From the
Laboratory of Molecular Cardiology and
¶Electron Microscopy Core, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892-1762 and
Division of Physical Biochemistry, National
Institutes for Medical Research, London NW71AA, United Kingdom
Received for publication, April 8, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpeded by nonproductive strongly bound myosin heads that have completed their powerstroke.
If a vesicle translocating myosin had similar kinetics, a patch of 20 or more myosins would have to be positioned to possibly interact with actin to keep the vesicle attached to the actin filament so that it could move processively. Alternatively, if the myosin ATPase kinetics were altered such that the myosin spent most of its kinetic cycle strongly bound to actin, then a small number or possibly even a single myosin could efficiently translocate its vesicle processively along an actin filament. Another consideration for vesicle movement is that the actin filament is a helical polymer composed of 5 nm monomers with a 36-nm half-repeat that is usually bound to the cytoskeleton. If the moving myosin stepped from one actin monomer to the next, the motor and vesicle would rotate around the actin filament and crash its cargo into the cytoskeleton before any appreciable distance could be covered. Alternatively, if the motor could take large steps that matched the actin pseudo-repeat, it could effectively walk across the "top" of the cytoskeletal-bound actin filament while holding its cargo above.
Experiments using optical trapping nanometry, electron microscopy, and single molecule motility studies demonstrated that myosin V moves processively along actin filaments, taking many 36-nm steps before dissociating (58). Kinetic analyses demonstrated that it is a high duty cycle motor that associates strongly with actin in the presence of physiological concentrations of ATP (9). The processive nature and long step size reflect the intracellular function of the molecule as a cargo motor. The processivity is necessary to keep the cargo tethered to the actin filament, whereas the long step size matches the pseudo-repeat of the actin filament, allowing myosin V to "walk" across the top of, rather than rotate around, the actin filament, which would occur if the molecule took steps either longer or shorter than 36 nm. This behavior is in marked contrast to the myosin II molecules that power muscle contraction. These low duty ratio myosins are designed to make single transient interactions with actin and spend most of their kinetic cycle dissociated from actin (10, 11).
To test whether the long neck is essential for processive movement and how neck length affects the working stroke, we engineered recombinant heavy meromyosin (HMM)-like myosin V molecules with shorter or longer necks than that of wild type (WT) myosin V HMM by altering the number of IQ motifs. The effect of neck length on step size was measured in an optical trap, and the processivity was measured by directly observing the movements of single, fluorescently labeled molecules on actin using total internal reflection fluorescence (TIRF) microscopy. The results strongly suggest that a neck containing more than two CaM subunits is necessary for efficient processive movement, and the step size is roughly proportional to the length of the neck.
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EXPERIMENTAL PROCEDURES |
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Preparations of ProteinsHMM-IQ molecules were co-expressed
with CaM in baculovirus/Sf9 cells and purified using FLAG-affinity
chromatography (12). The
concentration of purified WT and IQ mutants were measured using the following
extinction coefficients
(A280A320) calculated from
their cDNA sequences: S16IQ, 0.644; HMM-2IQ, 0.683; HMM-4IQ, 0.631;
HMM-6IQ, 0.607; HMM-8IQ, 0.591; HMM-2Ala-6IQ, 0.607. All values are for
1 mg/ml. Biotin labeling of actin, Alexa 488 labeling of CaM,
and exchange of Alexa 488-CaM into the HMM-IQ constructs were performed as
described previously (6). Actin
was purified from skeletal muscle
(13).
Rhodamine-phalloidin-labeled rabbit F-actin and
N-ethylmaleimide-modified rabbit myosin were prepared by standard
methods (14). Rabbit skeletal
muscle HMM was prepared according to Homsher et al.
(15)
MgATPase AssayThe MgATPase activity was measured using an NADH-coupled assay at 25 °C in 10 mM MOPS, pH 7.0, 2 mM MgCl2, 1 mM ATP, 50 mM KCl, 0.15 mM EGTA, 40 units/ml l-lactic dehydrogenase, 200 units/ml pyruvate kinase, 200 µM NADH, and 1 mM phospho(enol)pyruvate. Vmax and KATPase, were obtained by fitting the data to the Michaelis-Menten equation.
