Calcium Regulation of Myosin-I Tension Sensing
John H. Lewis, Michael J. Greenberg, Joseph M. Laakso, Henry Shuman, and E.
Pennsylvania Muscle Institute and Department of Physiology, Perelman School of Medicine at the
University of Pennsylvania, Philadelphia, Pennsylvania
Calcium sensitivity of the key steps in the ATPase cycle of myo1b from ensemble-level
transient kinetic experiments
We determined the effect of calcium on the rates of the individual steps of the myo1bIQ
ATPase cycle in the presence of 100 µM Ca2+F at 37 °C (Scheme 1; Table 1). For comparison,
we also provide the rate constants for myo1bIQ in the absence of free calcium, which we
published previously (Figure 2; Table 1; (1)). To correlate biochemical kinetic rates with
mechanical transitions observed in the optical trap, we determined the effect of calcium on key
steps on the ATPase cycle at 20 °C (Tables 1-2; Figure S2).
Pyrene-actin fluorescence was used to measure the rate of ATP binding and the
population of the weakly-bound states. Strong binding of myo1bIQ to pyrene-actin quenches the
pyrene fluorescence ~75%, while ATP binding to myo1bIQ results in the population of detached
or weakly bound myosin states and the restoration of pyrene fluorescence (A* in Scheme S1).
The fluorescence transients acquired at different ATP concentrations were best fit by the sum of
two exponential rates, with a fast phase (kf) hyperbolically related to the ATP concentration (0 -
2000 µM) and a slow phase (ks) independent of ATP at concentrations > 500 µM (Figure 2). The
data were modeled according to Scheme S1:
where nucleotide-free myo1b exists in two states (2), one that binds nucleotide (AM’), and
another that is nucleotide inaccessible (AM). The ATP dependence of kf can be described by:
][ / 1
where K1’ is a rapid equilibrium and k+2’ is a rate-limiting isomerization to the high fluorescence
AM.ATP state. At both 37 °C and 20 °C, K1’ and k+2’ are only moderately affected by calcium,
with k+2’ increased ~ 2-fold by calcium (Table 1, Figure 2) for myo1bIQ. Interestingly, a similar
2-fold change in k+2' upon the addition of calcium is seen in myo1ba at 20 °C. However, the
affinity of myo1ba for ATP (K1') is 1.4 times lower than in myo1bIQ. This lower ATP affinity
leads to a slower second-order rate constant for ATP binding in myo1ba than myo1bIQ. At
physiological ATP concentrations in the cell (>1 mM), the differences between ATP binding
affinities in myo1ba and in myo1bIQ are not consequential since this concentration of ATP is well
above the dissociation constant for ATP (1/K1') for both isoforms.
The rate of ks reports the rate of isomerization of AM to AM’ (k+α) at ATP concentrations
> 500 µM, and the ratio of the amplitudes determines the equilibrium constant, Kα (1, 2). At 37
°C, the rate of going from AM to AM’ (k+α) is relatively unaffected by the introduction of
calcium, whereas the reverse rate, k-α, is increased 1.9-fold (Table 1). At 20 °C, both myo1ba
and myo1bIQ show similar fold increases in k-α. However, the forward rate, k+α, is increased
approximately 3-fold upon the addition of calcium.
The binding of mantATP to myo1bIQ does not result in a fluorescence change upon direct
excitation of the mant fluorophore, but a fluorescence change is observed when the mantATP
fluorescence is excited via energy transfer from neighboring tryptophans (1). The rate of this
change reports the second-order rate of mantATP binding at concentrations < 50 µM, and reports
the rate of the conformational change that accompanies ATP hydrolysis (k3app = k+3 + k-3) at
higher mantATP concentrations (Scheme S2).
The maximum rates in the presence and absence of calcium at 37 °C are 41 ± 4.9 s-1 and 39 ± 3.2
s-1, respectively (Figure S1, Table 1; (1)), indicating that calcium binding does not affect ATP
Fluorescently labeled phosphate-binding protein was used to measure the rate of
phosphate release (k+4’; (1, 3, 4)) at 37 °C. Phosphate release is typically measured in
sequential-mix experiments where myosin and ATP are mixed and aged to form the M.ADP.Pi
state, followed by mixing with various actin concentrations. However, pre-equilibration of
myo1bIQ and 100 µM free calcium at 37° C in the absence of actin resulted in occasional protein
aggregation. Therefore, single-mix, single-turnover experiments were performed by mixing 1
µM ATP with pre-mixed 3 µM myo1bIQ, 100 µM free calcium, and 0 - 80 µM actin. Time
courses of the phosphate-binding transients contained a small lag phase due to ATP binding and
hydrolysis (Scheme 1). The transients were well fit by a double exponential function, where one
rate emerged as actin dependent (Figure 2 c,d). The actin concentration independent rate was
found to be consistent with the rate of ATP binding at 1 µM ATP (~3.3 s-1) in the presence of
100 µM free calcium for all actin concentrations (data not shown). The absence of a linear
component in the transient confirms that the myosin underwent a single turnover. The
experimental reaction mechanism can be simplified to Scheme S3 since the M.D.Pi complex is
formed much more rapidly relative to the rate of phosphate release (1).
