|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 47, 37167-37172, November 24, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Molecular Physiology and Biophysics,
University of Vermont, Burlington, Vermont 05405
Received for publication, July 19, 2000
Structural data led to the proposal that the
molecular motor myosin moves actin by a swinging of the light chain
binding domain, or "neck." To test the hypothesis that the neck
functions as a mechanical lever, smooth muscle heavy meromyosin (HMM)
mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary
displacements and forces in the laser trap assay. Two shorter necked
constructs had smaller unitary step sizes and moved actin more slowly
than WT HMM in the motility assay. A longer necked construct that
contained an additional essential light chain binding site exhibited a
1.4-fold increase in the unitary step size compared with its control.
Kinetic changes were also observed with several of the constructs. The
mutant lacking a neck produced force at a somewhat reduced level, while
the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that
the neck functions as a rigid lever, with the fulcrum for movement and
force located at a point within the motor domain.
Muscle contracts as a result of the cyclic interaction of the
molecular motor myosin with actin, powered by the hydrolysis of MgATP.
A simple mechanistic model by which myosin could move actin was
proposed based on the crystal structure of skeletal myosin subfragment
1 (1-3). The key feature was an 8.5-nm single Additional evidence in support of a lever arm rotation was obtained
from the crystal structure of a motor domain-essential light chain
complex with a transition state analog at the active site, which showed
the lever arm in a second position that may represent myosin in the
prepowerstroke state (4). The skeletal subfragment 1 structure is
likely to resemble the structure adopted at the end of the powerstroke.
A comparison of the two conformations shows that smaller changes that
originate at the active site are amplified into much larger movements
of the lever arm. This motion could accommodate a powerstroke on the
order of 10 nm, in the range of the 5-15 nm of displacement measured
using single molecule techniques (5-8).
The simplest mechanical model for the neck region predicts that myosin
with a shorter neck (i.e. shorter lever arm) should generate
smaller unitary displacements and move actin more slowly, whereas a
longer neck should lead to larger displacements and more rapid actin
movement (reviewed in Ref. 9). Several studies in which myosins of
various neck lengths were produced either by removing light
chains (10, 11) or by genetically adding or deleting light chain
binding sites (12, 13) showed that constructs with necks shorter than
wild type moved actin more slowly, while a construct with a longer neck
moved actin more quickly. A chimera in which two Here we test the lever arm hypothesis at the single molecule level by
measuring the displacement (d) and force (F) of a
series of smooth muscle heavy meromyosin (HMM) mutants in which light chain binding sites were either added to or deleted from the neck. The
laser trap data support the hypothesis that the neck acts as a lever
and are consistent with structural data that suggest that the fulcrum
for movement and force is located near the SH1 helix (4).
Protein Preparation and Expression--
Wild type (WT) smooth
muscle HMM (amino acids 1-1175) and neck length mutants of this heavy
chain backbone were co-expressed with the regulatory and essential
light chains using the baculovirus insect cell expression system (11).
The HMM backbone was chosen as the basis for the different neck-length
constructs so that monoclonal anti-rod antibody S2.2 could be used as a
common means of attaching the molecules to the nitrocellulose
substratum for both motility and laser trap studies. Proteins were
purified by binding to actin and release with MgATP as described
previously (11).
Design of Mutants--
Two constructs with neck lengths shorter
than WT HMM were cloned (see Fig. 1). An HMM with no light chain
binding region ("neckless") had heavy chain residues 791-848
deleted. This resulted in a dimeric construct with the motor domain
attached to the rod. The sequence of the region joining the motor
domain to the rod in the neckless construct was ERDLGPLLQV. An HMM
mutant lacking an RLC binding site ("-R site") had amino acids
820-848 deleted. The sequence of the joining region in this construct,
which contains the motor domain and ELC binding site attached to the
rod, was QQQLLGPLLQV.
A long necked mutant ("giraffe") in which a second ELC binding site
was added between the native ELC and RLC binding sites was also cloned
(Fig. 1). This mutant retains native contacts between the motor domain
and the ELC and between the ELC and the RLC but introduces a foreign
ELC-ELC interaction. The following sequence was introduced between
Leu819 and Thr820:
LGITDVIIAFQAQCRGYLARKAFAKRQQQL. This sequence is LG plus amino acids 792-819. To test if the orientation of the two ELCs with respect
to each other influences the properties of the giraffe construct, a
second variation was constructed ("giraffe's twisted sister").
This revised construct had one less amino acid in the added ELC site
than the previous construct; thus, the two ELCs would be rotated by
approximately 100°. This was accomplished by inserting the sequence
of amino acids 791-819 (i.e. KITDVIIAFQAQCRGYLARKAFAKRQQQL) between Leu819 and Thr820.
A phosphorylation-independent variant of giraffe HMM was also
engineered. Based on earlier studies, substitution of the 50/20-kDa Biochemical Characterization of Expressed
Constructs--
Actin-activated ATPase assays were performed in 10 mM imidazole, pH 7, 8 mM KCl, 1 mM
MgCl2, 1 mM EGTA, 1 mM
dithiothreitol, 1 mM NaN3 at 37 °C.
