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Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

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Titration of actin with cardiac myosin isoforms. a Fluorescence transients observed when 20 μM ATP was used to dissociate 30 nM actin from increasing concentrations of β-S1. The fluorescence was fitted to a single exponential, the kobs (= 18 s−1) was constant and the amplitude increased with increasing [S1]. b A plot of the amplitudes in A versus [β-S1] (open triangle) and similar data for β-S1 in the presence of 500 μM ADP (filled triangle). Note that plotted concentrations are before mixing. The result was fitted to the quadratic equation describing the binding isotherm (see “Experimental” section) resulted in a KA = 8 nM and KDA = 190 nM for β-S1. c Example traces used for the results in (d): 30 nM actin was incubated with 400 nM α-S1 or 400 nM α-S1·ADP before rapidly mixing with 20 μM ATP or 250 μM ATP. Without ADP the fluorescence transient was fitted to a single exponential with kobs = 29 s−1 and Amp = 30%. In the presence of ADP the fluorescence transient, fitted to a single exponential, resulted in kobs = 66 s−1 and Amp = 11%. The large difference in measured fluorescence amplitude is due to the weak affinity of α-S1-ADP for actin. d A similar plot as B for α-S1 (open square) and α- (filled square) in which ADP was 1 mM resulting in KA = 44 nM and KDA = 2.4 μM. Plotted concentrations are before mixing. Table 1 gives the average values of 2–3 independent measurements of KA and KDA
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Fig5: Titration of actin with cardiac myosin isoforms. a Fluorescence transients observed when 20 μM ATP was used to dissociate 30 nM actin from increasing concentrations of β-S1. The fluorescence was fitted to a single exponential, the kobs (= 18 s−1) was constant and the amplitude increased with increasing [S1]. b A plot of the amplitudes in A versus [β-S1] (open triangle) and similar data for β-S1 in the presence of 500 μM ADP (filled triangle). Note that plotted concentrations are before mixing. The result was fitted to the quadratic equation describing the binding isotherm (see “Experimental” section) resulted in a KA = 8 nM and KDA = 190 nM for β-S1. c Example traces used for the results in (d): 30 nM actin was incubated with 400 nM α-S1 or 400 nM α-S1·ADP before rapidly mixing with 20 μM ATP or 250 μM ATP. Without ADP the fluorescence transient was fitted to a single exponential with kobs = 29 s−1 and Amp = 30%. In the presence of ADP the fluorescence transient, fitted to a single exponential, resulted in kobs = 66 s−1 and Amp = 11%. The large difference in measured fluorescence amplitude is due to the weak affinity of α-S1-ADP for actin. d A similar plot as B for α-S1 (open square) and α- (filled square) in which ADP was 1 mM resulting in KA = 44 nM and KDA = 2.4 μM. Plotted concentrations are before mixing. Table 1 gives the average values of 2–3 independent measurements of KA and KDA

Mentions: For Fig. 5 the analysis of the titration of S1 binding to actin was performed as described by Kurzawa and Geeves [27]. Using a fixed concentration of pyrene-actin and increasing S1 concentrations, the concentration of the actin·S1 complex can be estimated from the amplitude of the observed fluorescence transient when ATP is added to dissociate the complex. The amplitude dependence on [S1] data was then fitted to the physically significant root of the following quadratic equation.4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {{\upalpha}} = \frac{{ [M ]+ K_{\text{D}} + [A ]_{ 0} - \sqrt {\left( {[M] + K_{\text{D}} + [A]_{0} } \right)^{2} - \frac{4}{{ [M ] [A ]_{ 0} }}} }}{{ 2 [A ]_{ 0} }} $$\end{document} α is the fraction of actin with myosin bound, [M] is the total concentration of S1 added, [A]0 is the concentration of actin and KD is the dissociation constant of S1 for actin (i.e., KA or KDA).


Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

Titration of actin with cardiac myosin isoforms. a Fluorescence transients observed when 20 μM ATP was used to dissociate 30 nM actin from increasing concentrations of β-S1. The fluorescence was fitted to a single exponential, the kobs (= 18 s−1) was constant and the amplitude increased with increasing [S1]. b A plot of the amplitudes in A versus [β-S1] (open triangle) and similar data for β-S1 in the presence of 500 μM ADP (filled triangle). Note that plotted concentrations are before mixing. The result was fitted to the quadratic equation describing the binding isotherm (see “Experimental” section) resulted in a KA = 8 nM and KDA = 190 nM for β-S1. c Example traces used for the results in (d): 30 nM actin was incubated with 400 nM α-S1 or 400 nM α-S1·ADP before rapidly mixing with 20 μM ATP or 250 μM ATP. Without ADP the fluorescence transient was fitted to a single exponential with kobs = 29 s−1 and Amp = 30%. In the presence of ADP the fluorescence transient, fitted to a single exponential, resulted in kobs = 66 s−1 and Amp = 11%. The large difference in measured fluorescence amplitude is due to the weak affinity of α-S1-ADP for actin. d A similar plot as B for α-S1 (open square) and α- (filled square) in which ADP was 1 mM resulting in KA = 44 nM and KDA = 2.4 μM. Plotted concentrations are before mixing. Table 1 gives the average values of 2–3 independent measurements of KA and KDA
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Fig5: Titration of actin with cardiac myosin isoforms. a Fluorescence transients observed when 20 μM ATP was used to dissociate 30 nM actin from increasing concentrations of β-S1. The fluorescence was fitted to a single exponential, the kobs (= 18 s−1) was constant and the amplitude increased with increasing [S1]. b A plot of the amplitudes in A versus [β-S1] (open triangle) and similar data for β-S1 in the presence of 500 μM ADP (filled triangle). Note that plotted concentrations are before mixing. The result was fitted to the quadratic equation describing the binding isotherm (see “Experimental” section) resulted in a KA = 8 nM and KDA = 190 nM for β-S1. c Example traces used for the results in (d): 30 nM actin was incubated with 400 nM α-S1 or 400 nM α-S1·ADP before rapidly mixing with 20 μM ATP or 250 μM ATP. Without ADP the fluorescence transient was fitted to a single exponential with kobs = 29 s−1 and Amp = 30%. In the presence of ADP the fluorescence transient, fitted to a single exponential, resulted in kobs = 66 s−1 and Amp = 11%. The large difference in measured fluorescence amplitude is due to the weak affinity of α-S1-ADP for actin. d A similar plot as B for α-S1 (open square) and α- (filled square) in which ADP was 1 mM resulting in KA = 44 nM and KDA = 2.4 μM. Plotted concentrations are before mixing. Table 1 gives the average values of 2–3 independent measurements of KA and KDA
Mentions: For Fig. 5 the analysis of the titration of S1 binding to actin was performed as described by Kurzawa and Geeves [27]. Using a fixed concentration of pyrene-actin and increasing S1 concentrations, the concentration of the actin·S1 complex can be estimated from the amplitude of the observed fluorescence transient when ATP is added to dissociate the complex. The amplitude dependence on [S1] data was then fitted to the physically significant root of the following quadratic equation.4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {{\upalpha}} = \frac{{ [M ]+ K_{\text{D}} + [A ]_{ 0} - \sqrt {\left( {[M] + K_{\text{D}} + [A]_{0} } \right)^{2} - \frac{4}{{ [M ] [A ]_{ 0} }}} }}{{ 2 [A ]_{ 0} }} $$\end{document} α is the fraction of actin with myosin bound, [M] is the total concentration of S1 added, [A]0 is the concentration of actin and KD is the dissociation constant of S1 for actin (i.e., KA or KDA).

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

Show MeSH
Related in: MedlinePlus