Function Of ATP Concentration Research Articles

Parallel path mechanisms lead to nonmonotonic force-velocity curves and an optimum load for molecular motor function.

Molecular motors convert chemical potential energy into mechanical work and perform a great number of critical biological functions. Examples include the polymerization and manipulation of nucleic acids, the generation of cellular motility and contractility, the formation and maintenance of cell shape, and the transport of materials within cells. The mechanisms underlying these molecular machines are varied, but are almost always considered in the context of a single kinetic pathway that describes motor stepping. However, the multidimensional nature of protein energy landscapes suggests the possibility of multiple reaction pathways connecting two states. Here we investigate the properties of a hypothetical molecular motor able to utilize parallel translocation mechanisms. We explore motor velocity and force dependence as a function of the energy landscape of each path and reveal the potential for such a mechanism to result in negative differential conductance. More specifically, regimes exist where increasing opposing force leads to increased velocity and an optimum load for motor function. We explore how the presence of this optimum depends on the rates of the individual paths and show that the distribution of stepping times characterized by the randomness parameter may be used to test for parallel path mechanisms. Last, we caution that experimental data consisting solely of measurements of velocity as a function of ATP concentration and force cannot be used to eliminate the possibility of such a parallel path mechanism.

Markov modeling of run length and velocity for molecular motors

ABSTRACT Molecular motors are nanometer scale proteins involved in various intracellular processes such as cargo transport, muscle contraction and cell division. The steps in the chemo-mechanical cycle that use ATP hydrolysis to generate motion along the cytoskeletal tracks can be characterized by stepping rates depending only on the current state, permitting modeling of the cycle as a Markov process. To learn more about the nature of motor motion, cell researchers have conducted in vitro experiments in which molecular motors pull beads along filaments attached to glass slides and their velocity and run length until detachment from the microtubule are recorded. A formula is derived for distance traveled until detachment. Four molecular motors for which there are run length and velocity measurements in the literature are considered. In each case the derived formulas for run length and velocity have a generalized Michaelis–Menten form as functions of ATP concentration. The degrees of the numerator and denominator polynomials increase with the complexity of chemo-mechanical cycle. The degrees of the Michaelis–Menten form determine the maximum number of reaction rates that can be determined from experimental data on run length and velocity, however consistency conditions may reduce this number and restrict which rates can be determined.

Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.

Processivity and Velocity for Motors Stepping on Periodic Tracks

Processive molecular motors enable cargo transportation by assembling into dimers capable of taking several consecutive steps along a cytoskeletal filament. In the well-accepted hand-over-hand stepping mechanism, the trailing motor detaches from the track and binds the filament again in the leading position. This requires fuel consumption in the form of ATP hydrolysis and coordination of the catalytic cycles between the leading and the trailing heads. Alternate stepping pathways also exist, including inchworm-like movements, backward steps, and foot stomps. Whether all the pathways are coupled to ATP hydrolysis remains to be determined. Here, to establish the principles governing the dynamics of processive movement, we present a theoretical framework that includes all of the alternative stepping mechanisms. Our theory bridges the gap between the elemental rates describing the biochemical and structural transitions in each head and the experimentally measurable quantities such as velocity, processivity, and probability of backward stepping. Our results, obtained under the assumption that the track is periodic and infinite, provide expressions that hold regardless of the topology of the network connecting the intermediate states, and are therefore capable of describing the function of any molecular motor. We apply the theory to myosin VI, a motor that takes frequent backward steps and moves forward with a combination of hand-over-hand and inchworm-like steps. Our model quantitatively reproduces various observables of myosin VI motility reported by four experimental groups. The theory is used to predict the gating mechanism, the pathway for backward stepping, and the energy consumption as a function of ATP concentration.

GroEL Allostery Illuminated by a Relationship between the Hill Coefficient and the MWC Model

A fundamental problem that has hindered the use of the classic Monod-Wyman-Changuex (MWC) allosteric model since its introduction is that it has been difficult to determine the values of its parameters in a reliable manner because they are correlated with each other and sensitive to the data-fitting method. Consequently, experimental data are often fitted to the Hill equation, which provides a measure of cooperativity but no insights into its origin. In this work, we derived a general relationship between the value of the Hill coefficient and the parameters of the MWC model. It is shown that this relationship can be used to select the best estimate of the true combination of the MWC parameter values from all the possible ones found to fit the data. Here, this approach was applied to fits to the MWC model of curves of the fraction of GroEL molecules in the high-affinity (R) state for ATP as a function of ATP concentration. Such curves were collected at different temperatures, thereby providing insight into the hydrophobic effect associated with the ATP-promoted allosteric switch of GroEL. More generally, the relationship derived here should facilitate future thermodynamic analysis of other MWC-type allosteric systems.

