Saturating ATP Research Articles

A model of processive walking and slipping of kinesin-8 molecular motors

Kinesin-8 molecular motor can move with superprocessivity on microtubules towards the plus end by hydrolyzing ATP molecules, depolymerizing microtubules. The available single molecule data for yeast kinesin-8 (Kip3) motor showed that its superprocessive movement is frequently interrupted by brief stick–slip motion. Here, a model is presented for the chemomechanical coupling of the kinesin-8 motor. On the basis of the model, the dynamics of Kip3 motor is studied analytically. The analytical results reproduce quantitatively the available single molecule data on velocity without including the slip and that with including the slip versus external load at saturating ATP as well as slipping velocity versus external load at saturating ADP and no ATP. Predicted results on load dependence of stepping ratio at saturating ATP and load dependence of velocity at non-saturating ATP are provided. Similarities and differences between dynamics of kinesin-8 and that of kinesin-1 are discussed.

The temperature dependence of kinesin motor-protein mechanochemistry

Biophysical studies of the mechanochemical cycle of kinesin motors are essential for understanding the mechanism of energy conversion. Here, we report a systematic study of the impact of temperature on velocity and run length of homodimeric Drosophila kinesin-1, homodimeric C. elegans OSM-3 and heterodimeric C. elegans kinesin-II motor proteins using in vitro single-molecule motility assays. Under saturated ATP conditions, kinesin-1 and OSM-3 are fast and processive motors compared to kinesin-II. From in vitro motility assays employing single-molecule fluorescence microscopy, we extracted single-motor velocities and run lengths in a temperature range from 15 °C to 35 °C. Both parameters showed a non-Arrhenius temperature dependence for all three motors, which could be quantitatively modeled using a simplified, two-state kinetic model of the mechanochemistry of the three motors, providing new insights in the temperature dependence of their mechanochemistry.

A Generalized Kinetic Model for Coupling between Stepping and ATP Hydrolysis of Kinesin Molecular Motors.

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.

Single-Molecule Studies of ATP Binding to the Sodium Pump

The sodium pump uses energy from the hydrolysis of 1 ATP to move 3Na+ out of and 2K+ into the cell via transitions between phosphorylated and unphosphorylated forms of two major conformations (E1 and E2). In earlier experiments, we had found that ATP binds with low and high affinity to the E1 conformation of pump isolated from nasal glands of juvenile ducks. We attributed this heterogeneity to interactions between ATP binding sites on different protomers within multiprotomeric aggregates. We report here on experiments designed to examine ATP binding to pump protomers using single-molecule fluorescence measurements. Pump labeled with Cy3-maleimide was solubilized with C12E8. Protomers were separated from aggregates using FPLC and reconstituted into lipid vesicles at an average concentration of 1 protomer/vesicle. The fluorescence of vesicles tethered to a glass cover slip using a biotin-streptavidin linkage was measured as a function of time using single-molecule total internal reflection fluorescence microscopy under conditions where the pump was in the E1 conformation. Vesicles where fluorescence photobleached in a single-step were identified as containing single protomers. These traces were either dynamic (fluorescence fluctuated between two states) or nondynamic (no fluctuations). The fraction of protomers that showed dynamic behavior increased saturably with ATP from ∼40% at 0 ATP to ∼70% at 1 mM (saturating) ATP. This titration curve could not be fitted with a single binding isotherm. A fit to a model that assumed a heterogeneous mixture of pump molecules with high- and low-affinity ATP binding showed: (a) high- and low-affinity apparent dissociation constants of 0.1 and 40 μM; and (b) that the preparation had equal amounts of high- and low-affinity forms of the pump. Our data suggest that binding heterogeneity is an intrinsic property of the protomer and is not attributable to aggregation.