In Vitro Motility AssaysThe motility assay was performed and quantified as described previously in a motility buffer containing 20 mM MOPS, pH 7.4, 2 mM MgCl2, 0.1 mM EGTA, 50 mM KCl, 1 mM ATP, 1 µM CaM, 25 µg/ml glucose oxidase, 45 µg/ml catalase, 2.5 mg/ml glucose, and 50 mM dithiothreitol at 30 °C (12, 15).
The TIRF assay was performed on an Olympus IX70 microscope equipped for objective type TIRF using a PlanApo (x60, numerical aperture 1.45) objective lens and a relay lens (PE5, x5 or PE2.5, x2.5; Olympus) connected to an image-intensified (KS1380; Videoscope) neuvicon camera (model C2400; Hamamatsu Photonics). Excitation light sources were an argon laser (488 nm, model 163-C0201; Spectra Physics) and a diode green laser (532 nm, model GCC-025-C; Crystal Laser). To prevent nonspecific binding of fluorescently labeled-protein, the coverslips (top, 18 x 18 mm and bottom, 24 x 50 mm) comprising the flow cell were prepared as follows. Coverslips were soaked in 5 N NaOH for 2 days, washed extensively with warm water, and placed into a sealed plastic box containing an open Eppendorf tube of acryloyl silanes (prepared by mixing 3-methacryloxypropyltrimethoxy-silane (Sigma) and 3-aminopropyltriethoxysilane (Pierce) in a 10:1 ratio) for 1 h at 50 °C. Unbound silane was removed by a brief rinse in 100% methanol. A solution of 2% acrylamide, 0.5% bisacrylamide, 0.1% cystamine, 0.1% ammonium persulfate, and 1% TEMED, was dropped onto the silane-coated coverslips, which were then inverted and placed on top of a 25 x 100-mm microscope slide with the wet surface adhering to the slide. After 45 min at 37 °C the coverslip and slide were separated with a razor, and unbound acrylamide and cystamine were washed away by filtered (0.45-µm pore diameter) deionized water (dH2O). Coverslips were then immersed in 1% 2-mercaptoethanol for 15 min to reduce disulfide bonds in the cystamine and then washed with a gentle flow of filtered dH2O. Biotin-maleimide (2 mM) in 20 mM TES, pH 7.0, was dropped onto the acrylamide-coated coverslip and incubated for 4 h at 37 °C. Upper coverslips (18 x 18 mm) were treated the same as the bottom one except that no cystamine was used. The flow cell, 15 µl in volume, was prepared using double-sticky cellophane tape. For TIRF assays, 30 µl streptavidin (47 µg/ml) solution was applied to the flow cell, incubated for 4 min, and washed with motility assay buffer (1 ml). Biotin-labeled, rhodamine-phalloidin-labeled rabbit F-actin filaments (1 µM) were applied to the flow cell and incubated for 2 min. Unbound actin was washed off with 1 ml of motility assay buffer. Fluorescently labeled HMM in 100 µl of motility assay buffer containing 1 µM CaM was introduced into the flow cell, which was then mounted onto the microscope for imaging. Data were captured on a sVHS videotape, and the run length and the velocity of single fluorescently labeled myosin V HMM molecules were measured using MetaMorph (Universal Imaging).
Landing assays were performed in the motility assay buffer described above, except containing 2 mM ATP (5). The sample was illuminated by laser at an angle less than the critical angle for TIRF to produce conditions in which higher concentrations of rhodamine-phalloidin-labeled rabbit F-actin (21 µg/ml) could be used than with conventional epifluorescence illumination, yet actin filaments could be imaged farther away from the surface than when using conventional TIRF.