Inorganic phosphate was determined colorimetrically at six time points
per actin concentration, using SDS to stop the reaction (17). The
concentration of active heads was determined by
NH4+-ATPase activity relative to a
myosin standard (25 mM Tris, pH 7.5, at 37 °C, 0.4 M NH4Cl, 2 mM EDTA, 0.2 M sucrose, 1 mM dithiothreitol, 1 mg/ml bovine
serum albumin).
Electron Microscopy--
Rotary-shadowed platinum images were
obtained in 0.5 M ammonium acetate, 66% glycerol and
observed with a Philips EM301 electron microscope operated at 60 kV
(18). Actin was decorated with the expressed constructs, negatively
stained, and observed over holes on carbon-coated grids (19).
In Vitro Motility--
The motility assay was performed at
30 °C in 25 mM imidazole, pH 7.5, 25 mM KCl,
4 mM MgCl2, 1 mM EGTA, 0.5%
methylcellulose, as described by Trybus and Chatman (20). CABL-giraffe
was also assayed at a higher ionic strength in 25 mM
imidazole, pH 7.5, 60 mM KCl, 4 mM
MgCl2, 1 mM EGTA, 0.7% methylcellulose.
Monoclonal anti-rod antibody S2.2 was used to ensure a uniform site of
attachment to the nitrocellulose surface (21). Fifteen or more
filaments were used to calculate the mean and S.D. of the velocity of
movement. The average velocity for a given filament was calculated by
dividing the distance traveled by the filament during a 1-s period
between video snapshots for 7-15 consecutive snapshots as described
previously (22). To control for sporadic motility potentially
compromising the velocity estimate, velocities for a paired preparation
of CABL-HMM and CABL-giraffe were determined without averaging over numerous short intervals (1 s) and then plotted in a histogram (see
Fig. 4C).
Laser Trap Studies--
Technical details of our laser trap
techniques have been published elsewhere (7, 23, 24). Briefly, a pair
of 1-µm polystyrene microspheres coated with
N-ethylmaleimide-modified myosin were held in solution using
two independent laser traps. A fluorescently labeled
(tetramethylrhodamine isothiocyanate-phalloidin) actin filament was
strung between the two microspheres and pulled taut (>4 piconewtons).
The actin filament is then lowered onto the surface of a 2.0-µm
diameter silica bead adhered to a coverslip. The silica bead acts as a
platform on which a sparse coating of HMM is applied, so that on
average only one HMM molecule interacts with the actin filament at any
time. HMM was adhered to the nitrocellulose-coated surface through an
anti-subfragment 2 antibody applied at 100 µg/ml (21) before
surface blocking with bovine serum albumin (500 µg/ml). HMM was then
applied in concentrations varying from 2.5-10 µg/ml and allowed to
bind to the antibody for 2 min. Thus, HMM was bound to the surface with
random orientation but at a defined point on the molecule. All
experiments were performed at low ionic strength (25 mM
KCl), limiting ATP concentration (10 µM), and 25 °C,
to prolong the unitary event durations.
To measure the displacements imparted to the actin filament by a single
HMM molecule, the bright field image of one of the microsphere handles
is projected onto a quadrant photodiode detector, providing
x and y bead position data. Myosin's unitary
displacements were recorded under "unloaded" conditions (0.02-0.04
piconewtons/nm/trap). To measure force, a nearly isometric condition
was obtained under feedback control by increasing the effective trap
stiffness approximately 100-fold. The feedback loop contained an
acousto-optic modulator, which was used to deflect the laser trap and
thus counterbalance the myosin-generated forces (7). The acousto-optic
modulator control signal was calibrated and served as the force signal. Force measurements in this assay may not represent the true maximum unitary force due to compliance in the actin filament-bead attachment and the low bandwidth of the feedback system (i.e. ~140
Hz). Although the forces measured are surely underestimates, they are
still meaningful for comparative purposes across mutants.
The technique of "mean-variance" (MV) analysis (25) was used to
derive estimates of force and displacement from the laser trap data. MV
analysis begins with a model-independent transformation of the time
record, thus giving an alternative view of the data (a MV histogram),
which emphasizes intervals of constant properties within the data.
Generation of the MV histogram requires no assumptions about or
interpretation of the underlying data, and quantitative descriptions of
the data are derived from curve fits to the histogram. Thus, MV
analysis is less prone to the biases introduced by manual scoring
methods and may be used to estimate the size (i.e.
d and F), distribution, number, and duration of
events (ton) in the data as described previously
(7, 24).
Characterization of Shorter Necked Constructs--
Two
double-headed constructs with neck lengths shorter than that of wild
type HMM were engineered and expressed in the baculovirus/insect cell
system (Fig. 1). One construct lacked an
RLC binding site (-R site), and SDS-gels confirmed that this construct
did not bind RLC (Fig. 2C).
