mantADP Release Research Articles

Myosins Active Site Closure via Switch-1 Allosterically Regulates the Motor-Actin Interaction during the ATPase Cycle for Productive Force Generation

Myosins are essential for many cellular processes including cell motility and muscle contraction. These motors have active site motifs (P-loop, switch-1, switch-2) that bind ATP and a divalent metal cofactor to coordinate ATP hydrolysis. ATP hydrolysis is activated through allosteric communication between the ATP and polymer binding sites to couple the free energy of ATP hydrolysis to force generation. Our lab seeks to understand how changes in enzymatic properties leads to different mechanical properties in molecular motors. We hypothesize that myosins depend on active site closure via switch-1 to regulate the motor-actin interaction during the ATPase cycle for productive force generation. We developed a strategy to control enzymatic activity and motility of myosin by substituting a switch-1 serine with cysteine, diminishing its interaction with the Mg+2 metal thus inhibiting ATPase activity. Substituting Mg+2 with Mn+2, which strongly interacts with cysteine, restores metal interaction and ATPase activity. Thus swapping of the divalent metals provides a direct and experimentally reversible link between switch-1 and the actin binding cleft. We have shown that the S237C mutant of non-muscle myosin II has rapid basal steady-state ATP turnover, but upon binding actin, its MgATPase is inhibited. Mn+2 relieves this inhibition and restores ATP turnover near WT values. The acto•S237C complex has slow, rate limiting MgATP binding which is rescued to near WT activity in the presence of Mn+2. Mant-ADP release experiments show the need for switch-1 interaction with the metal for tight ADP binding. Finally, to test whether this allosteric communication is necessary for force generation, we will test S237C motility in the presence of Mg2+ and Mn2+. These results support a strong reciprocal coupling of nucleotide and F-actin binding in myosin.

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

Kinetic Characterization of Rice Plant Specific Kinesin E11 using Fluorescent ATP Analogue

Kinesin is an ATP-driven motor protein that 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, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures.In this study, we characterized rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E. coli expression system and purified with Co-chelate column in order to characterize biochemical and ATPase kinetic properties. The fluorescent ATP analogues, Mant-ATP was employed for the kinetic characterization. We have successfully observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters of initial binding of Mant-ATP to E11 and release of Mant-ADP from E11 were analyzed by monitoring the FRET using stopped flow apparatus and compared with other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are slower than those of other rice plant specific kinesin.

Strain-Dependent Regulation of the Kinesin-1's Catalytic Activity as Studied by Disulfide-Crosslinking of the Neck Linker

Kinesin-1 walks along microtubules by alternately hydrolyzing ATP and moving two motor domains. Several recent studies have suggested that the strain developed through the neck linker is essential for the coordination between two motor domains, although the mechanism for the regulation of the motor domain's activity remains unknown. At the last annual meeting we employed disulfide-crosslinking between cysteine residues introduced into the neck linker and the motor domain to constrain the neck linker of a monomer in the forward or backward extended conformation, and using single molecule fluorescent observation we showed that detachment rate from the microtubule dramatically decreased when the neck linker is constrained in a backward extended conformation. To further investigate the effect of crosslinking on the ATP hydrolysis kinetics, we measured mant-ADP release after rapid mixing with microtubule and subsequent mant-ATP binding rates of the monomers. Stopped flow measurements showed that ADP release and ATP binding rates did not change after constraining in the backward extended conformation, suggesting that either ATP hydrolysis or Pi release step may be the rate-limiting. When the neck linker is constrained in a forward extended conformation, ATP binding rate did not changed but the ADP release rate dramatically decreased. These results suggest that ATP hydrolysis cycle of the motor domain can be differently regulated depending on the direction of the neck linker tension, explaining the alternate catalysis in the dimer.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Mitotic Kinesin Kar3Cik1 Interaction with Microtubules

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet crosslinks interpolar MTs (ipMTs) during anaphase. Kar3 contains ATP and MT binding sites, yet Cik1 lacks the nucleotide binding site. Presteady-state kinetic and thermodynamic studies were pursued using paclitaxel-stabilized MTs to define the Kar3Cik1 interactions with the MT lattice expected during ipMT crosslinking. The results reveal that Kar3Cik1's association with the MT occurs at 4.9 μM-1s-1 followed by a 5 s-1 structural transition that limits mantADP release to 4-5 s-1. However, the intrinsic rate of mantADP release from Kar3Cik1 was observed at 109 s-1. ATP binding to nucleotide-free MT•Kar3Cik1 occurred at 2.1 μM-1s-1 followed by an ATP-promoted isomerization at 64 s-1. ATP hydrolysis was a fast step observed at 25 s-1, yet the reduced burst amplitude indicates reversals at this step. ATP hydrolysis was required for ATP-promoted Kar3Cik1 detachment from the MT at 12.7 s-1. The rate constant of phosphate release at 10 s-1 was similar to the rate constant of ATP-promoted MT•Kar3Cik1 dissociation, suggesting that these two steps are coupled. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change after Cik1 collision with the MT leading to Kar3 association with the MT followed by ADP release. These findings provide a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 MT association followed by Kar3 binding with regulation through Kar3 nucleotide state and mechanical strain between the Kar3-Cik1 heads. Supported by NIH grant R01-GM54141 to SPG.