High-Speed Observations of the Unbinding/Binding Motions of the Leading Head of Kinesin-1 From/To Microtubule Provide Kinetic Evidence for the Front-Head Gating Mechanism

Kinesin-1 is a motor protein that moves processively along microtubule by alternately moving two motor domains (“heads”). To move in the hand-over-hand manner, ATP hydrolysis cycles of the two heads should be coordinated such that the trailing head hydrolyses ATP and detaches from the microtubule before the leading head does. To explain such coordination, the front-head gating model has been proposed that ATP binding or hydrolysis in the leading head is prohibited due to the backward tension posed through the neck linker. However, it has been technically difficult to directly measure the detachment rate of the leading head. Here, we utilized a heterodimer composed of the wild-type head and a E236A mutant head that hydrolyses ATP very slowly. Single molecule FRET observations between dyes attached to two heads showed that the wild-type head spent majority of time in the leading position under saturating ATP condition. Then we attached a 40 nm-ϕ gold particle specifically to the wild-type head and observed its motion using total internal reflection-based dark-field microscopy at 50 μs temporal resolution. At this resolution, we could clearly distinguish microtubule-bound and -unbound states by the difference in the magnitude of fluctuation of the gold probe. The dwell time of the leading head in the microtubule-bound state was 160 ms on average at 1 mM ATP condition, which was significantly larger than the dwell time of a head of wild-type homodimer in the bound state at trailing position (10 ms; Isojima et al. Nat. Chem. Biol. 2016). These results provide kinetic evidence for the front-head gating model.

Biased Binding Mechanism Alone Can Explain the Preferential Forward Stepping of Kinesin-1

Kinesin-1 moves along microtubule by alternately moving two motor domains (“heads”); the unbound trailing head is prohibited from rebinding to the rear-binding site and is allowed to binds to the forward-binding site 16-nm ahead. The “neck linker-docking (or biased-diffusion) model” has been proposed to explain the preferential forward stepping, however this model cannot explain how the rebinding of the unbound head is prohibited while kinesin waits for ATP-binding. Alternate “biased-binding model” proposes that an unfavorable increase in the tension on the neck linker prohibits the unbound head from rebinding to the rear-binding site. To distinguish between these two models, we previously engineered an asymmetrically joined two-headed monomer, in which the neck linker of the first head (N-head) is connected to N-terminus of second head (C-head), such that this motor alternately uses biased-binding (when N-head steps forward) and biased-diffusion (when C-head steps) mechanisms to move in a hand-over-hand manner. In this study we observed the motion of each heads during the processive movement of the two-headed monomer using the total internal reflection-based dark-field microscopy with 50 µs temporal resolution. We found that the stepping of C-head was less efficient because the unbound C-head frequently rebound to the rear-binding site even at saturating ATP condition, while N-head stepped forward efficiently because the unbound N-head rarely showed rebinding under various ATP conditions. These results indicate that the diffusional-biased of the unbound head is not necessary, and that the biased-binding mechanism that prohibits rebinding of the unbound head is sufficient, for the preferential forward stepping of kinesin-1, at least under low external load.

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

Study of Bloom resolving G-quadruplex process by using high resolution magnetic tweezer with illumination of total internal reflection

