Fluorescent ATP Analogue Research Articles

Analysis of Conformation of the Skeletal Muscle Myosin Modified by F2DNB Using FRET

Previously biochemical studies have demonstrated that the highly reactive cysteine residues SH1 and SH2 can be crosslinked by variety of bifunctional reagents with different spans (3-14 A) in the presence of nucleotides, suggesting that the region is highly flexible. The SH1-SH2 region is believed to play a key role in the conformational changes that occur in the myosin head during the force generation coupled to ATP hydrolysis. We have previously shown that the skeletal muscle heavy mero-myosin (HMM), which SH1-SH2 was crosslinked by 1,5-difluoro-2, 4-dinitrobenzene (F2DNB) in the presence of ADP, have a novel conformation using quick freeze deep etch electron microscopy (QFDE-EM). We have also demonstrated that conformational change of the myosin motor domain during ATP hydrolysis can be monitored by measuring the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we analyzed the conformation of the myosin crosslinked by F2DNB using FRET between the ATP binding site and the essential light chain (ELC) A1. We prepared skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by 6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluorescent ADP analogue NBD-ADP was trapped within the ATPase site of S1 labeled by BD. The FRET efficiency was estimated by measuring the change of fluorescence intensity of BD comparing with control BD-S1. The FRET efficiency of F2DNB-S1-NBD-ADP was lower than S1-NBD-ADP state. This suggests that the F2DNB-S1-ADP states form more kinked conformation than S1-ADP state.

Abstract A139: Discovery and biological profiling of potent fibroblast growth factor receptor (FGFR) kinase inhibitors derived from in silico design with in vivo antitumor activity against FGFR2-dependent human tumors

Abstract Dysregulation of members of the FGFR tyrosine kinase family has been increasingly implicated in a number of human cancers, including gastric, breast, endometrial, and bladder carcinomas. A proprietary structure-based design paradigm was employed to identify inhibitors which favor a mode of binding that is distinct from the commonly described ATP competitive inhibitors. A molecular template was identified (ARQ 523) which, upon further modification, provided molecules that were shown to inhibit FGFR kinases in the low micromolar range, to bind to FGFR2 in an enantiomeric-specific fashion, to bind to unphosphorylated FGFR2 with a KD of 5 µM; and, after pre-incubation with inactive FGFR2, to prevent a fluorescent analogue of ATP from binding to the enzyme. We have designated this novel type of kinase inhibitor as “ATP-exclusionary” or “Type IV” to differentiate these inhibitors from extant molecules. Following a lead optimization campaign, biochemical potencies of 1 nM or less against FGFR kinases with corresponding increases in binding affinities to FGFR2 in biophysical assays were documented in several compounds in two distinct series. These compounds showed sub-micromolar activity in both FGFR2-dependent pharmacodynamic and cytotoxicity assays, and demonstrated a moderately high degree of selectivity across the human kinome. A representative compound showed marked FGFR2 pharmacodynamic suppression and corresponding growth inhibition in KATO III and SNU-16 human gastric carcinoma cells. In addition, growth of SNU-16 tumor xenografts in athymic mice was markedly suppressed (58% regression as compared to vehicle-treated controls) after daily intraperitoneal administration for 9 days. A paired Ba/F3 xenograft model was also employed, using both Ba/F3 cells transfected with either FGFR2 or the unrelated insulin receptor. Significant tumor growth inhibition (77% inhibition as compared to vehicle-treated controls) was observed in treated mice bearing FGFR2-transfected Ba/F3 tumors but not in the insulin receptor transfectants. In summary, we have achieved proof-of-principle of a structure-based kinase inhibitor design paradigm for the identification of a series of FGFR kinase inhibitors that exhibits a preference for the inactive form of the kinase and excludes ATP upon binding. One advanced lead molecule demonstrated the ability to exert profound anti-tumor effects against cancer cell lines in which FGFR2 is the dominant oncogenic driver. The opportunity now exists to significantly expand the number of new chemotypes available for drug discovery against disease-relevant kinases in oncology and in other therapeutic areas, since it is predicted that approximately half of the human kinome is amenable to this novel mode of inhibition. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A139.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·Pi being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than Pi release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.

Alternating-Site Mechanism of Kinesin-1 Characterized by Single-Molecule FRET Using Fluorescent ATP Analogues

Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of ∼1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.

Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coli FoF1-ATP Synthase

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

Fluorescence Quenching and Fluorescence Resonance Energy Transfer Studies in the Recombinant N-domain from the Plasma Membrane H(+)-ATPase, Pma1

Upon substrate binding the isolated plasma membrane H(+)-ATPase (Pma1) from Kluyveromyces lactis displays large changes in fluorescence intensity (Sampedro et al, 2007 Biochemistry 46:5616-5622). The nucleotide binding domain (N-domain) contains one Trp505 residue, that seems to be responsible for the variations in intrinsic fluorescence. The N-domain was cloned and the protein expressed in E. coli. The purified N-domain displayed nucleotide-dependent (ATP and ADP) quenching of fluorescence similar to that observed in the whole Pma1. The dissociation constants (Kd) for ATP and ADP were 100 and 110 uM respectively. Fluorescence resonance energy transfer (FRET) studies were also performed by using mantATP; a fluorescent ATP analog (Ex. 337 nm, Em. 423nm). The absorbance spectra of mantATP overlaps the fluorescence spectra of the N-domain, and thus FRET was observed by exciting at 280 nm. FRET efficiency was 100% indicating a close proximity between Trp505 and the nucleotide. Therefore, in this domain there is a Trp residue located near the substrate binding site which is of high value to determine Kds and molecular distances using fluorescence.