Optical Trapping ConditionsThe optical trapping apparatus, detection, and quantification of events and solution conditions were as described previously (8, 14, 16). In brief, a single actin filament was captured between two N-ethylmaleimide-myosin coated latex beads (1 µM diameter) held in two independent optical traps. The actin was positioned over a third surface-bound bead that was sparsely coated with HMM-IQ molecules. The surface density was adjusted by serial dilution of the HMM-IQ solutions such that only one in three beads gave binding events. Mechanical interactions were measured by monitoring the positions of the beads holding the actin filament using two photo-detectors (14).
Electron MicroscopyRotary shadowed images of HMM-IQ mutants were obtained as described previously (12, 17).
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RESULTS |
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The electrophoretic mobility of the HMM-IQ mutants varied in proportion to the number of IQ motifs (Fig. 2b). Rotary shadowed electron microscopic images of the molecules were consistent with the neck lengths, but no obvious differences were observed between the images of HMM-2Ala-6IQ and HMM-6IQ in terms of neck flexibility (Fig. 2c).
ATPase ActivityThe steady-state MgATPase at various actin
concentrations was measured using an NADH-coupled assay to prevent ADP build
up. The MgATPase activities of all HMM-IQ constructs were <0.02
s1 in the absence of actin and were activated
750-fold by actin (Table
I). The Vmax of myosin V S16IQ (15.3
s1) was consistently higher than that of all
HMM-IQ molecules (between 10 and 12 s1
head1). The KATPase of HMM-6IQ (WT),
HMM-8IQ, and HMM-2Ala-6IQ were slightly lower than that of HMM-2IQ and
HMM-4IQ, whereas that of myosin V S16IQ was much higher than those of
the HMM constructs.
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Sliding Actin In Vitro Motility AssayIn vitro actin sliding assays under conditions where multiple HMM molecules can interact with actin showed that the velocity increased with increasing neck length for the HMM-2IQ, HMM-4IQ, HMM-6IQ, and HMM-8IQ (Table I). The velocity of HMM-2Ala-6IQ was intermediate between that of HMM-2IQ and HMM-4IQ. The velocity of S1 was slower than that of HMM-6IQ (Table I) (12).
Optical TrappingThe mechanical properties of the HMM-IQ constructs undergoing single step interactions with actin were observed using a dual optical trapping apparatus (8) (Fig. 3). The amplitudes of single displacement events of HMM-2IQ, HMM-4IQ, HMM-6IQ, and HMM-8IQ were proportional to neck length. The displacements for HMM-4IQ and HMM-6IQ were similar to those observed for S14IQ and S16IQ constructs (18), suggesting that they represent the working stroke produced by a single head. HMM-2Ala-6IQ gave a significantly shorter step size than expected from its neck length, which may be because of extra flexibility in the lever arm caused by the insertion of the two alanines (Fig. 3). The lifetimes of attachment (t50) for all constructs were about 0.22 s at 5 µM ATP.
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Landing AssayThe actin landing rate, a measure of how many actin filaments land on the surface in a given period of time and move longer than 0.2 µm as the myosin surface density is decreased, distinguishes between processive motors requiring only a single myosin molecule to move actin filaments and nonprocessive ones requiring multiple myosins to translocate actin filaments (5). The data of HMM-6IQ, HMM-4IQ, HMM-8IQ, and HMM-2Ala-6IQ were well fitted with n = 1 for the landing equation, consistent with these molecules being processive motors (Fig. 4). In contrast, the data for HMM-2IQ was fitted with an n = 2 suggesting that this molecule is not processive. Myosin V S16IQ and skeletal muscle HMM did not support movement at surface densities less than 3000 and 9000 molecules/µm2 and were fitted by n = 3 and n = 6, respectively (Fig. 4). At very low surface density (less than 20 molecules per µm2), HMM-4IQ, HMM-6IQ, HMM-8IQ, and HMM-2Ala-6IQ supported movement of landed actin filaments, but the moving filaments often pivoted about a single attachment point as described for intact myosin V and kinesin at low surfaces densities (5, 12, 19). This behavior was not seen for HMM-2IQ and S16IQ.