G-quadruplex (G4) is a DNA structure which commonly exists in human genome, and it is considered as an important structure in DNA metabolism such as replication, transcription and homologous recombination. The G-quadruplex helicases have been widely investigated these years. Of them, the Bloom (BLM) helicase is most thoroughly studied. However, there are some basic problems that are still unclear. Most of previous studies of G4 are performed by single molecule fluorescence resonance energy transfer technique. The G4 is in a free state in these experiments, which is different from the physiological environment in cells. The traditional magnetic tweezers have a limitation of spatial resolution in a low force circumstance. Thus here we use high resolution magnetic tweezer under the illumination of total internal reflection fluorescence to study the process of BLM resolving G4. Our modification of magnetic tweezer is to separate the measurements of force and distance of magnetic tweezer in order to improve the spatial resolution, which allows us to observe the unfolding process of G4. With a 2-3 pN force we find that the process of BLM unfolding G4 in low ATP concentration is stepwise, and the G4 is mainly in the state between G-quadruplex and G-triplex. We also find that the BLM could interact with G4 for a long time. Our apparatus is also able to obtain the long time observation results compared with the single molecule fluorescence technique. So we perform experiments with a nearly saturated ATP concentration. We find that the BLM has two ways to maintain G4 dissolution in this condition. The BLM could unfold the G4 repetitively in a long period and it could also keep the G4 in unfolding state for a long time after it has opened the G4. Finally, we also perform single molecule fluorescence resonance energy transfer experiment in the same condition, and we find that the 2-3 pN force in magnetic tweezers has a rare influence on the process of BLM interacting with G4. The results of single molecule fluorescence resonance energy transfer experiments are corresponding to the results of magnetic tweezer in the same conditions. All of our experimental results show that ATP dependent BLM has a high affinity with G4 and BLM has a different way to resolve G4 in high ATP concentration. These results could provide new ideas of the mechanism of BLM resolving G4. Our modified magnetic tweezer shows its capacity in G4 single molecule study, and it could be a useful tool in the future single molecule studies.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.

The Acidosis-Induced Slowing of Regulated Thin Filament Velocity in a Motility Assay Disappears at Low ATP

Acidosis (low pH) is a putative agent of muscle fatigue that slows contraction velocity but the mechanisms underlying this effect are unclear. It may directly affect myosin by slowing a step associated with detachment or it may act indirectly by affecting the ability of Ca++ to activate the thin filament, through effects on the regulatory proteins troponin/tropmyosin. We measured the effect of manipulating Ca++ and the lifetime rigor binding (by lowering the [ATP]) to examine the relative role of each of these mechanisms. At saturating Ca++ (pCa 4) and ATP concentrations (1mM) decreasing the pH from 7.4 to 6.8 decreased regulated actin filament velocity (Vrtf) by 50%, however at low [ATP] (<200 μM) Vrtf was unaffected by acidosis. Repeating these experiments in the absence of Ca++ (pCa 9) confirmed the phenomenon of rigor activation at normal pH (7.4), with filaments moving fastest at 50 μM ATP (0.5 μm/s). At low pH the filaments moved slower but still demonstrated rigor activation at [ATP] <75 μM. Single molecule laser trap assay experiments are under way to determine whether the slowed velocity was due slowed attachment or detachment kinetics. The acidosis-induced decrease in Vrtf at saturating Ca++ and ATP are consistent with a slowing of the steps in the cross-bridge cycle associated with ADP-release. However this effect disappears at low ATP where the attached lifetime is more dependent on the duration of rigor. Therefore the data suggest that acidosis does not affect the formation of a rigor bond or its ability to activate the thin filament. Inhibition of Ca++ binding to troponin might still contribute to slowing at saturating ATP and submaximal Ca++ levels.

Kinetics of Nucleotide-Dependent Structural Transitions in the Kinesin-1 Hydrolysis Cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. The high spatiotemporal resolution of this method enabled real-time recording of structural changes in the protein as it walked at ∼100 steps per second. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying the structural transitions to and from this one-head-bound intermediate were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, meaning that there is one rate-limited step in each the one- and two-heads bound states. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor, and show that the mechanism underlying stepping is a two-step process. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

High Speed Microscopy for Observing the Stepping Behavior of Kinesin-1 Motors at Saturating ATP