Expression and Characterization of Novel Rice Kinesin E15

Kinesin is an ATP driven motor protein that moves along microtubules. Kinesin plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, only a few kinesis have been characterized in plants. E15 is one of the kinesin encoded on rice genome. E15 has COOH-terminal motor domain and exhibits a high homology with the kinesin-14 family in Arabidopsis thaliana. However, this kinesin is not similar to other kinesin -14 family kinesins derived from animal, e.g., DmNcd, ScKar3, and CeKlp. Consequently, kinesin E15 may be plant-specific kinesin. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, E15, in Escherichia coli. and studied its enzymatic characteristics and compared with other related kinesins. Molecular weight of the E15 motor domain was 37.6 kD. The MT-dependent ATPase activity was higher and the affinity for MT was weaker than rice kinesin K16 that we have previously reported. The optimum pH was pH 6.0–6.5, which is similar to K16. Interaction of E15 with fluorescent ATP analogues was also studied for the kinetic characterization. E15 showed weaker affinity for nucleotide than other kinesins. Currently, we are preparing E15 dimer for the motility assay.

Analysis of Conformation of Skeletal Muscle Myosin Cross-linked by pPDM Using FRET

Previously biochemical studies have demonstrated that the highly reactive cysteine residues SH1 and SH2 can be crosslinked by variety of bifunctional reagents with different spans (3-14 A) in the presence of nucleotides, suggesting that the region is highly flexible. The SH1-SH2 region is believed to play a key role in the conformational changes that occur in the myosin head during the force generation coupled to ATP hydrolysis. We have previously shown that the HMM, which SH1-SH2 was crosslinked by p-Phenylene-dimaleimide (pPDM) in the presence of ADP, have a novel conformation using quick freeze deep etch electron microscopy (QFDE-EM). We have also demonstrated that conformational change of the myosin motor domain during ATP hydrolysis can be monitored by measuring the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we analyzed the conformation of the myosin crosslinked by pPDM using the FRET between the ATP binding site and the A1 essential light chain (ELC) and compared with the 3D structure models of ATPase intermediates derived from electron microscopic analysis. We prepared the skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by 6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluorescent ADP analogue NBD-ADP was trapped in the ATPase site of S1 labeled by BD. The FRET efficiency was estimated by measuring the change of fluorescence intensity of BD comparing with control BD-S1. FRET efficiency of pPDM-S1-NBD-ADP was apparently different from other nucleotides and nucleotide analogues bound states.

PcrA Helicase ATPase Mechanism: RepD Modulation During Unwinding

Helicases catalyse the unwinding of double stranded DNA or RNA for a variety of functions through various mechanisms. The monomeric, bacterial helicase PcrA is well characterized: it translocates ssDNA with discrete steps of one base per ATP. The ATPase activity is enhanced by DNA and the cycle includes a rate limiting cleavage step, followed by rapid phosphate release.PcrA and the plasmid encoded replication initiator protein, RepD, act together during replication of some plasmids, containing antibiotic resistance. RepD is a dimer and binds to a specific origin sequence (ori) containing inverted complementary repeat (ICR) elements. ICRIII provides affinity and plasmid specificity, whereas ICRII is conserved amongst ori family members and contains the nick site for initiation. RepD binds to ICRIII and then nicks at a specific site in ICRII exposing a single-stranded region. PcrA helicase then binds to the ssDNA and begins unwinding. RepD has been shown to increase PcrA helicase activity; in the absence of RepD PcrA is a poor helicase. Measurements have shown that unwinding occurs at a reduced rate but the coupling ratio is unaltered.We have analyzed the mechanism of the PcrA ATPase cycle with ssDNA and extended this to include probing how DNA junctions and RepD affect this cycle. The individual rate constants were determined to see how RepD modulates the ATPase rate. This included utilizing the fluorescent ATP analogue mantATP to monitor fluorescence intensity in rapid-reaction, kinetic experiments, allowing the initial binding and release kinetics to be explored. The analogue was further used for analysis of the hydrolysis step using quenched-flow measurements. Additionally, the fluorescent phosphate binding protein (MDCC-PBP) independently measures the phosphate release step.