The second shorter necked construct lacked both an RLC and an ELC
binding site (neckless). Metal-shadowed images showed that the two
globular domains closely abut the rod, as would be expected from a
neckless construct that lacked the light chain binding domain (Fig.
2A). These characterizations, as well as the functional
assays that follow, are necessary to ensure that the constructs that
are analyzed in the laser trap retain certain essential and
characteristic features of myosin.
To assess if the mutations altered the kinetics of the cross-bridge
cycle, actin-activated ATPase measurements were performed. Since both
shorter necked constructs lacked the RLC, these molecules did not
require phosphorylation for activation. The actin-activated ATPase
activity of -R site (open squares) and neckless
(filled triangles) was similar to that of
phosphorylated WT HMM (filled circles) (Fig.
3A).
The mutants were also tested by an in vitro motility assay,
which serves as a simplified model system to assess the
motion-generating capacity of myosin at the molecular level. Both
shorter necked constructs moved actin more slowly than WT HMM. The
neckless construct moved actin at ~25% of the velocity (0.26 ± 0.04 µm/s) of phosphorylated WT HMM (1.1 ± 0.19 µm/s), while
-R site moved actin at ~45% the rate (0.49 ± 0.18 µm/s) of
phosphorylated WT HMM (Fig.
4A).
Unitary Displacements and Forces of Shorter Necked
Constructs--
To understand the molecular basis for the differences
in actin filament velocities between the various constructs, the
mechanical and kinetic properties of these constructs were
characterized in the laser trap. At the single molecule level, actin
filament velocity (vmax) is related to
d, the unitary displacement generated by myosin during the
powerstroke, and ton, the time spent attached to
actin following the powerstroke by the equation
vmax
Based on this analysis, neckless generated an average displacement of
2.0 nm, significantly smaller than the 10.5-nm displacement obtained
with WT HMM. The -R site mutant, which has half the neck length of WT
HMM, also produced smaller displacements
(d = 6.2 nm) than WT HMM (Figs. 5 and 6 and Table
I). The d values obtained here
for WT HMM are similar to those previously reported for smooth muscle
myosin as well as expressed WT HMM under similar conditions (7, 24,
26). Analyses of mean displacement event durations, ton, showed that the -R site mutant had shorter
event durations than WT HMM (Table I).
Unitary forces were also recorded (Fig. 5, Table I). The appearance of
force events within the time series data was similar to that of
displacement events and thus analyzed by MV analysis. The neckless
construct generated forces that were somewhat lower than that of WT HMM
(~1.1 versus ~1.6 piconewtons, respectively).
Characterization of Longer Necked Constructs--
If the lever arm
model appropriately describes the mechanical properties of the neck
region, then a longer neck should result in larger unitary
displacements. Therefore, a longer necked "giraffe" construct with
an additional ELC binding site was expressed. This type of construct is
a more stringent test of the lever arm hypothesis (Fig. 1), given that
a gain rather than a loss in function is expected.
Visual inspection of giraffe HMM by metal-shadowing showed that the
construct appeared normal except for having an apparently longer neck
region (Fig. 2A). In addition, the construct decorated actin
in a regular manner, suggesting that its actin-binding properties were
intact (Fig. 2B). The ratio of ELC/RLC was 1.7-1.9 times larger in the giraffe construct compared with WT HMM (average slope
from four gel loadings, two independent preparations), consistent within experimental error with the construct containing an
additional ELC binding site. However, despite having actin-activated
ATPase activity, less than half of the actin filaments moved in the
motility assay, and they moved at a rate at least 2-fold slower
than phosphorylated WT HMM. The rates of motility were 0.49 ± 0.13 µm/s for phosphorylated giraffe HMM and 0.23 ± 0.04 µm/s
for unphosphorylated giraffe HMM. The addition of exogenous ELC to the
preparation had no effect on motility. When three independent
preparations of the phosphorylated giraffe HMM were used in an attempt
to obtain unitary displacement data, the data were of insufficient
quality to perform the MV analysis.
Given that smooth muscle myosin's ability to move actin is strictly
dependent on light chain phosphorylation, we hypothesized that light
chain phosphorylation was not able to fully activate the giraffe HMM
construct. If this is true, then a constitutively active mutant that
contains the same elongated neck region as the "wild type" giraffe
construct should circumvent the problem of poor motility. We had
previously shown that mutation of the actin-binding loop to the
sequence found in cardiac myosin produced a constitutively active
molecule (16), and thus we expressed a long necked mutant,
CABL-giraffe, that was active even when unphosphorylated (see
"Experimental Procedures"). CABL-HMM with a native neck served as
the control. Both constructs were analyzed in the unphosphorylated
state. The actin-activated ATPase of CABL-giraffe (filled
triangles) (Vmax = 6.7 ± 0.6 s
When four independent preparations of CABL-giraffe were compared in
parallel with CABL-HMM, there were no significant differences in
motility, as would have been predicted from a simple lever arm model
(Fig. 4B). One pair of preparations (Fig. 4B,
1) was also analyzed by determining the velocities during
1-s intervals instead of obtaining an average velocity for a longer
run, but still no significant differences emerged (Fig. 4C).