Kinesin-1 is the most well studied member of the kinesin superfamily of molecular motors and is responsible for the trafficking of vesicles and organelles towards terminal branches of axons. This motor hydrolyzes ATP in a tightly coupled fashion in order to take directed 8 nm steps along a single protofilament of a microtubule with high processivity. However, the comprehensive mechanochemical cycle of kinesin-1 remains elusive, due largely to the technical limitations of ensemble experiments and fluorescence microscopy approaches at the single-molecule level. Questions remain in identifying the rate limiting step in the hydrolysis cycle as well as the time spent in the one head bound state. To probe the mechanochemical cycle, we employ a fluorescence-free imaging technique, interferometric scattering microscopy (iSCAT), to track kinesin-1 at saturating ATP in a reconstituted system with greatly improved spatiotemporal resolution. With point spread function fitting, 8 nm and 16 nm steps are measured for C-terminus and N-terminus labeled motors, respectively. Using novel differences of means and Gaussian mixture model algorithms for the identification of step and sub-step regimes, we investigate the information present in both on-axis and off-axis positional data. Overall, these results point towards new insights into mechanochemical cycle of kinesin-1. The optical system and data analysis tools used present a new platform for investigating the stepping cycles of diverse molecular motors.

Varying the Number of Heads in Phosphorylated Smooth Muscle Myosin Filaments Provides Evidence for Attachment Limited Kinetics of in vitro Actin-Sliding Velocities

Previously we have shown that the ATP-dependence of EDC-stabilized fluorescent smooth muscle myosin (SMM) filament motility is very similar to solution ATPase values (KATP = 8.5 and 9.2 µM, respectively). This suggests both processes are influenced by the same kinetic step, acto-myosin attachment not detachment, presumably linked to Pi release. This mechanism predicts that velocities will be linearly related to the number of working heads. Therefore, we prepared SMM- rod co-filaments that contain fewer molecules per filament length as well as longer SMM filaments that contain more molecules per filament to determine the relationship between the number of heads and velocity. SMM:rod co-filaments (at 50:50, 25:75, and 15:25 ratios) were approximately the same length as normal SMM filaments (∼0.6 µm), and the number of molecules incorporated was directly related to the ratio of SMM to rods. As the number of heads decreased, the maximal velocity at saturating ATP also decreased, and the KATP was lower for all of the co-filaments compared to the normal filaments. These findings provide evidence that the velocity of SMM filaments is influenced by attachment limited kinetics. The longer SMM filaments (average length of 2.3 ± 0.81 µm) will allow us to visualize the geometry of acto-myosin interactions during motion. Also, the relationship between velocity and filament length will be determined at various [ATP]. The long filaments also better demonstrate our previously described “parking” behavior where filaments move to the end of actin filaments but remain attached for long periods even at saturating [ATP] (Haldeman et al, JBC, 2014). Interestingly, most of the SMM filament hangs off the end of the actin suggesting the parking is mediated by a small fraction of the heads.

Role of protons in the pump cycle of KdpFABC investigated by time-resolved kinetic experiments.

The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 μM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P → P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Direct Observation of the Forward Stepping Motion of Kinesin-1 using Dark-Field Microscopy with 50-Micro Second Temporal Resolution

Kinesin-1 moves along microtubule by alternately moving two motor domains, in which the trailing head detaches and displaces toward the 16-nm forward tubulin-binding site upon ATP binding to the partner head. However, the detailed process of the forward stepping motion is still unclear, mainly due to the lack of temporal resolution of the single-molecule measurements. Here we employed dark-field microscopy with perforated mirror, and observed the movement of one of kinesin heads labeled with 40-nm gold particle with ∼2 nm spatial and ∼55 μs temporal resolutions. In the presence of saturating ATP, the gold particles showed ∼16 nm discrete steps toward the microtubule long axis, which is consistent with the previous observations. However, our measurements also revealed that, just before 16 nm step, the gold particle displaced rightward, perpendicular to the microtubule axis, accompanied with a significant increase in the fluctuation of the gold particle. The rightward asymmetry is consistent with the geometry that the proximal end of the neck linker is located right side of the head. Then the gold particles showed rapid left-forward displacement followed by decrease in the fluctuation, although we could not detect clear sub-step at this temporal resolution. The duration of the highly fluctuating period increased as the ATP concentration was decreased, indicating that the period represents ATP-waiting state. These results provide direct clue to understand how kinesin takes a step forward.

