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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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Binding of ATP or ADP to cardiac S1. a Tryptophan fluorescence traces observed upon rapidly mixing 0.2 μM α- or β-S1 with 500 μM ATP. For α-S1 the fluorescence traces were best fit by a single exponential, kobs = 151 s−1 (amp = 5.1%), whereas for β-S1 the fluorescence traces (offset by −0.02) were best fit by a double exponential (solid line), kobs = 124 s−1 (amp = 8.4%) and 19 s−1 (amp = 0.8%). Note that a single exponential fit (dashed linekobs = 117 s−1) is also shown for comparison. b The dependence of kobs on [ATP] yields K1k+2 = 2.7 μM−1 s−1 for α-S1 (filled square) and K1k+2 = 1.23 μM−1 s−1 for the fast phase of β-S1 (filled triangle). At high ATP-concentrations kobs saturates at 196 s−1 (α-S1) and 158 s−1 (β-S1). The slow phase measured for β-S1 saturates at ~26 s−1. c Tryptophan fluorescence traces observed after incubating 0.2 μM β-S1 with variable [ADP] (0–1.6 μM) before rapidly mixing with 100 μM ATP. The data fit best to a sum of two exponentials with kobs = 112 s−1 (fast phase) and 0.8 s−1 (slow phase). d Dependence of the relative amplitudes of the two exponentials measured in Fig. 6c on ADP concentration (before mixing). The data are fitted to Eqs. 5A and 5B (“Experimental” section) with a K5 = 0.53 μM (fast phase, filled square) and 0.8 μM (slow phase, filled circle)
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Fig6: Binding of ATP or ADP to cardiac S1. a Tryptophan fluorescence traces observed upon rapidly mixing 0.2 μM α- or β-S1 with 500 μM ATP. For α-S1 the fluorescence traces were best fit by a single exponential, kobs = 151 s−1 (amp = 5.1%), whereas for β-S1 the fluorescence traces (offset by −0.02) were best fit by a double exponential (solid line), kobs = 124 s−1 (amp = 8.4%) and 19 s−1 (amp = 0.8%). Note that a single exponential fit (dashed linekobs = 117 s−1) is also shown for comparison. b The dependence of kobs on [ATP] yields K1k+2 = 2.7 μM−1 s−1 for α-S1 (filled square) and K1k+2 = 1.23 μM−1 s−1 for the fast phase of β-S1 (filled triangle). At high ATP-concentrations kobs saturates at 196 s−1 (α-S1) and 158 s−1 (β-S1). The slow phase measured for β-S1 saturates at ~26 s−1. c Tryptophan fluorescence traces observed after incubating 0.2 μM β-S1 with variable [ADP] (0–1.6 μM) before rapidly mixing with 100 μM ATP. The data fit best to a sum of two exponentials with kobs = 112 s−1 (fast phase) and 0.8 s−1 (slow phase). d Dependence of the relative amplitudes of the two exponentials measured in Fig. 6c on ADP concentration (before mixing). The data are fitted to Eqs. 5A and 5B (“Experimental” section) with a K5 = 0.53 μM (fast phase, filled square) and 0.8 μM (slow phase, filled circle)

Mentions: For Figs. 3b, 4d, and 6b, the dependence of kobs on ATP concentration is defined by:3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ k_{\text{obs}} = k_{ \max } \left[ {\text{ATP}} \right] /\left( {K_{ 0. 5} { + }\left[ {\text{ATP}} \right]} \right) $$\end{document}where kmax is the maximum value of kobs and K0.5 is the nucleotide concentration required to give kobs = kmax/2.


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)

Binding of ATP or ADP to cardiac S1. a Tryptophan fluorescence traces observed upon rapidly mixing 0.2 μM α- or β-S1 with 500 μM ATP. For α-S1 the fluorescence traces were best fit by a single exponential, kobs = 151 s−1 (amp = 5.1%), whereas for β-S1 the fluorescence traces (offset by −0.02) were best fit by a double exponential (solid line), kobs = 124 s−1 (amp = 8.4%) and 19 s−1 (amp = 0.8%). Note that a single exponential fit (dashed linekobs = 117 s−1) is also shown for comparison. b The dependence of kobs on [ATP] yields K1k+2 = 2.7 μM−1 s−1 for α-S1 (filled square) and K1k+2 = 1.23 μM−1 s−1 for the fast phase of β-S1 (filled triangle). At high ATP-concentrations kobs saturates at 196 s−1 (α-S1) and 158 s−1 (β-S1). The slow phase measured for β-S1 saturates at ~26 s−1. c Tryptophan fluorescence traces observed after incubating 0.2 μM β-S1 with variable [ADP] (0–1.6 μM) before rapidly mixing with 100 μM ATP. The data fit best to a sum of two exponentials with kobs = 112 s−1 (fast phase) and 0.8 s−1 (slow phase). d Dependence of the relative amplitudes of the two exponentials measured in Fig. 6c on ADP concentration (before mixing). The data are fitted to Eqs. 5A and 5B (“Experimental” section) with a K5 = 0.53 μM (fast phase, filled square) and 0.8 μM (slow phase, filled circle)
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Fig6: Binding of ATP or ADP to cardiac S1. a Tryptophan fluorescence traces observed upon rapidly mixing 0.2 μM α- or β-S1 with 500 μM ATP. For α-S1 the fluorescence traces were best fit by a single exponential, kobs = 151 s−1 (amp = 5.1%), whereas for β-S1 the fluorescence traces (offset by −0.02) were best fit by a double exponential (solid line), kobs = 124 s−1 (amp = 8.4%) and 19 s−1 (amp = 0.8%). Note that a single exponential fit (dashed linekobs = 117 s−1) is also shown for comparison. b The dependence of kobs on [ATP] yields K1k+2 = 2.7 μM−1 s−1 for α-S1 (filled square) and K1k+2 = 1.23 μM−1 s−1 for the fast phase of β-S1 (filled triangle). At high ATP-concentrations kobs saturates at 196 s−1 (α-S1) and 158 s−1 (β-S1). The slow phase measured for β-S1 saturates at ~26 s−1. c Tryptophan fluorescence traces observed after incubating 0.2 μM β-S1 with variable [ADP] (0–1.6 μM) before rapidly mixing with 100 μM ATP. The data fit best to a sum of two exponentials with kobs = 112 s−1 (fast phase) and 0.8 s−1 (slow phase). d Dependence of the relative amplitudes of the two exponentials measured in Fig. 6c on ADP concentration (before mixing). The data are fitted to Eqs. 5A and 5B (“Experimental” section) with a K5 = 0.53 μM (fast phase, filled square) and 0.8 μM (slow phase, filled circle)
Mentions: For Figs. 3b, 4d, and 6b, the dependence of kobs on ATP concentration is defined by:3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ k_{\text{obs}} = k_{ \max } \left[ {\text{ATP}} \right] /\left( {K_{ 0. 5} { + }\left[ {\text{ATP}} \right]} \right) $$\end{document}where kmax is the maximum value of kobs and K0.5 is the nucleotide concentration required to give kobs = kmax/2.

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