This similarity in rates held true under standard motility conditions
(25 mM KCl) as well as at higher salt concentrations (60 mM KCl). As previously reported, unphosphorylated CABL-HMM
moved actin at ~50% the rate of phosphorylated WT HMM, and
phosphorylation of CABL-HMM had only a slight effect, increasing this
value to ~75% that of phosphorylated WT HMM (16).
A second long necked construct, CABL-giraffe's twisted sister (see
"Experimental Procedures") was expressed in order to test if the
relative orientation of the two ELCs had an impact on the rate of
motility. The motility observed with this construct was 0.54 ± 0.07 µm/s (number of filaments = 26). This is within the range
of values observed for both CABL-HMM and the original CABL-giraffe construct (0.36-0.55 µm/s; see Fig. 4).
Unitary Displacements and Force of a Longer Necked
Construct--
Despite the fact that CABL-giraffe did not show an
increased velocity in the in vitro motility assay (Table I),
it is still possible that its unitary displacements could be larger.
This in fact proved to be the case. CABL-giraffe generated an average displacement of 12.4 nm, 1.4-fold greater than its control, CABL-HMM, which produced 9.1-nm steps. The mutation at the actin binding loop did
not affect step size per se, since the 9.1-nm step of CABL-HMM is statistically indistinguishable from the 10.5-nm unitary step size of WT HMM. The ton for CABL-giraffe
was marginally longer than for its control CABL-HMM, but both CABL
constructs have significantly shorter values than obtained for WT
HMM.
Unitary forces produced by CABL-giraffe were approximately twice that
generated by CABL-HMM (Table I). Given the higher force-generating capacity of the CABL-giraffe, it is unlikely that the addition of an
ELC site introduced a significant compliance within the neck, which
potentially could have explained why the rate of motility of
CABL-giraffe was not faster than its control CABL-HMM.
The goal of this study was to provide a stringent test for whether
the myosin neck region acts as a mechanical lever that transmits force
and displacements originating within the motor domain. We therefore
characterized the molecular mechanics of smooth muscle HMM mutants with
shorter and longer neck regions by measuring in vitro
motility velocities, unitary displacements, and unitary forces. If the
neck acts as a simple lever arm, one prediction is that under unloaded
conditions in the laser trap, the magnitude of d should be
directly related to lever arm length. Unitary displacements of
two short necked constructs were 20% (neckless) and 60% (-R site) of
the values obtained with WT HMM. Strikingly, the long necked giraffe
mutant generated displacements that were 1.4 times that of its control.
The relationship between unitary displacements and lever arm length is
linear, strongly supporting the hypothesis that the neck acts as a
lever arm (Fig. 6).
Another conclusion of this study is that the lever must extend into the
motor domain, since our neckless mutant generated significant force and
motion. Motion was also observed for other neckless myosin species
created genetically in Dictyostelium myosin (27) or
proteolytically from skeletal muscle myosin (28). The portion of the
long The lever hypothesis also makes specific predictions about the
relationship between lever arm length (L) and unitary force (see Fig. 7), but these are
model-dependent. In contrast, all three models described
below predict a linear relationship between lever arm length and
unitary displacement (Fig. 6). Our data showed that neckless generated
somewhat less force than WT HMM, while the giraffe construct generated
more force than its control. These data rule out the simplest lever arm
model, which would have force inversely related to L. A
second model, suggested by Howard and Spudich (Appendix in 13),
proposed that the neck region acts more like an elastic cantilever
rather than a rigid lever and predicts that force (F) should
be inversely related to L2. Our data also do not
fit this model, which was recently used to explain changes in x-ray
diffraction intensity data with small changes in muscle length (29),
where the intensity changes were assumed to be the result of both a
rotation and bending of the neck region. A third mechanical model, that
we previously proposed to explain the force- and motion-generating
capacity of light chain-deficient skeletal muscle myosins (10, 30),
predicts that force should be linearly related to L. In this
model, a torque generated in the motor domain produces force at the end
of the neck, which acts as a rigid lever. This force would then stretch an elastic element, presumably in the subfragment 2 region (30). A comparison of the force data from the laser trap with the predictions for the three models (see Fig. 7) suggests that the third model may be
the most appropriate. Both the displacement and the force data support
the conclusion that the neck region acts as a rigid lever and that much
of the myosin elasticity is external to the neck. It should be noted
that force measurements in this assay are underestimates, given the
stray compliances within the experimental system (see "Experimental
Procedures"), and thus conclusions based on these data should be
viewed in light of this concern.
An alternative view of the neck has been championed by Yanagida and
co-workers (31). Although they also obtained a reduced velocity with a
similar neckless construct of Dictyostelium myosin, they
showed that the unitary step size was unaffected by the change in neck
length and therefore concluded that the slower velocity was due only to
an increase in the time of attachment following the powerstroke (31).