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Single Molecule Motility Assay (TIRF Assay)The endogenous CaMs of HMM-IQ mutants were partially exchanged for Alexa-488-labeled CaM, and single-fluorescently labeled HMM-IQ molecules were visualized using TIRF microscopy (6). The ratio between the fluorescence intensity and the HMM concentration in solution was used to determine the average number of fluorophores per HMM molecule, and the quantal photobleaching characteristics of the individual fluorescent molecules bound to the surface were then measured as a function of time (data not shown). The labeling ratio of all HMM-IQ constructs corresponded to the average number of photobleaching events observed in single spots in TIRF microscopy, suggesting that each fluorescent spot is a single HMM molecule. The average number of photobleaching events did not increase linearly with the number of IQ motifs of the HMM-IQ constructs (HMM-2IQ, 3.1 events; HMM-4IQ, 3.1; HMM-6IQ, 4.5; HMM-8IQ, 6.0; S1, 2), suggesting that not all CaMs exchange uniformly.
Single HMM-6IQ molecules bound to and moved in a highly processive manner along actin filaments bound to the surface, often moving all the way to the end of the actin filament before detaching (Fig. 5a). Fig. 5b plots the "full run frequency" defined as the probability that, upon attachment, an HMM molecule moves all the way to the end of the actin filament without detachment. The mean length of the actin filaments bound to the surface is 4.3 ± 2.5 µm. At 25 mM KCl and 2 mM ATP, the full run frequency for HMM-6IQ was about 80% (Fig. 5b). Under the same conditions, HMM-4IQ, HMM-8IQ, and HMM-2Ala-6IQ also moved highly processively with full run frequencies of more than 50%. In marked contrast, almost no HMM-2IQ molecules were able to move the entire remaining length of the actin filament under these conditions. When the ATP concentration in the assay was lowered, the full run frequency increased for all of the HMM-IQ molecules. At low ATP concentrations (1 µM and 10 µM) even HMM-2IQ exhibited a significant full run frequency.
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Another way to demonstrate the difference between the behaviors of HMM-2IQ and HMM-6IQ is to examine movement frequency, the number of molecules that attach and have detectable movement as opposed to merely attaching and detaching. At 25 mM KCl and 10 µM ATP, only 7% of the HMM-2IQ molecules that attached to actin moved, whereas 98% of the HMM-6IQ molecules that bound moved (Table II). At the same KCl concentration and 2 mM ATP, greater than 99% of the HMM-6IQ molecules that attached to actin moved, whereas in the same observation period no movement events were observed for HMM-2IQ. Fig. 5, ch shows a distribution of the actual measured run lengths. This distribution will be blunted for a highly processive motor as its run length will be limited by the available length of the actin filament. The bar on the far left represents HMM molecules that attached but moved less than 200 nm. At 25 mM KCl, all of the HMM-IQ molecules except the HMM-2IQ exhibited long processive runs (>0.5 µm). The mean run length of HMM-6IQ (1.81 µm) was longer than that of HMM-4IQ (0.74 µm), HMM-8IQ (1.38 µm), and HMM-2Ala-6IQ (0.76 µm). HMM-2IQ exhibited some movements longer than 200 nm, but the frequency was low, and the length of the run was typically shorter (Fig. 5, a and c). At 100 mM KCl concentration very few processive movements were seen for HMM-4IQ (Fig. 5e), but HMM-6IQ and HMM-8IQ molecules still showed long processive run lengths (data not shown).
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The velocity of single HMM-IQ mutants on actin filaments in the TIRF assay was roughly proportional to neck length at 10 µM ATP (Fig. 6). However at 2 mM ATP, single molecules of HMM-6IQ moved faster than any other mutant, including the HMM-8IQ. No movements of HMM-2IQ were observed under these conditions, and HMM-2Ala-6IQ moved slower than HMM-6IQ.
