Investigation into the mechanism of thin filament regulation by transient kinetics and equilibrium binding: Is there a conflict?

Figures

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  Figure 1.. Schematic of double-mixing stopped flow method. Double-mixing stopped-flow apparatus. First mix: myosin-S1 (M) in syringe I is mixed with NTP (either ATP or md-ATP) in syringe II and allowed to incubate for 1–2 s in the delay line (a) to attain steady-state binding and hydrolysis of the substrate by myosin alone. The time of the delay was determined from knowledge of rate constants of nucleotide binding and steady-state hydrolysis determined from the rate of formation and composition of intermediates in the delay line as analyzed by quench flow (White et al., 1997). Second mix: the steady-state intermediates are combined with actin (A) or thin filaments (actin plus tropomyosin and troponin) contained in syringe III. After the second mix, the solution enters the flow cell (b), where the rate of phosphate dissociation is measured either from phosphate binding to (N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MCDD) phosphate binding protein (excitation, 430 nm; emission >450 nm) or the observed rate of md-ADP dissociation from actomyosin-md-ADP-Pi (excitation, 360 nm; emission >420 nm). Product dissociation from myosin and actomyosin is an ordered mechanism in…

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  Figure 2.. Examples of time course of phosphate release from regulated acto-myosin-NDP-Pi as monitored with phosphate binding protein or md-ATP. Time course of fluorescence change (A–D). The residuals at the top of each trace show the difference between the fit curve and the data. (A and B) Double mixing experiments were performed with fluorescent PBP and skeletal muscle proteins. Data taken from Heeley et al. (2002). All solutions contained ∼10-fold excess of PBP versus ATP to ensure binding of any liberated Pi, as well as a Pi scavenging system (Brune et al., 1994). The intervening period (delay time) between the first and second mixes was carefully selected so as to have the maximal amount of M-ADP-Pi and minimal M-ADP before the second mix. The effect of rigor S1 binding was investigated by having a surplus of myosin-S1A1 versus ATP in the first mix so that excess was available to bind to the thin filaments, before Pi dissociation, in the second mix. Ionic strength, ∼12 mM. A fuller description of the conditions is to be found in Heeley et al. (2002). (A) Phosphate dissociation from “turned-off” (no bound ligands) regulated AM-ADP-Pi. First mix, 2 μM myosin-S1A1 + 2 μM ATP. Delay, 1 s.…

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  (Scheme 1).

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  Figure 3.. Dependence of the rates of phosphate dissociation from pre–power stroke myosin at different ionic strengths on the concentration of cardiac thin filaments and ligand binding. Native thin filaments were isolated from porcine cardiac muscle. Different myosin-S1s were used at the different ionic strengths: Cardiac-S1 (zero added salt; A); Skeletal-S1A1 (22 mM Kac; B). After the second mix, rigor S1 (if present) was at a 1:7 molar ratio to the actin monomer concentration. T, 20°C, pH 7. The solid lines dissecting the data points are best fits to a hyperbolic equation: k obs = k(1 + (KTF/[TF])). Data from Houmeida et al. (2010). (A) Phosphate dissociation from regulated AM-ADP-Pi-md-ADP-Pi at low ionic strength (∼13 mM, zero added salt) was measured from the fast component as seen in Fig. 2, C and D. Thin filaments with zero rigor S1 contained 2 mM ATP. Note: Only the fast component of the biphasic fluorescence change from md-ADP dissociation is plotted. No bound ligands, kfast = 0.49 s−1, K = 16 μM; Ca(II) alone, kfast = 27 s−1, K = 19 μM; rigor myosin-S1 alone, kfast = 24 s−1, K = 11 μM; Ca(II), and rigor myosin-S1, kfast = 36 s−1, K = 11 μM. Error bars, ≤15% (Fig. 4 legend in…

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  Figure 4.. Dependence of the rates of product dissociation from post–power stroke myosin on the concentration of cardiac thin filaments and ligand binding. md-ADP release from regulated AM-md-ADP (post–power stroke configuration) at low ionic strength (∼13 mM) and at 20°C. Conditions same as in Fig. 3 A except md-ATP was replaced by md-ADP in the first mix. Second mix, thin filaments (of varying concentration) plus 2 mM MgATP. The observed time course of the fluorescence change at each individual concentration conformed to a single exponential process (Houmeida et al., 2010). The rate constants for md-ADP dissociation are as follows: no bound ligands, k = 64 s−1, K = 32 μM; Ca(II) alone, k = 357 s−1, K = 12 μM; rigor myosin-S1 alone, k = 332 s−1, K = 27 μM; Ca(II) and rigor myosin-S1, k = 359 s−1, K = 6 μM. Note: The rates of dissociation of md-ADP from cardiac actomyosin-S1-md-ADP are about six times faster than those for native ADP at 15°C (Siemankowski and White, 1984).

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  Figure 5.. Comparison of the activation of different types of thin filaments by Ca(II) and rigor myosin-S1. The maximum rate of phosphate dissociation from regulated actomyosin-NDP-Pi is expressed as a percentage of the maximum unregulated rate. The ligand condition is indicated. All measurements were with md-ATP except those represented by black bars. Black bars, measurements using fluorescent PBP with all skeletal proteins at low ionic strength (zero added salt; Heeley et al., 2002); gray bars, all cardiac proteins at low ionic strength (zero added salt; Houmeida et al., 2010). Striped and checkered bars, native cardiac thin filaments (bound Ca[II] only) in, respectively, 22 and 100 mM potassium acetate and using skeletal myosin-S1A1 (Risi et al., 2017). Error, ≤15%.

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  Scheme 2.. Two-state model taken from Houmeida et al. (2010) for cardiac myosin-S1 and cardiac native thin filaments. A, active thin filament conformation (accelerates product dissociation from M-ADP-Pi); I, inactive thin filament conformation (does not accelerate product release from M-ADP-Pi); subscripts to equilibrium constants i = inactive thin filament conformation and a = active thin filament conformation; if neither i nor a is indicated, the equilibrium constant is the apparent equilibrium constant [A(Ca,M)]/[I(Ca,M)], with the indicated ligands bound to the thin filament (e.g., KCa is the equilibrium between A and I with calcium bound to the thin filament, and KiCa is the association constant for calcium binding to the inactive filaments (I); M, cardiac S1; Ca, calcium; the second subscript is the associating/dissociating ligand. ∑A and ∑I are the sum of the active and inactive thin filament regulatory units. The values for the equilibrium constants or ratios of equilibrium constants assuming that the affinity is only a function of the thin filament conformation and not of the bound ligands (i.e., KiCa = KiMCa, KaCa = KaMCa, KiM = KiCaM, and KaM = KaCaM) are shown in parentheses.