This view favors the idea that the neck region serves to regulate the
kinetics of the cross-bridge cycle. In contrast, our unitary
displacement data support the idea that the neck acts as a mechanical
lever arm. However, we also have evidence that kinetic changes have
occurred with several of the neck length constructs, both in the
unitary event durations (Table I) and in steady-state actin-activated
ATPase activity (Fig. 3B). Thus, the assumption that
ton remains constant and that all changes in
velocity are due to changes in unitary displacement (vmax We conclude that changes in the length of the light chain binding
domain affect the unitary displacement and force of both shorter and
longer necked constructs in a way that is consistent with the neck
acting as a lever arm. The fulcrum for the rotation is located ~3 nm
internal to the C-terminal end of the motor domain, far from the
actomyosin interface. Perturbations to the neck region also appear to
modulate the kinetics of several constructs, as judged by changes in
steady state ATPase activity and by changes in the unitary displacement
event durations. It is possible that kinetic changes are accentuated in
these smooth muscle myosin constructs, where myosin activity is tightly
regulated by light chain phosphorylation. Thus, the neck region acts as
a lever arm but may also serve to transmit mechanical strain to the
catalytic site within the motor domain.
We thank Eric Hayes, Janet Vose, and Greta
Ouyang for technical assistance, Anne-Marie Lauzon for assisting in
gathering data early on in the study, Don Gaffney for programming
expertise, and Guy Kennedy for instrumentation skills that were needed
to keep the trap functioning throughout this study.
*
This work was supported by National Institutes of Health
Grant HL54568 (to K. T. and D. W.) and the Totman Fund for
Cerebrovascular Research (to D. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Whitehead Institute for Biomedical Research, Massachusetts
Institute of Technology, Cambridge, MA 02142.
¶
Dept. of Biology, San Diego State University, San Diego, CA 92182.
**
To whom correspondence should be addressed: Molecular Physiology
and Biophysics, University of Vermont, Burlington, VT 05405. Tel.:
802-656-8750; Fax: 802-656-0747; E-mail:
trybus@salus.med.uvm.edu.
Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M006438200
The abbreviations used are:
ELC, essential light
chain;
RLC, regulatory light chain;
HMM, heavy meromyosin;
WT, wild
type;
CABL,
The Light Chain Binding Domain of Expressed Smooth Muscle Heavy
Meromyosin Acts as a Mechanical Lever*
,
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix, stabilized by
the essential and regulatory light chains (ELC and
RLC),1 which formed an
elongated neck region that emerged from the globular motor domain. It
was suggested that a substantial portion of the myosin motor domain
maintains a fixed orientation when attached to actin, while the neck
region pivots about a fulcrum within the motor domain, thus
generating a power stroke.
-actinin repeats
were fused to the Dictyostelium motor domain showed a higher
average velocity than a similar construct with only one -actinin
repeat, suggesting that nonnative structures can mimic some aspects of
the native neck (14). Although these studies are consistent with a
simple lever arm model, they all relied on the assumption that no
kinetic changes resulted from these biochemical or genetic
perturbations. Since velocity in the motility assay,
vmax, is dependent upon both step size
(d) and the time spent attached to actin following the
powerstroke (ton) (i.e.
vmax 
d/ton) (15), changes in either parameter could equally well account for the observed differences in motility.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cardiac actin binding loop (CABL) for the native sequence (residues 626-653) activated unphosphorylated smooth muscle HMM (16). Thus, the
double mutants, CABL-giraffe and CABL-giraffe's twisted sister, were
also constructed. The movement of these constructs was more consistent
than the original giraffe construct, and thus the single-molecule
studies could be more readily performed. Unphosphorylated CABL-HMM with
a native neck was used as the control for the unphosphorylated long
necked CABL-giraffe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Schematic diagram of the constructs used in
this study.
View larger version (45K):
[in a new window]
Fig. 2.
Characterization of expressed myosin mutants.
A, metal-shadowed images of neckless, giraffe, and WT HMM.
In some of the images, it can be clearly seen that neckless has a
shorter neck than WT and that giraffe has a longer neck than WT.
Magnification was × 110,000. B, negative stained
images of actin decorated with the long necked giraffe construct. The
typical arrowhead appearance indicates regular binding despite
the mutation of the neck region. Magnification was × 150,000. C, SDS-gel of tissue purified myosin (lane 1), and the
mutant construct lacking the RLC binding site (-R site)
(lane 2).
View larger version (13K):
[in a new window]
Fig. 3.
Actin-activated ATPase activity of expressed
constructs. A, rate of ATP hydrolysis as a function of
actin concentration for phosphorylated WT HMM ( 
), dephosphorylated
WT HMM (
), -R site (
), and neckless (
). The solid
line through the points is a fit with a
Vmax = 4.6 ± 1.7 s
1, but this value is approximate, since the
Km values were ~75 µM. B,
rate of ATP hydrolysis as a function of actin concentration for
dephosphorylated CABL-HMM () and dephosphorylated CABL-giraffe HMM
(). The fits to the curves are Vmax = 2.9 ± 0.3 s1, Km = 16 ± 5 µM for CABL-HMM and
Vmax = 6.7 ± 0.6 s1, Km = 6 ± 3 µM for CABL-giraffe HMM.