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Single myosin V S16IQ molecules could bind to actin filaments, but no movements were observed (Fig. 7). The attachment lifetime at 2 mM ATP (0.12 s) is shorter than that at 1 µM ATP concentration (0.52 s). The value at 2 mM ATP is consistent with the Vmax of the actin-activated Mg-ATPase and the expected dwell time (0.11 s) from Tanaka et al. (20) if S1 is not a processive molecule. At 1 µM ATP the lifetime is longer as expected for a second order reaction at low substrate concentration.
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DISCUSSION |
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Two different assays were used to probe whether the neck length affected processivity. One involved watching single fluorescently labeled HMM-IQ molecules interact with actin using TIRF microscopy. At 100 mM KCl and 2 mM ATP only the WT HMM-6IQ and the HMM-8IQ showed long processive movements. When the KCl concentration was lowered to 25 mM, the HMM-4IQ also moved processively for significant distances. The only conditions in which HMM-2IQ showed significant processive movements in the TIRF assay were at very low ATP concentrations (1 and 10 µM ATP) and at low ionic strength. Even here, the frequency with which HMM-2IQ moved on actin filaments was markedly lower than with the other mutants. The lack of robust processivity of HMM-2IQ cannot be attributed to the geometry of the assay. Purcell et al. (18) used optical tweezers to measure the step size of a myosin V HMM-4IQ construct undergoing processive movements and found a value of 25 nm compared with 36 nm obtained from the WT HMM-6IQ. They proposed that the two heads of HMM-4IQ must bind to actin monomers 25 nm apart and be rotated about 90° around the actin filament. For this molecule to move processively, it would have to rotate around the actin filament in a right-handed direction or meander between the two strands as it advanced. In our experiments, actin filaments are bound to a streptavidin-coated surface via biotin-groups coupled to actin. By varying the ratio of unlabeled to biotin-labeled actin in a filament, we vary the tightness with which the filament is tacked down to the surface. The processivity of HMM-4IQ and HMM-8IQ dramatically decreases when the ratio of unlabeled actin to biotinylated actin decreases from 100:1 to 10:1, whereas the processivity of HMM-6IQ is not affected (data not shown). This suggests that the former group of molecules must rotate around the actin filament to some extent to move processively and that this rotation is inhibited if the actin filaments are tightly tacked down. The low frequency of processive movements observed with the HMM-2IQ, however, is probably not due to steric hindrance of the expected rotation because of the actin filament being tightly tacked down to the surface, since HMM-4IQ molecules move processively under these conditions.
Another way of addressing processivity is to measure the landing rate of actin filaments as a function of myosin surface density. When the experimental data were fitted to an equation derived to determine the minimal number of molecules, n, required to translocate an actin filament, the HMM-2IQ data were best fit to an n of 2, whereas the other HMM-IQ molecules showed an n of 1. Thus a neck length of greater than 2IQ motifs is required for efficient processive movements.
The size of single displacements measured using optical tweezers and the rate of actin filament sliding at moderate myosin V surface densities were roughly proportional to the number of IQ motifs with the exception of HMM-2Ala-6IQ, which moved more slowly and showed smaller single displacements than did the HMM-6IQ. The insertion of two amino acids into a helical segment should result in a 200° rotation of the next IQ motif, which would likely change the normal CaM-CaM interactions and possibly create a less rigid neck.
The rate of movement of single molecules of each mutant was measured at both low (10 µM) and high ATP (2 mM) concentrations using TIRF microscopy. At 2 mM ATP and 100 mM KCl, HMM-6IQ moves faster than HMM-8IQ suggesting that, under unloaded conditions, the rate-limiting process for HMM-8IQ may be the time to search for a suitable actin binding site. No movements were observed with HMM-2IQ under these conditions. At 10 µM ATP the movement of single molecules of HMM-2IQ, HMM-4IQ, HMM-6IQ, and HMM-8IQ are all roughly proportional to neck length. At this ATP concentration, the ATP binding rate (23) would limit the rate of stepping, and hence velocity would be proportional to neck length (8).
