATP.

F(1)-ATPase, a water-soluble portion of F(o)F(1)-ATP synthase, is a rotary motor driven by ATP hydrolysis. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, catalytic dwell, and rapid 40 degrees substep rotation. In the catalytic dwell, at least two catalytic reactions occur-cleavage of the enzyme-bound ATP and presumably release of the hydrolyzed product(s) from the enzyme-but we found that a slow ATP cleavage mutant of F(1)-ATPase from thermophilic Bacillus PS3 rotates at low ATP concentration without substeps and the catalytic dwell. Analysis indicates that in this alternative reaction pathway the two catalytic reactions occur during the preceding long ATP-binding dwell. Thus, F(1)-ATPase can operate through (at least) two competing reaction pathways, not necessarily through a simple consecutive reaction.

Adenosine Triphosphate↗

PubMed2005

The characteristics of the (alpha V371C)3(beta R337C)3 gamma double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha TP-beta TP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP.

In the MF(1) crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 A of the alpha-oxygen of alphaS(370) and 3.7 A of a methyl group of alphaV(371) at the alpha(E)-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF(1)-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl(2) cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl(3) and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 microM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl(3) and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 microM MgADP irreversibly inactivated ATPase activity with a common rate constant of approximately 10(-4) min(-1). Correlation of these results with crystal structures of MF(1) suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleotide-depleted F(1)-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.

Adenosine Diphosphate↗

PubMed2004

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.

F(1)-ATPase is a rotary molecular motor in which the central gamma-subunit rotates inside a cylinder made of alpha(3)beta(3)-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F(1) rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F(1) molecules that carried a bead duplex on the gamma-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 10(7) M(-1)s(-1). A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F(1) produced the same torque of approximately 40 pN.nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.

Adenosine Triphosphate↗

PubMed2004

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.

Adenosine Triphosphate↗

PubMed2003

Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation.

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha 3 beta 3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, interim dwell, and rapid 40 degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (alpha 3 beta 3 gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATP gamma S (adenosine 5'-[gamma-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP gamma S hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Adenosine Triphosphate↗

PubMed2003

Origin of apparent negative cooperativity of F(1)-ATPase.

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.

Adenosine Triphosphate↗

PubMed2003

Evidence for rotation of V1-ATPase.

V(o)V(1)-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to F(o)F(1)-ATP synthase, V(o)V(1)-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V(1)-ATPase, a catalytic subcomplex of V(o)V(1)-ATPase. V(1)-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that V(o)V(1)-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Adenosine Triphosphate↗

PubMed2002

F1-ATPase changes its conformations upon phosphate release.

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.

Adenosine Diphosphate↗

PubMed2002

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum.

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.

Biophysical Phenomena↗

PubMed2002

The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F(1)-ATPase.

F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P(i). Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F(1)-ATPase (alpha W463F/beta Y341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P(i) interfered with the binding of MgATP to F(1)-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F(1)-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alpha W463F/beta E190Q/beta Y341W), also gave the same results. The half-maximal concentrations of P(i) that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximately 20 mm. It is likely that the presence of P(i) at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P(i) complex, an active intermediate in the catalytic cycle.

Adenosine Diphosphate↗