View larger version (18K):
[in a new window]
Fig. 4.
Motility of expressed neck-length
mutants. A, velocity of movement (mean and S.D.) for
the two shorter neck-length constructs compared with WT HMM. All three
constructs were attached to the nitrocellulose substratum via
monoclonal antibody S2.2. B, velocity of movement (mean and
S.D.) of the longer necked giraffe construct (striped
bars) compared with its WT control (solid
bars). Four independent paired preparations of CABL-HMM and
CABL-giraffe are shown. C, histogram of filament velocities
(preparation 1, B). Velocities during
1-s intervals for at least 20 filaments are plotted. 213 1-s intervals
were used for CABL-HMM, and 194 1-s intervals were used for
CABL-giraffe.
d/ton. Estimates of d and
ton were obtained for the mutants and compared with WT HMM by analyzing displacement time series data obtained in the
laser trap assay (Fig. 5). Displacement
records were characterized by periods of Brownian noise in which
HMM-generated displacement events were interspersed. The Brownian noise
is reduced upon attachment of HMM to actin (5-7), which serves as a
means of identifying events through the mean-variance analysis
technique (see "Experimental Procedures"). This approach becomes a
critical tool particularly for analyzing records from shorter necked
mutants, which generate displacement events that are small in amplitude
and well within the Brownian noise of the system.
View larger version (48K):
[in a new window]
Fig. 5.
Laser trap time series data of single HMM
molecule displacements and forces. Shown are 5-s records of
displacement and force from WT HMM, a neckless mutant, CABL-WT
(which is the control for a long necked mutant), and CABL-giraffe (see
"Experimental Procedures" for construct details). For data
analysis, records were typically 30-60 s long for a given HMM molecule
and contained tens to hundreds of events per record. These raw data are
shown for illustrative purposes. To estimate the displacement and force
amplitudes from full-length records, mean-variance analysis was
applied, and the results are reported in Table I. The noise is due to
the Brownian motion of the bead-actin-bead assembly in solution.
Individual events are seen as positive deflections with occasional
negative deflections apparent (see force records for neckless and
CABL-giraffe for examples).
View larger version (14K):
[in a new window]
Fig. 6.
Unitary step size is a linear function of
relative lever arm length. Unitary displacements (d)
for neck length mutants are plotted as mean ± S.D. against
relative lever arm length (L). WT HMM or CABL-HMM, which
contain two light chains, were assigned a relative lever arm length of
1. Assuming that each light chain encompasses 50% of the lever length,
then an L value of 0.0, 0.5, and 1.5 were assigned to
neckless, -R site, and CABL-giraffe, respectively. The linear
regression for the data does not pass through zero but intersects the
x axis at 
0.34L, suggesting that the lever may
extend ~3 nm (i.e. 0.34 * 8.5-nm neck length) into the
motor domain.
Summary of mechanical data
1, Km = 6 ± 3 µM) was slightly more than twice than of CABL-HMM
(filled circles) (Vmax = 2.9 ± 0.3 s1, Km = 16 ± 5 µM) (Fig. 3B). This result shows
that the kinetics of the interaction with actin have been altered by
the mutation in the neck.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix that remains in the neckless construct (residues
778-790), which abuts the more compact domain of the converter region
(residues 721-777), could provide an additional piece of lever arm for
generating force and motion in this construct. The crystal structures
of skeletal subfragment 1 and smooth muscle MDE show that the rotation
of the converter and the lever arm occurs because of two perpendicular
rotations around two conserved glycines that are located at either end
of the SH1 helix (4). In agreement with the structural data, the
correlation between neck length and d (Fig. 6) suggests that
the fulcrum for movement is ~3 nm within the motor domain. Therefore,
the bulk of the motor domain remains in a relatively fixed orientation
with respect to actin, and the point at which myosin undergoes a major
rotation is closer to the converter region than to the actomyosin interface.
View larger version (18K):
[in a new window]
Fig. 7.
Model relationships for the effect of lever
arm length on force. Unitary forces (F) for neck length
mutants normalized to their respective controls (i.e. WT HMM
for neckless and CABL-HMM for CABL-giraffe) are plotted as mean ± S.D. against relative lever arm length. The solid
lines are the predicted relationships for the dependence of
unitary force on lever arm length (see "Discussion" for details).
#1, the prediction for a simple lever (i.e.