Kinetics and Thermodynamics of Nucleotide Binding Pocket Opening/closing in Myosin V Monitored with FRET

Kinetic and structural studies of both muscle and non-muscle myosins have revealed that the enzymatic cycle of these motors frequently contains more than one actomyosin ADP state. Interestingly, the rate of ADP release in myosin motors is thought to be the main determinant of sliding velocity in muscle, suggesting strain dependent ADP release may be a critical mechanism of mechanochemical coupling. Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH (MV FlAsH) can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides. We also determined that this donor-acceptor pair likely monitors opening/closing of the nucleotide binding pocket. Currently, we utilized the FRET signal to examine the kinetics of nucleotide binding pocket opening during the process of mantADP release from acto-MV FlAsH. We obtained evidence that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP. We also explored the temperature dependence of the closed to open transition and nucleotide release steps. We find that at lower temperatures the closed conformation is favored while at higher temperature the open conformation is favored. The more rapid ADP release step which follows nucleotide binding pocket opening is also temperature dependent. Therefore, since both steps are temperature-dependent they likely require significant conformational changes. We also compared our FRET results to the rate of ATP-induced dissociation from actin in the presence of ADP monitored by light scatter. Understanding how strain alters either of these two steps may be critical for elucidating the structural mechanism of strain-dependent ADP release in myosins.

ADP but Not Pi Dissociation Contributes to Rate Limitation for Escherichia coli Rho

To define the molecular mechanism by which ATP hydrolysis powers the 5'-->3' travel of homohexameric Escherichia coli transcription termination factor Rho along RNA, rates for association and dissociation of non-RNA substrates and products were measured. Rapid mix/chemical quench and stopped-flow spectrofluorometry measurements were carried out with Rho and [gamma-(32)P]ATP, mantADP, or fluorescently tagged E. coli phosphate-binding protein. The results indicate that the P(i) off-rate is not rate limiting, but at approximately 90 s(-1), the ADP dissociation rate is comparable to the 30 s(-1) k(cat). Previous results indicate that the chemistry step of ATP hydrolysis by Rho is at least 10-fold faster than the overall catalytic cycle. The as yet unmeasured RNA dissociation step, which could be associated with a protein conformation change, might also be a rate-limiting factor.

Interactions between Subunits in Heterodimeric Ncd Molecules

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acid-quench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P(i) release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.

S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app) <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-trityl-L-cysteine binds more tightly than monastrol because it has both an approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-1) s(-1) and 15 s(-1) for monastrol). S-trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC(50) of 500 nm. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.

The Kinetic Mechanism of the SufC ATPase: THE CLEAVAGE STEP IS ACCELERATED BY SufB

Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.

Mechanistic Analysis of the Mitotic Kinesin Eg5

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

Rapid double 8-nm steps by a kinesin mutant.

The mechanism by which conventional kinesin walks along microtubules is poorly understood, but may involve alternate binding to the microtubule and hydrolysis of ATP by the two heads. Here we report a single amino-acid change that affects stepping by the motor. Under low force or low ATP concentration, the motor moves by successive 8-nm steps in single-motor laser-trap assays, indicating that the mutation does not alter the basic mechanism of kinesin walking. Remarkably, under high force, the mutant motor takes successive 16-nm displacements that can be resolved into rapid double 8-nm steps with a short dwell between steps, followed by a longer dwell. The alternating short and long dwells under high force demonstrate that the motor stepping mechanism is inherently asymmetric, revealing an asymmetric phase in the kinesin walking cycle. Our findings support an asymmetric two-headed walking model for kinesin, with cooperative interactions between the two heads. The sensitivity of the 16-nm displacements to nucleotide and load raises the possibility that ADP release is a force-producing event of the kinesin cycle.

Ionic Interactions Play a Role in the Regulatory Mechanism of Scallop Heavy Meromyosin

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 can be used to define the equilibrium constant ( K eq) between on and off conformations. The data show that K eq is markedly ionic strength dependent, with high salt (≥200 mM) abolishing the off state even in the absence of calcium and low salt (<50 mM) favoring the off state even in the presence of calcium. Debye-Huckel plots of the equilibrium constant ( K eq) for the on and off forms gave parallel slopes (5.94 ± 0.33 and 6.36 ± 0.17 M −0.5) in the presence and absence of calcium. The presence of an equilibrium mixture of two conformations was confirmed by sedimentation data and the effects of ADP, calcium and ionic strength were in qualitative agreement. Thus scHMM exists in two conformations that can be distinguished in sedimentation profiles and by the rate of release of mant-ADP. Increasing salt concentrations biases the system toward the on state, suggesting a role for ionic interactions in stabilizing the off state.

Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

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 transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.

Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity.

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 s(-1)), but release is accelerated by added ADP or ATP. The maximum rate of ADP-stimulated dissociation of tethered DKH405 from the microtubule is approximately 12 s(-1) as determined by turbidity. Parallel measurements of ADP-stimulated release of 2'(3')-O-(N-methylanthraniloyl)-ADP (mantADP) from the tethered intermediate by fluorescence indicate that the reaction is biphasic with a fast phase that occurs at a rate that is similar to dissociation. The rate of the slow phase is dependent on the concentrations of salt and microtubules and is equal in each case to the rate for bimolecular stimulation of ADP release by microtubules as measured independently. These results are consistent with a scheme in which the fast phase, with approximately one-third of the total amplitude change, is due to ADP-stimulated release of mantADP from the tethered intermediate at approximately 6 s(-1). This direct release of mantADP continues until terminated by dissociation of DKH405 from the microtubule at approximately 12 s(-1). The majority of the amplitude change thus occurs through bimolecular recombination of DKH405.mantADP with microtubules following initial dissociation. Analysis of a simple scheme indicates that hydrolysis of ATP at the attached head before the tethered head can release its ADP and become tightly bound may be the principal limitation to processivity.

Actin Cross-Linking and Inhibition of the Actomyosin Motor

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.

Interaction of Mant-Adenosine Nucleotides and Magnesium with Kinesin

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 the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.