The data above and published experiments (18, 22, 24) have shown that both the working stroke measured from single interactions and the step size obtained when myosin V is moving processively along actin is proportional to the length of the neck region. Interestingly, the rates of movement of vesicles in vivo in yeast expressing mutant myosin V molecules were also found to be proportional to neck length (25). Together these studies provide strong support for the lever arm model of myosin motility where the neck region is thought to act as a rigid lever arm in undergoing large scale movements in response to more subtle changes in the motor domain. However, one study with myosin V neck mutants reached a different conclusion. Tanaka et al. (20) engineered a chimeric clone with the motor domain and first IQ motif of myosin V fused to the rod of smooth muscle myosin. Trace amounts of this chimera were co-polymerized with skeletal muscle myosin rod fragments. The chimeric myosin V construct showed multiple 36-nm steps per encounter with actin, which the authors interpreted in terms of a model where the neck region does not act as a lever arm. The choice of smooth muscle myosin rod for the chimera may introduce several complexities. First, the N-terminal portion of smooth muscle myosin rod forms a weakly dimerizing coiled-coil (2628), thus it is possible that even though the chimeric myosin V has a short neck, the coiled-coil smooth muscle S2 segment may be flexible or able to unwind to allow the second head to search for a favorably positioned actin monomer 36 nm away. Second, smooth muscle myosin is thought to assemble via addition of myosin dimers to growing filaments (29, 30). Thus, it is possible that the chimeric myosin was forming dimers consisting of four closely spaced myosin heads when co-polymerizing with skeletal muscle rod fragments, which would increase the likelihood that an actin filament would be moved processively. No other assays for processivity were used in their study. In contrast, we examined processivity using both single molecule motility studies and landing assays. HMM-2IQ was not very processive at high ATP concentrations in either of these assays. A similar conclusion was reached by Purcell et al. (18) using HMM-1IQ in their optical trap assays.
Processivity can be viewed as a race between the time needed for the unattached head to find a suitable actin binding site and the ATP cycle time for the attached head. If the bound head dissociates before the unbound head finds a target the myosin dissociates, and the processive run terminates. Veigel et al. (8) proposed a model wherein the wild type myosin V spends most of its kinetic cycle attached to actin with a brief stepping time in between periods of two-headed attachment. They further proposed that the kinetic cycles of the two heads are gaited in favor of keeping at least one head attached. Electron microscopic studies show that most of the time the two heads of HMM-6IQ are 13 actin monomers apart (a 36-nm spacing), but some molecules attach with an 11- or 15-actin monomer separation (7) indicating that the leading head has some flexibility in its binding. The virtual lack of processivity of HMM-2IQ at saturating ATP concentrations may be because of a prolonged search time of the unattached head for actin, because we show that its ATP cycling time is not greatly different from that of wild type HMM-6IQ. Because this short neck cannot sample a large landscape, the average time to finding a binding site may be longer than the cycle time of the bound head at saturating ATP. As the ATP concentration is lowered, the time for binding of ATP to the attached apo head would increase, which might then allow the free head more time to search for a binding site. Thus, the long neck of myosin V may not only have evolved to allow for a powerstroke size coincident with the pseudo-repeat of actin but also to have a high probability of finding a suitable target site.
In summary, our data provide strong support for the lever arm model for myosin movement and demonstrate that the neck length of myosin V has evolved to take advantage of the spacing between actin monomer within the filament. A neck containing six CaM molecules is not absolutely required for processivity, but it is necessary to have a neck with more than two CaMs bound to be able to ensure that at least one head is bound at any given instance under physiological ionic conditions.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Bldg. 10, Rm. 8N202, NHLBI, National Institutes of Health, Bethesda, MD 20892-1762. Tel.: 301-496-6887; Fax: 301-402-1542; E-mail: sellersj{at}nhlbi.nih.gov.
1 The abbreviations used are: CaM, calmodulin; HMM, heavy meromyosin; WT,
wild type; TIRF, total internal reflection fluorescence; MOPS,
4-morpholinepropanesulfonic acid; TEMED,
N,N,N',N'-tetramethylethylenediamine; TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino}ethanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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