Assuming a rapid equilibrium for actin binding, K9, the intrinsic rate k+4’ could then be
determined according to equation S2:
The rate of phosphate release, k+4’ at 37 °C, was found to be 2.8 times faster in the presence of
calcium than in its absence (Table 1, Figure 2). Furthermore, the affinity of the M.ADP.Pi
complex for actin, K9, was increased 11-fold.
The rate of ADP release (k+5’) was determined by ATP-induced dissociation of myo1bIQ
from pyrene-actin. When actomyo1bIQ active-sites are saturated with ADP, ATP binding is rate-
limited by the slow dissociation of ADP (k+5’), as described in Scheme S4.
At 37 °C with ADP concentrations ≥ 10 µM, pyrene transients are best fit to a single
exponential function, yielding ADP release rates of 6.7 s-1 and 11 s-1 at the highest ADP
concentration (30 μM) in the absence or presence of 100 µM free calcium, respectively (Table 1,
Figure 2e and 2f). A similar ~2-fold increase in the rate of ADP release with calcium was also
seen at 20 °C for both myo1ba and myo1bIQ. At non-saturating concentrations of ADP, the
transient is best fit with the sum of two exponential rates, where the fast phase is ATP binding
and the slow phase is ADP release (or the rate of the nucleotide free isomerization, k+α, at low
concentrations of ADP). The relative amplitudes of both the fast and the slow phases have a
hyperbolic relationship to ADP concentration that reports the dissociation constant for ADP
binding, K5’ (1). This affinity was similar both in the presence and absence of 100 µM free
calcium with a value of 0.85 µM (Table 1). At 20 °C, both myo1bIQ and myo1ba show a 2-fold
change in the dissociation constant for ADP upon the addition of free calcium. The larger
change in the affinity at 20 °C compared to 37 °C is due to the fact that at 20 °C, the ADP
binding rate does not increase by the same magnitude as the ADP release rate, as it does at 37
1. Lewis, J. H., T. Lin, D. E. Hokanson, and E. M. Ostap. 2006. Temperature dependence of
nucleotide association and kinetic characterization of myo1b. Biochemistry 45:11589-
Geeves, M. A., C. Perreault-Micale, and L. M. Coluccio. 2000. Kinetic analyses of a
truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide
binding. The Journal of biological chemistry 275:21624-21630.
White, H. D., B. Belknap, and M. R. Webb. 1997. Kinetics of nucleoside triphosphate
cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured
using a novel fluorescent probe for phosphate. Biochemistry 36:11828-11836.
Brune, M., J. L. Hunter, J. E. Corrie, and M. R. Webb. 1994. Direct, real-time
measurement of rapid inorganic phosphate release using a novel fluorescent probe and its
application to actomyosin subfragment 1 ATPase. Biochemistry 33:8262-8271.
Supporting Figure Legends
Figure S1. Rate of ATP hydrolysis by myo1bIQ as measured by mantATP fluorescence.
MantATP concentration dependence of the rate of change in mant fluorescence upon binding and
fluorescence energy transfer from the intrinsic tryptophans of myo1bIQ at 37° C in the (orange,
) absence and (red, ) presence of 100 µM free calcium. (○) Rate of actomyo1bIQ dissociation
as a function of mantATP as measured by light scattering at 37°C. The maximum rate of the
mantATP fluorescence change is not reporting a conformation change due to mantATP binding,
since light scatting measurements do not saturate at high mantATP concentrations. Thus the
plateau value of the mantATP fluorescence change provides a measure of ATP hydrolysis. The
data sets acquired in the absence of calcium were published previously (1).
Figure S2: Transient kinetics of myo1ba and myo1bIQ at 20 °C. All experiments were
conducted with 0.5 µM myosin, 0.5 µM pyrene-actin, and 1 µM free calmodulin. Experiments
were conducted with either 0 or 100 µM free calcium. Values for the best fit parameters are
found in Tables 1 and 2.
Movie S1: Movies showing in vitro motility assays conducted at 37 °C using myo1ba.
Experiments were conducted as described in the Materials and Methods with a frame rate of 0.2
frames per second. Filament gliding rates are quantified in Fig. 1. The addition of calcium
causes a reduction in actin filament sliding velocity. At high calcium concentrations, directed
motility is lost and the filaments reptate in a non-directional manner.
Figure S1: ATP hydrolysis
Figure S2: Transient kinetics of myo1ba and myo1bIQ at 20 °C.