Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein. Studies with ribose and base-modified fluorescent nucleotide analogs.
AuthorsBujalowski W,Klonowska MM
JournalBiochemistry
PubMed ID8161526
Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (epsilon ADP) and the ribose-modified fluorescent analogs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP), 3'-O-(N-methylantraniloyl)deoxyadenosine 5'-diphosphate (MANT-dADP), 3'-O-(N-methylantraniloyl)-deoxyadenosine 5'-triphosphate (MANT-dATP), ... More
A mechanistic model for Ncd directionality.
AuthorsFoster KA, Mackey AT, Gilbert SP
JournalJ Biol Chem
PubMed ID11278404
Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in ... More
Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
AuthorsMackey AT, Gilbert SP
JournalBiochemistry
PubMed ID10684615
'Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor ... More
Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein.
AuthorsGalletto R, Rajendran S, Bujalowski W
JournalBiochemistry
PubMed ID11041861
'Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and ... More
The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.
AuthorsMurphy CT, Spudich JA
JournalBiochemistry
PubMed ID10090768
'We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ... More
Kinetic mechanism of kinesin motor domain.
AuthorsMa YZ, Taylor EW
JournalBiochemistry
PubMed ID7548087
'The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed ... More
An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.
AuthorsZhuang Z, Berdis AJ, Benkovic SJ
JournalBiochemistry
PubMed ID16800623
'In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form ... More
'While most of the sequence of myosin''s motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop ... More
Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.
AuthorsFoster KA, Gilbert SP
JournalBiochemistry
PubMed ID10677228
'Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. ... More
Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.
AuthorsKurzawa-Goertz SE, Perreault-Micale CL, Trybus KM, Szent-Györgyi AG, Geeves MA
JournalBiochemistry
PubMed ID9585566
'The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding ... More
Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.
AuthorsRosenfeld SS, Xing J, Cheung HC, Brown F, Kar S, Sweeney HL
JournalJ Biol Chem
PubMed ID9786863
'In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally ... More
Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes.
AuthorsWoodward SK, Eccleston JF, Geeves MA
JournalBiochemistry
PubMed ID1824820
'We have used a fluorescent analogue of ATP, mantATP [2''(3'')-O-(N-methylanthraniloyl)-adenosine 5''-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, ... More
X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.
AuthorsBauer CB, Kuhlman PA, Bagshaw CR, Rayment I
JournalJ Mol Biol
PubMed ID9405148
'Mant (2''(3'')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching ... More
Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain.
AuthorsShimizu T, Sablin E, Vale RD, Fletterick R, Pechatnikova E, Taylor EW
JournalBiochemistry
PubMed ID7548090
'ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate ... More
A biosensor for fluorescent determination of ADP with high time resolution.
AuthorsKunzelmann S, Webb MR,
JournalJ Biol Chem
PubMed ID19801632
'Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological ... More
Actin and light chain isoform dependence of myosin V kinetics.
AuthorsDe La Cruz EM, Wells AL, Sweeney HL, Ostap EM
JournalBiochemistry
PubMed ID11087368
'Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V ... More
Interacting head mechanism of microtubule-kinesin ATPase.
AuthorsMa YZ, Taylor EW
JournalJ Biol Chem
PubMed ID8995356
'Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) ... More
The conformation of the active site of myosin probed using mant-nucleotides.
AuthorsFranks-Skiba K, Cooke R
JournalBiophys J
PubMed ID7787057
'Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced ... More
Kinetic characterization of a monomeric unconventional myosin V construct.
AuthorsTrybus KM, Krementsova E, Freyzon Y
JournalJ Biol Chem
PubMed ID10488077
'An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 ... More
Ionic interactions play a role in the regulatory mechanism of scallop heavy meromyosin.
AuthorsNyitrai M, Stafford WF, Szent-Györgyi AG, Geeves MA
JournalBiophys J
PubMed ID12885652
'Heavy meromyosin from scallop (scHMM) striated muscle is regulated by calcium binding to the essential light chain. The regulation can be modeled with a calcium-dependent equilibrium between on and off scHMM conformations. The observed rate constant for mant-ADP dissociation from scHMM is calcium dependent, and we show here that it ... More
Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.
AuthorsSun M, Oakes JL, Ananthanarayanan SK, Hawley KH, Tsien RY, Adams SR, Yengo CM
JournalJ Biol Chem
PubMed ID16377637
'The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding ... More
Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity.