An investigation into the role of ATP in the mammalian pre-mRNA 3' cleavage reaction.

RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. Invitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(β,γ-methylene)triphosphate (AMP-PCP) is an effective invitro 3' cleavage inhibitor with an IC50 of ∼300μM, but that most other ATP analogs, including adenosine 5'-(β,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation invitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage.

Torque Generation by Axonemal Outer-Arm Dynein

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αβγ) as well as proteolytically generated two-headed (βγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αβγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed βγ did not display this sensitivity. In the assays on lawns containing mixtures of α and βγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αβγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

Ensemble velocity of non-processive molecular motors with multiple chemical states.

We study the ensemble velocity of non-processive motor proteins, described with multiple chemical states. In particular, we discuss the velocity as a function of ATP concentration. Even a simple model which neglects the strain dependence of transition rates, reverse transition rates and nonlinearities in the elasticity can show interesting functional dependencies, which deviate significantly from the frequently assumed Michaelis-Menten form. We discuss how the order of events in the duty cycle can be inferred from the measured dependence. The model also predicts the possibility of velocity reversal at a certain ATP concentration if the duty cycle contains several conformational changes of opposite directionalities.

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

The molecular chaperone GroEL exists in at least two allosteric states, T and R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, approximately 50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

Regulation of Escherichia coli Polynucleotide Phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.

Conformational Dynamics of Self-thiophosphorylating RNA

The Kin.46 kinase ribozyme was previously selected from a random-sequence RNA library that was loosely based on the sequence of a known ATP aptamer, based on its ability to catalyze transfer of the thiophosphate from ATPgS to its own 5' hydroxyl end. The reaction requires an oligonucleotide effector that is complementary to the 3' end of the ribozyme and that served as reverse transcription primer during the amplification steps of the original selection for activity (Fig. 1). Omitting the oligonucleotide reduces the observed catalytic rate constant (kobs) by 10 3 to 10-fold, indicating that the oligo acts as an allosteric effector that is necessary for full catalytic activity. The activator helix is separated from the substrate-binding internal guide sequence by a 5nt “linker” that appears to form long-range baseparing interaction with nucleotides within the catalytic core (S. Rhee, unpublished results). Binding of small ligands, such as metal ions and ATP, can also potentially influence the folded structure of the RNA. Divalent metal ions are well known for their abilities to stabilize nucleic acid secondary and tertiary structures, and for their potential catalytic roles in ribozyme-catalyzed reactions. For example, we recently found that topologically rearranged versions of Kin.46 exhibit markedly altered Mg dependence. Similarly, nucleotide binding by aptamers nucleates formation of the folded structure and induces order in the NTP binding pocket (adaptive binding). ATP recognition by Kin.46 is clearly distinct from ATP recognition by the original aptamer; key nucleotides required for ATP recognition are mutated in Kin.46, and the values of Km ATPgS and Km ATP for Kin.46 (~3 mM) are >10 times higher than Kd ATP for the aptamer (~1 μM). In this work, fluorescence emission from cy5-labeled ribozymes was monitored in order to investigate conformational dynamics as a function of ATP concentration and Mg ion. Truncated versions of Kin.46 ribozyme were obtained by internal cleavage or deletions (Fig. 1). Ribozyme130 was derived from the Kin.46 by just cleaving the most right-side loop, ribozyme119 was obtained by deletion of the loop and

Cys-113 and Cys-422 Form a High Affinity Metalloid Binding Site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.

External mechanical force as an inhibition process in kinesin's motion

We analysed published force-velocity data for kinesin using classical Michaelis-Menten kinetic theory and found that the effect of force on the stepping rate of kinesin is analogous to the effect of a mixed inhibitor in classical inhibition theory. We derived an analytical expression for the velocity of kinesin (the stepping rate, equal to the ATP turnover rate) as a function of ATP concentration and force, and showed that it accurately predicts the observed single molecule stepping rate of kinesin under a variety of conditions.

Biochemical Analysis of the DNA Unwinding and Strand Annealing Activities Catalyzed by Human RECQ1*[boxs

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.

Stability of the Topoisomerase II Closed Clamp Conformation May Influence DNA-stimulated ATP Hydrolysis

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Myo1c is designed for the adaptation response in the inner ear.

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES.

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.

Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis.

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1.

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.