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

Kinesin is a dimeric motor protein that hydrolyzes ATP and moves along microtubules in a hand-over-hand manner. To walk by alternately moving two motor heads, the trailing head should detach from the microtubule prior to the leading head and the detached head should preferentially bind to the forward tubulin-binding site. To explain these mechanisms, we hypothesized that ATP hydrolysis reaction of kinesin motor domain can be regulated depending on the direction of the tension posed to the neck linker: backward strain posed to the neck linker suppresses ATP hydrolysis in the leading head and the forward strain posed to the neck linker suppresses ADP release at the trailing position. To test this hypothesis, we constrained the neck linker in the forward or backward extended conformation using disulfide-crosslinking between cysteine residues on the head and the neck linker, and examined these effects on the microtubule affinity and ADP release kinetics. Single molecule fluorescent observation of the GFP-fused monomeric kinesin showed that when the neck linker was constrained in a backward extended conformation, the dwell time on the microtubule in the presence of saturating ATP was increased by a factor of 15 compared to unconstrained condition. In contrast, stopped-flow measurement showed that when the neck linker was constrained in a forward extended conformation, ADP release rate after microtubule-binding was significantly decreased. These results support the idea that ATP hydrolysis cycle of kinesin's motor domain can be differently regulated depending on the direction of the neck linker extension.

Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

Muscle contraction results from cyclic interaction of myosin and actin, driven by ATP hydrolysis, where the actomyosin complex undergoes transitions between weakly and strongly bound structural states. Rabbit skeletal muscle myosin has two isoforms of the essential light chain (ELC), designated A1 and A2. These isoforms have significant effects on myosin's interaction with actin, resulting in higher catalytic efficiency for A1 but higher in vitro motility and muscle shortening velocity for A2. In order to understand the structural basis of these differences, we have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of ELC isoforms on the microsecond dynamics of actin during interaction with skeletal muscle myosin in the absence and presence of saturating ATP. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes containing A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy similarly, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (rigor) state of actomyosin. In contrast, in the presence of saturating ATP, the final anisotropy of actin was substantially higher during interaction with S1A1 than with S1A2. Thus, S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin, suggesting greater population of the strong-binding state of actomyosin. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, and thus modulates the contractile function of actomyosin.

Structural Transitions within the Actomyosin Complex in the Presence of Saturating ATP, Detected by Time-Resolved FRET

Myosin generates force by cycling between actin-attached (strong binding) and actin-detached (weak binding) states during the actomyosin ATPase cycle. However, it remains unclear what specific conformational changes in myosin take place upon binding to actin, and how these structural changes lead to product release and force. To characterize these changes, we have used site-directed FRET to determine distance between specific sites in actin and myosin subfragment 1 (S1) in the presence and absence of ATP. Rabbit skeletal muscle myosin has two essential light chain (ELC) isoforms, A1 and A2.The ELC isoform has significant effects on the interaction of S1 with actin, resulting in higher catalytic efficiency for the S1A1-isoenzyme but higher in vitro motility and muscle shortening velocity for S1A2.We have focused on S1A1, since it binds to actin in the presence of ATP with higher affinity. Actin was labeled at C374 with a donor, AlexaFluor 568. A1 light chain, was expressed,purified and labeled at C177 with an acceptor, AlexaFluor 647. The labeled A1 was exchanged into myosin S1, and S1A1 was isolated using a Talon affinity resin. The efficiency of energy transfer between C374 of actin and C177 of A1 was measured by time-resolved fluorescence, allowing us to detect directly the FRET within the actin-S1A1 complex, even in the presence of saturating ATP. Addition of ATP to the strongly bound complex resulted in a significant decrease in FRET within the weakly bound complex, corresponding to a significant increase in the donor-acceptor distance. We conclude that the transition between strongly and weakly bound states of acto-S1 is associated with a structural transition in the acto-S1 complex that affects the distance from actin to the light-chain domain of myosin.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors

In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.