AuthorsHackney DD
JournalBiochemistry
PubMed ID11914091
'Kinesin binds to microtubules with half-site ADP release to form a tethered intermediate with one attached head without nucleotide and one tethered head that retains its bound ADP. For DKH405 containing amino acid residues 1-405 of Drosophila kinesin, release of the remaining ADP from the tethered head is slow (0.05 ... More
The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding.
AuthorsJault JM, Fieulaine S, Nessler S, Gonzalo P, Di Pietro A, Deutscher J, Galinier A
JournalJ Biol Chem
PubMed ID10636874
'Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on ... More
New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.
AuthorsHiratsuka T
JournalBiochim Biophys Acta
PubMed ID6132622
'The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3''-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence ... More
A kinetic study of the kinesin ATPase.
AuthorsSadhu A, Taylor EW
JournalJ Biol Chem
PubMed ID1534560
'The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2''-(3'')-O-(N-methylanthraniloyl)-ATP and mant-ADP. ... More
Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket.
AuthorsSweeney HL, Rosenfeld SS, Brown F, Faust L, Smith J, Xing J, Stein LA, Sellers JR
JournalJ Biol Chem
PubMed ID9497352
'A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops ... More
Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.
AuthorsJekabsons MB, Echtay KS, Brand MD
JournalBiochem J
PubMed ID12030845
'Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and ... More
A structural change in the kinesin motor protein that drives motility.
AuthorsRice S, Lin AW, Safer D, Hart CL, Naber N, Carragher BO, Cain SM, Pechatnikova E, Wilson-Kubalek EM, Whittaker M, Pate E, Cooke R, Taylor EW, Milligan RA, Vale RD
JournalNature
PubMed ID10617199
'Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region ... More
Interaction of mant-adenosine nucleotides and magnesium with kinesin.
AuthorsCheng JQ, Jiang W, Hackney DD
JournalBiochemistry
PubMed ID9548760
'Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3''-isomer, followed by ... More
Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism.
AuthorsHeitz F, Morris MC, Fesquet D, Cavadore JC, Dorée M, Divita G
JournalBiochemistry
PubMed ID9125522
The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and ... More
Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.
AuthorsCrevel I, Carter N, Schliwa M, Cross R
JournalEMBO J
PubMed ID10545098
We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at ... More
A kinetic model for the action of a resistance efflux pump.
AuthorsWalmsley AR, Zhou T, Borges-Walmsley MI, Rosen BP
JournalJ Biol Chem
PubMed ID11096086
ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting ... More
Docking and rolling, a model of how the mitotic motor Eg5 works.
AuthorsRosenfeld SS, Xing J, Jefferson GM, King PH
JournalJ Biol Chem
PubMed ID16115880
Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? ... More
The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility.
AuthorsMackey AT, Gilbert SP
JournalJ Biol Chem
PubMed ID12446697
Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with ... More
A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.
AuthorsBrendza KM, Sontag CA, Saxton WM, Gilbert SP
JournalJ Biol Chem
PubMed ID10767290
Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) ... More
Membrane-catalyzed nucleotide exchange on DnaA. Effect of surface molecular crowding.
AuthorsAranovich A, Gdalevsky GY, Cohen-Luria R, Fishov I, Parola AH
JournalJ Biol Chem
PubMed ID16517983
DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ... More
The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis.
AuthorsVertommen D, Bertrand L, Sontag B, Di Pietro A, Louckx MP, Vidal H, Hue L, Rider MH
JournalJ Biol Chem
PubMed ID8663445
All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide ... More
A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1.
AuthorsPearson DS, Holtermann G, Ellison P, Cremo C, Geeves MA
JournalBiochem J
PubMed ID12010120
Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume ... More
Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release.
AuthorsXing J, Wriggers W, Jefferson GM, Stein R, Cheung HC, Rosenfeld SS
JournalJ Biol Chem
PubMed ID10852922
Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor ... More
Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.
AuthorsMihashi K, Ooi A, Hiratsuka T
JournalJ Biochem (Tokyo)
PubMed ID2140356
In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at ... More
Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle.
AuthorsCremo CR, Geeves MA
JournalBiochemistry
PubMed ID9485324
Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP ... More
Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle.
AuthorsYengo CM, Chrin LR, Rovner AS, Berger CL
JournalJ Biol Chem
PubMed ID10827189
To examine the structural basis of the intrinsic fluorescence changes that occur during the MgATPase cycle of myosin, we generated three mutants of smooth muscle myosin motor domain essential light chain (MDE) containing a single conserved tryptophan residue located at Trp-441 (W441-MDE), Trp-512 (W512-MDE), or Trp-597 (W597-MDE). Although W441- and ... More
Chemical decoupling of ATPase activation and force production from the contractile cycle in myosin by steric hindrance of lever-arm movement.
AuthorsMuhlrad A, Peyser YM, Nili M, Ajtai K, Reisler E, Burghardt TP
JournalBiophys J
PubMed ID12547786
The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5'-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy ... More
Kinetic mechanism of a monomeric kinesin construct.
AuthorsMa YZ, Taylor EW
JournalJ Biol Chem
PubMed ID8995355
The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s(-1) at 20 degrees C; Km(Mt) is ... More
Kinetic characterization of myosin head fragments with long-lived myosin.ATP states.
AuthorsFriedman AL, Geeves MA, Manstein DJ, Spudich JA
JournalBiochemistry
PubMed ID9657680
We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ... More
Transient kinetic analysis of the 130-kDa myosin I (MYR-1 gene product) from rat liver. A myosin I designed for maintenance of tension?
AuthorsColuccio LM, Geeves MA
JournalJ Biol Chem
PubMed ID10419463
The 130-kDa myosin I (MI(130)), product of the myr-1 gene, is one member of the mammalian class I myosins, a group of small, calmodulin-binding mechanochemical molecules of the myosin superfamily that translocate actin filaments. Roles for MI(130) are unknown. Our hypothesis is that, as with all myosins, MI(130) is designed ... More
Pathway of ATP hydrolysis by monomeric and dimeric kinesin.
AuthorsMoyer ML, Gilbert SP, Johnson KA
JournalBiochemistry
PubMed ID9454569
The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate ... More
Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence.
AuthorsEspinosa V, Kettlun AM, Zanocco A, Cardemil E, Valenzuela MA
JournalPhytochemistry
PubMed ID12657291
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), ... More
Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin.
AuthorsRosenfeld SS, Xing J, Rener B, Lebowitz J, Kar S, Cheung HC
JournalJ Biol Chem
PubMed ID7982925
The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain. The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) ... More
Interactions of Escherichia coli primary replicative helicase DnaB protein with nucleotide cofactors.
AuthorsJezewska MJ, Kim US, Bujalowski W
JournalBiophys J
PubMed ID8889182
Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of ... More
Mechanism of microtubule kinesin ATPase.
AuthorsMa YZ, Taylor EW
JournalBiochemistry
PubMed ID7548088
A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with ... More
Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues.
AuthorsBujalowski W, Jezewska MJ
JournalBiochemistry
PubMed ID10684661
The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and ... More
Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase.
AuthorsPechatnikova E, Taylor EW
JournalJ Biol Chem
PubMed ID9388211
The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, ... More
Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues.
AuthorsMoore KJ, Lohman TM
JournalBiochemistry
PubMed ID7981217
The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the ... More
Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking.
AuthorsCremo CR, Neuron JM, Yount RG
JournalBiochemistry
PubMed ID2110475
The environment near the ribose binding site of skeletal myosin subfragment 1 (S1) was investigated by use of two adenosine 5'-diphosphate analogues with fluorescent groups attached at the 2'- and 3'-hydroxyls of the ribose ring. We have compared steady-state and time-resolved fluorescent properties of the reversibly bound S1-nucleotide complexes and ... More
The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase.
AuthorsAlahyan M, Webb MR, Marston SB, El-Mezgueldi M
JournalJ Biol Chem
PubMed ID16540476
Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin ... More
Alternating site mechanism of the kinesin ATPase.
AuthorsGilbert SP, Moyer ML, Johnson KA
JournalBiochemistry
PubMed ID9454568
The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results ... More
ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility.
AuthorsShimizu T, Thorn KS, Ruby A, Vale RD
JournalBiochemistry
PubMed ID10819995
Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed ... More
Prodan fluorescence reflects differences in nucleotide-induced conformational states in the myosin head and allows continuous visualization of the ATPase reactions.
AuthorsHiratsuka T
JournalBiochemistry
PubMed ID9585528
The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex ... More
Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin.
Authors
JournalCurr Biol
PubMed ID31280993
Hexameric helicase G40P unwinds DNA in single base pair steps.
Authors
JournalElife
PubMed ID30688211
Nup159 Weakens Gle1 Binding to Dbp5 But Does Not Accelerate ADP Release.