F 
L1);
#2, the prediction (i.e. F L2) based on the model of Howard
and Spudich (Appendix in 13); #3, the prediction
(i.e. F L) based on the model of
VanBuren et al. (30). The mutant data are offset on the
x axis by 0.34 lever arm lengths based on the prediction in
Fig. 6 as to the origin of the lever arm. The mutant data are best fit
by model 3, where the neck region acts like a rigid lever that is
coupled to a torque motor within the motor domain and to an
elastic element presumably in the subfragment 2 segment (30).
d/ton), which was made in several
previous studies with altered neck length mutants (12-14), is probably
not justified. For example, CABL-giraffe moved actin at the same rate
as its control, CABL-HMM, although the step size was increased, in
contrast to a previous study, which reported that a long necked myosin showed faster motility than WT myosin (13). A kinetic contribution to
changes in velocity because of neck alterations has also been inferred
from several previous studies. Changes in the unloaded duty cycle
contributed to the reduced velocity and force production of
ELC-deficient myosin (30). Point mutations in the RLC have been shown
to affect in vitro motility rates although ATPase activity remains high (32). Finally, a RLC point mutant, with weakened divalent
cation binding, modulates the kinetics of cross-bridge attachment and
detachment (33). Assuming that the unitary step size was unchanged by
this point mutation, these data also argue for the potential modulation
of velocity via kinetic mechanisms as a result of structural
perturbations to the neck.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Biomedical Engineering, University of
Virginia, Charlottesville, VA 22908.
Dept. of Molecular, Cellular, and Developmental Biology, Yale
University, New Haven, CT 06511.
ABBREVIATIONS
-cardiac actin binding loop;
MV, mean-variance.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Rayment, I.,
Rypniewski, W. R.,
Schmidt-Base, K.,
Smith, R.,
Tomchick, D. R.,
Benning, M. M.,
Winkelmann, D. A.,
Wesenberg, G.,
and Holden, H. M.
(1993)
Science
261,
50-58
2.
Rayment, I.,
Holden, H. M.,
Whittaker, M.,
Yohn, C. B.,
Lorenz, M.,
Holmes, K. C.,
and Milligan, R. A.
(1993)
Science
261,
58-65
3.
Fisher, A. J.,
Smith, C. A.,
Thoden, J.,
Smith, R.,
Sutoh, K.,
Holden, H. M.,
and Rayment, I.
(1995)
Biophys. J.
68,
19S-26S
4.
Dominguez, R.,
Freyzon, Y.,
Trybus, K. M.,
and Cohen, C.
(1998)
Cell
94,
559-571
5.
Finer, J. T.,
Simmons, R. M.,
and Spudich, J. A.
(1994)
Nature
368,
113-119
6.
Molloy, J. E.,
Burns, J. E.,
Kendrick-Jones, J.,
Tregear, R. T.,
and White, D. C. S.
(1995)
Nature
378,
209-212
7.
Guilford, W. H.,
Dupuis, D. E.,
Kennedy, G.,
Wu, J.,
Patlak, J. B.,
and Warshaw, D. M.
(1997)
Biophys. J.
72,
1006-1021
8.
Kitamura, K.,
Tokunaga, M.,
Iwane, A. H.,
and Yanagida, T.
(1999)
Nature
397,
129-134
9.
Block, S.
(1996)
Cell
87,
151-157
10.
Lowey, S.,
Waller, G. S.,
and Trybus, K. M.
(1993)
Nature
365,
454-456
11.
Trybus, K. M.
(1994)
J. Biol. Chem.
269,
20819-20822
12.
Uyeda, T. Q. P.,
and Spudich, J. A.
(1993)
Science
262,
1867-1870
13.
Uyeda, T. Q.,
Abramson, P. D.,
and Spudich, J. A.
(1996)
Proc. Natl. Acad. Sci.
93,
4459-4464
14.
Anson, M,
Geeves, M. A.,
Kurzawa, S. E.,
and Manstein, D. J.
(1996)
EMBO J.
15,
6069-6074
15.
Huxley, H. E.
(1990)
J. Biol. Chem.
265,
8347-8350
16.
Rovner, A. S.,
Freyzon, Y.,
and Trybus, K. M.
(1995)
J. Biol. Chem.
270,
30260-30263
17.
White, H. D.
(1982)
Methods Enzymol.
85,
698-708
18.
Trybus, K. M.,
and Lowey, S.
(1984)
J. Biol. Chem.
259,
8564-8571
19.
Craig, R.,
Szent-Gyorgyi, A. G.,
Beese, L.,
Flicker, P.,
Vibert, P.,
and Cohen, C.
(1980)
J. Mol. Biol.
140,
35-55
20.
Trybus, K. M.,
and Chatman, T. A.
(1993)
J. Biol. Chem.
268,
4412-4419
21.
Trybus, K. M.,
and Henry, L.
(1989)
J. Cell Biol.
109,
2879-2886
22.
Work, S. S.,
and Warshaw, D. M.
(1992)
Anal. Biochem.
202,
275-285
23.
Dupuis, D. E.,
Guilford, W. H.,
Wu, J.,
and Warshaw, D. M.
(1996)
J. Muscle Res. Cell Motil.
18,
17-30
24.
Lauzon, A.-M.,
Tyska, M. J.,
Rovner, A. S.,
Freyzon, Y.,
Warshaw, D. M.,
and Trybus, K. M.
(1998)
J. Muscle Res. Cell Motil.
19,
825-837
25.
Patlak, J. B.
(1993)
Biophys. J.
65,
29-42
26.
