Actomyosin energy turnover declines while force remains constant during isometric muscle contraction.
AuthorsWest TG, Curtin NA, Ferenczi MA, He ZH, Sun YB, Irving M, Woledge RC
JournalJ Physiol
PubMed ID14565999
'Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish (Scyliorhinus canicula) at 12 degrees C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (Pi) release by permeabilized fibres was ... More
Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate.
AuthorsShrestha S, Salins LL, Mark Ensor C, Daunert S
JournalBiotechnol Bioeng
PubMed ID12115121
'Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the ... More
Real-time in vitro measurement of GTP hydrolysis.
AuthorsShutes A, Der CJ
JournalMethods
PubMed ID16288887
'Small GTPases require an active GTPase activity to function correctly in their cellular environment. Mutation of key residues involved in this activity renders the GTPase defective and the small G-protein constitutively active (GTP-locked). The GTPase activity is also a target for GTPase-activating proteins (GAPs) which act to attenuate GTPase signalling ... 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
The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release.
AuthorsBaird CL, Gordon MS, Andrenyak DM, Marecek JF, Lindsley JE
JournalJ Biol Chem
PubMed ID11353771
'DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange ... More
A new method for the time-resolved measurement of phosphate release in permeabilized muscle fibers.
AuthorsFerenczi MA, He ZH, Chillingworth RK, Brune M, Corrie JE, Trentham DR, Webb MR
JournalBiophys J
PubMed ID7787066
'A new method for the measurement of phosphate release in contracting and relaxed permeabilized muscle fibers is described. The assay is based on a genetically engineered phosphate-binding protein labeled with a coumarin fluorescent probe, which binds inorganic phosphate tightly and shows a fourfold increase in fluorescence upon binding. Measurements of ... More
Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.
AuthorsBrune M, Hunter JL, Howell SA, Martin SR, Hazlett TL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID9671505
'The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence ... More
ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.
AuthorsBarman T, Brune M, Lionne C, Piroddi N, Poggesi C, Stehle R, Tesi C, Travers F, Webb MR
JournalBiophys J
PubMed ID9635765
'Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ... More
Time resolved measurements show that phosphate release is the rate limiting step on myofibrillar ATPases.
AuthorsLionne C, Brune M, Webb MR, Travers F, Barman T
JournalFEBS Lett
PubMed ID7750544
'The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of Pi formation, due to AM.ADP.Pi or Pi, as shown in the scheme: AM+ATP<-->A.M.ATP<-->AM.ADP.Pi<-->AM.ADP+Pi<-->AM+ADP. Therefore, in the steady state, either AM.ADP.Pi or AM.ADP ... More
Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed.
AuthorsDillingham MS, Wigley DB, Webb MR
JournalBiochemistry
PubMed ID10625495
'Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ... More
Phosphate release during microtubule assembly: what stabilizes growing microtubules?
AuthorsVandecandelaere A, Brune M, Webb MR, Martin SR, Bayley PM
JournalBiochemistry
PubMed ID10387063
'The molecular mechanism underlying microtubule dynamic instability depends on the relationship between the addition of tubulin-GTP to a growing microtubule and its hydrolysis in the microtubule lattice to tubulin-GDP, with release of inorganic phosphate (Pi). Since this relationship remains controversial, we have re-examined the release of Pi upon microtubule assembly ... More
Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate.
AuthorsHirshberg M, Henrick K, Haire LL, Vasisht N, Brune M, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID9671506
Crystal structures are presented for the A197C mutant of Escherichia coli phosphate binding protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both proteins are complexed with inorganic phosphate. The latter molecule, MDCC-PBP, exhibits a large increase in fluorescence on binding inorganic phosphate. The resulting high-fluorescence state ... More
A new paradigm for DNA polymerase specificity.
AuthorsTsai YC, Johnson KA
JournalBiochemistry
PubMed ID16893169
We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the ... More
The mechanism of Ras GTPase activation by neurofibromin.
Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage ... More
Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers.
AuthorsHe Z, Stienen GJ, Barends JP, Ferenczi MA
JournalBiophys J
PubMed ID9788934
Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In ... More
Kinetic mechanism and regulation of myosin VI.
AuthorsDe La Cruz EM, Ostap EM, Sweeney HL
JournalJ Biol Chem
PubMed ID11423557
Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine ... More
The interaction between rac1 and its guanine nucleotide dissociation inhibitor (GDI), monitored by a single fluorescent coumarin attached to GDI.
The interaction of rac with guanine nucleotide dissociation inhibitor protein (rhoGDI) is described, using GDI fluorescently labeled on its single cysteine with N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC). The labeled GDI shows a 70% decrease in fluorescence emission on binding geranylgeranylated rac1.GDP and has an affinity for rac1 within a factor of 2 of ... 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
Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA.
AuthorsBertram JG, Bloom LB, Hingorani MM, Beechem JM, O'Donnell M, Goodman MF
JournalJ Biol Chem
PubMed ID10874049
Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy ... More
Class-selective drug detection: fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants.
AuthorsDouglass PM, Salins LL, Dikici E, Daunert S
JournalBioconjug Chem
PubMed ID12440852
A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific ... More
A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp.
AuthorsAnderson SG, Williams CR, O'donnell M, Bloom LB
JournalJ Biol Chem
PubMed ID17210572
Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by ... More
A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.
AuthorsAson B, Bertram JG, Hingorani MM, Beechem JM, O'Donnell M, Goodman MF, Bloom LB
JournalJ Biol Chem
PubMed ID10644772
The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. ... More
Phosphate binding protein as the biorecognition element in a biosensor for phosphate.
AuthorsSalins LL, Deo SK, Daunert S
JournalSens Actuators B Chem
PubMed ID14997877
This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, ... More
Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.
AuthorsBrune M, Hunter JL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID8031761
A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant ... More
ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.
AuthorsHe ZH, Chillingworth RK, Brune M, Corrie JE, Trentham DR, Webb MR, Ferenczi MA
JournalJ Physiol
PubMed ID9174999
1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was ... More
Mechanism of regulation of phosphate dissociation from actomyosin-ADP-Pi by thin filament proteins.
AuthorsHeeley DH, Belknap B, White HD
JournalProc Natl Acad Sci U S A
PubMed ID12486217
Regulation by calcium and myosin-S1 of the acceleration of the rate of phosphate release from myosin-ADP-inorganic phosphate (M-ADP-Pi) by the thin filament actin-tropomyosin (Tm)-troponin (Tn), was measured directly by using double mixing stopped-flow experiments with fluorescent phosphate binding protein. At low calcium and without rigor myosin-S1, saturating concentrations of thin ... More
A fluorescent sensor of the phosphorylation state of nucleoside diphosphate kinase and its use to monitor nucleoside diphosphate concentrations in real time.
AuthorsBrune M, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID11305926
A sensor for purine nucleoside diphosphates in solution based on nucleoside diphosphate kinase (NDPK) has been developed. A single cysteine was introduced into the protein and labeled with the environmentally sensitive fluorophore, N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide. The resultant molecule shows a 4-fold fluorescence increase when phosphorylated on His117; this phosphorylation is on the ... More
EF-G-dependent GTPase on the ribosome. conformational change and fusidic acid inhibition.
AuthorsSeo HS, Abedin S, Kamp D, Wilson DN, Nierhaus KH, Cooperman BS
JournalBiochemistry
PubMed ID16489743
Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements ... More
ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition.
AuthorsHe ZH, Bottinelli R, Pellegrino MA, Ferenczi MA, Reggiani C
JournalBiophys J
PubMed ID10920025
Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects ... More
Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.
AuthorsWhite HD, Belknap B, Webb MR
JournalBiochemistry
PubMed ID9305974
We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were ... More