2P-116 新規ATP誘導体の合成およびモータータンパク質との相互作用(分子モーター,第47回日本生物物理学会年会)

2P-116 新規ATP誘導体の合成およびモータータンパク質との相互作用(分子モーター,第47回日本生物物理学会年会)

Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c

The alternative sigma factors are regulated by a phosphorylation-mediated signal transduction cascade involving anti-sigma factors and anti-anti-sigma factors. The proteins regulating Mycobacterium tuberculosis sigma factor F (SigF), anti-SigF and anti-anti-SigF have been identified, but the factors catalyzing phosphorylation-dephosphorylation have not been well established. We identified a distinct pathogenic species-specific multidomain protein, Rv1364c, in which the components of the entire signal transduction cascade for SigF regulation appear to be encoded in a single polypeptide. Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of various domains, namely a phosphatase (RsbU) domain, an anti-SigF (RsbW) domain, and an anti-anti-SigF (RsbV) domain. We report that the RsbU domain of Rv1364c bears all the conserved features of the PP2C-type serine/threonine phosphatase family, whereas its RsbW domain has certain substitutions and deletions in regions important for ATP binding. Another anti-SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even more unfavorable substitutions in the kinase domain. Biochemical assay with the purified RsbW domain of Rv1364c and UsfX showed the loss of ability of autophosphorylation and phosphotransfer to cognate anti-anti-SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and UsfX protein display very weak binding with fluorescent ATP analogs, despite showing functional interactions characteristic of anti-SigF proteins. In view of conservation of specific interactions with cognate sigma and anti-anti-sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation-dependent interaction involved in SigF regulation in Mycobacterium.

Just When You Thought It Was Pseudo

Approximately 10% of all known kinases are thought to be catalytically inactive because they have modifications of conserved motifs in their kinase domains, leading to their being dubbed pseudokinases. The pseudokinase Ca 2+ /calmodulin (CaM)-activated serine-threonine kinase (CASK) has an altered DFG motif in its N-terminal CaM-kinase domain. This motif is important for the binding of Mg 2+ , which helps to coordinate the binding of ATP, the source of the phosphoryl group for the kinase. CASK binds to synaptic adhesion molecules, including neurexin, and deletion of CASK in mice leads to synaptic defects and perinatal death. Mukherjee et al . determined the crystal structure of the CaM-kinase domain of recombinant CASK and compared it with those of other kinases. The N- and C-terminal lobes of the CASK CaM-kinase domain in the absence of ATP were arranged in a similar fashion as those of other kinases when in their active states, indicating that CASK adopts a constitutively active conformation. In vitro experiments showed that a fluorescent ATP analog bound to recombinant CASK in the absence of coordinating Mg 2+ and that the addition of Mg 2+ inhibited this interaction. In vitro kinase assays showed that the CASK CaM-kinase domain exhibited autophosphorylation activity, that the kinetics of this phosphorylation were similar to those of true kinases, and that Mg 2+ and other divalent cations inhibited this activity. Overexpression of wild-type CASK but not a catalytically inactive CASK mutant in rat hippocampal neurons resulted in increased phosphorylation of β-neurexin compared with that in control neurons. As Kannan and Taylor discuss in commentary, this study challenges the idea that pseudokinases act merely as inactive scaffold proteins and should stimulate reinvestigation of their potential catalytic activity. K. Mukherjee, M. Sharma, H. Urlaub, G. P. Bourenkov, R. Jahn, T. C. Südhof, M. C. Wahl, CASK functions as a Mg 2+ -independent neurexin kinase. Cell 133 , 328-339 (2008). [PubMed] N. Kannan, S. S. Taylor, Rethinking pseudokinases. Cell 133 , 204-205 (2008). [PubMed]

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the Kms for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5′-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.

2P-170 新規イネキネシンL05,D04のNBD-ATPによる速度論的解析(分子モーター(2),第46回日本生物物理学会年会)

2P-170 新規イネキネシンL05,D04のNBD-ATPによる速度論的解析(分子モーター(2),第46回日本生物物理学会年会)

3P-150 新規蛍光標識ATP誘導体の開発(分子モーター(3),第46回日本生物物理学会年会)

3P-150 新規蛍光標識ATP誘導体の開発(分子モーター(3),第46回日本生物物理学会年会)

Cloning, E. coli expression, refolding, and ATP-binding properties of a WD40-deleted Apaf-1 isoform

The apoptotic protease activating factor (Apaf-1) is central to the regulatory mechanism by which procaspase-9 is activated in the cytochrome c-mediated pathway of apoptosis. For a detailed biochemical and structural investigation of Apaf-1 function, we have cloned and expressed in Escherichia coli inclusion bodies the WD40-deleted protein ( ΔWD40Apaf-1) from HepG2 cell. The construct contains an N-terminal His 6 tag derived from the cloning vector so that the mass of the protein and the tag together is 51,594 Da, as determined by TOF/TOF mass spectrometric analysis. An optimized refolding protocol has allowed protein recovery in highly pure form. Basic fluorescence and CD probes indicate that the refolded protein retains secondary and tertiary structures, and unfolds in the presence of higher concentration of denaturant. The equilibrium ATP binding property of the protein has been measured by changes in fluorescence emission due to the fluorescent ATP analog, mant-ATP (2′(3′)- O-( N-methylanthraniloyl) adenosine 5′-triphosphate). The results demonstrate a tight Apaf-1−ATP interaction, the binding affinity being 380 nM.

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.

Coupling of Rotation and Catalysis in F 1-ATPase Revealed by Single-Molecule Imaging and Manipulation

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.