Tyska, M. J.,
Dupuis, D. E.,
Guilford, W. H.,
Patlak, J. B.,
Waller, G. S.,
Trybus, K. M.,
Warshaw, D. M.,
and Lowey, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4402-4407
27.
Itakura, S.,
Yamakawa, H.,
Toyoshima, Y. Y.,
Ishijima, A.,
Kojima, T.,
Harada, Y.,
Yanagida, T.,
Wakabayashi, T.,
and Sutoh, K.
(1993)
Biochem. Biophys. Res. Commun.
196,
1504-1510
28.
Waller, G. S,
Ouyang, G.,
Swafford, J.,
Vibert, P.,
and Lowey, S.
(1995)
J. Biol. Chem.
270,
15348-15352
29.
Dobbie, I.,
Linari, M.,
Piazzesi, G.,
Reconditi, M.,
Koubassova, N.,
Ferenczi, M. A.,
Lombardi, V.,
and Irving, M.
(1998)
Nature
396,
383-387
30.
VanBuren, P.,
Waller, G. S.,
Harris, D. E.,
Trybus, K. M.,
Warshaw, D. M.,
and Lowey, S.
(1994)
Proc. Natl. Acad. Sci.
91,
12403-12407
31.
Tanaka, H.,
Kitamura, K.,
Iwane, E. H.,
and Yanagida, T.
(2000)
Biophys. J.
78,
3A (abstr.)
32.
Chaudoir, B. M.,
Kowalczyk, P. A.,
and Chisholm, R. L.
(1999)
J. Cell Sci.
112,
1611-1620
33.
Diffee, G. M.,
Patel, J. R.,
Reinach, F. C.,
Greaser, M. L.,
and Moss, R. L.
(1996)
Biophys. J.
71,
341-350
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Iwai and T. Q. P. Uyeda Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor PNAS, November 4, 2008; 105(44): 16882 - 16887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Laakso, J. H. Lewis, H. Shuman, and E. M. Ostap Myosin I Can Act As a Molecular Force Sensor Science, July 4, 2008; 321(5885): 133 - 136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Debold, S. E. Beck, and D. M. Warshaw Effect of low pH on single skeletal muscle myosin mechanics and kinetics Am J Physiol Cell Physiol, July 1, 2008; 295(1): C173 - C179. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Szczesna-Cordary, M. Jones, J. R. Moore, J. Watt, W. G. L. Kerrick, Y. Xu, Y. Wang, C. Wagg, and G. D. Lopaschuk Myosin regulatory light chain E22K mutation results in decreased cardiac intracellular calcium and force transients FASEB J, December 1, 2007; 21(14): 3974 - 3985. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ito, M. Ikebe, T. Kashiyama, T. Mogami, T. Kon, and K. Yamamoto Kinetic Mechanism of the Fastest Motor Protein, Chara Myosin J. Biol. Chem., July 6, 2007; 282(27): 19534 - 19545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Bryant, D. Altman, and J. A. Spudich The power stroke of myosin VI and the basis of reverse directionality PNAS, January 16, 2007; 104(3): 772 - 777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shima, T. Kon, K. Imamula, R. Ohkura, and K. Sutoh From the Cover: Two modes of microtubule sliding driven by cytoplasmic dynein PNAS, November 21, 2006; 103(47): 17736 - 17740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Guo and W. H. Guilford Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction PNAS, June 27, 2006; 103(26): 9844 - 9849. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Borejdo, J. Talent, and I. Akopova Measuring Rotations of a Few Cross-Bridges in Skeletal Muscle Experimental Biology and Medicine, January 1, 2006; 231(1): 28 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Sherwood, G. S. Waller, D. M. Warshaw, and S. Lowey A point mutation in the regulatory light chain reduces the step size of skeletal muscle myosin PNAS, July 27, 2004; 101(30): 10973 - 10978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Watanabe, H. Tanaka, A. H. Iwane, S. Maki-Yonekura, K. Homma, A. Inoue, R. Ikebe, T. Yanagida, and M. Ikebe A one-headed class V myosin molecule develops multiple large ({approx}32-nm) steps successively PNAS, June 29, 2004; 101(26): 9630 - 9635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sakamoto, F. Wang, S. Schmitz, Y. Xu, Q. Xu, J. E. Molloy, C. Veigel, and J. R. Sellers Neck Length and Processivity of Myosin V J. Biol. Chem., August 1, 2003; 278(31): 29201 - 29207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kohler, C. Ruff, E. Meyhofer, and M. Bahler Different degrees of lever arm rotation control myosin step size J. Cell Biol., April 28, 2003; 161(2): 237 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Alpert, C. Brosseau, A. Federico, M. Krenz, J. Robbins, and D. M. Warshaw Molecular mechanics of mouse cardiac myosin isoforms Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1446 - H1454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Moore, E. B. Krementsova, K. M. Trybus, and D. M. Warshaw Myosin V exhibits a high duty cycle and large unitary displacement J. Cell Biol., November 12, 2001; 155(4): 625 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mehta Myosin learns to walk J. Cell Sci., January 6, 2001; 114(11): 1981 - 1998. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |