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Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation.

Skórzewski R, Sliwińska M, Borys D, Sobieszek A, Moraczewska J - Biochim. Biophys. Acta (2008)

Bottom Line: Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms.Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state.We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

View Article: PubMed Central - PubMed

Affiliation: Kazimierz Wielki University in Bydgoszcz, Department of Experimental Biology, Chodkiewicza 30, 85-064 Bydgoszcz, Poland.

ABSTRACT
Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

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Effect of actin truncation on its affinity for tropomyosin isoforms. Binding of smTM (A), TM2 (B), TM1b9a (C), and TM5a (D) to native F-actin (closed symbols) or F-actin−3 (open symbols) was assayed as described in Materials and methods. Conditions: 5 mM imidazole, 1 mM DTT, 150 mM NaCl, 2 mM MgCl2, with pH adjusted to 7.0, in the case of D) concentrations of NaCl and MgCl2 were respectively 30 and 0.5 mM. Data are from 3 to 5 independent experiments. Binding curves were drawn by fitting the experimental points to Hill equation (Eq. 1); (solid line) native F-actin; (dashed line) F-actin−3.
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fig3: Effect of actin truncation on its affinity for tropomyosin isoforms. Binding of smTM (A), TM2 (B), TM1b9a (C), and TM5a (D) to native F-actin (closed symbols) or F-actin−3 (open symbols) was assayed as described in Materials and methods. Conditions: 5 mM imidazole, 1 mM DTT, 150 mM NaCl, 2 mM MgCl2, with pH adjusted to 7.0, in the case of D) concentrations of NaCl and MgCl2 were respectively 30 and 0.5 mM. Data are from 3 to 5 independent experiments. Binding curves were drawn by fitting the experimental points to Hill equation (Eq. 1); (solid line) native F-actin; (dashed line) F-actin−3.

Mentions: Removal of three residues from actin C-teminus had a small effect on the binding of each of the TM isoforms used (Fig. 3). The equilibrium binding constants (Kapp) obtained by fitting the experimental points to the Hill equation (Eq. 1) are summarized in Table 1 and show that actin truncation reduced the isoforms' affinity by a factor ranging between 1.2 and 1.5. The cooperativity coefficient (αH) of TMs binding to F-actin−3 was slightly greater than that for the binding to F-actin. The only TM isoform which bound to F-actin−3 with lower cooperativity was TM2 (Table 1). This might suggest that actin modification affects the affinity of TM either through changing direct TM–actin contacts or through distortion of binding cooperativity.


Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation.

Skórzewski R, Sliwińska M, Borys D, Sobieszek A, Moraczewska J - Biochim. Biophys. Acta (2008)

Effect of actin truncation on its affinity for tropomyosin isoforms. Binding of smTM (A), TM2 (B), TM1b9a (C), and TM5a (D) to native F-actin (closed symbols) or F-actin−3 (open symbols) was assayed as described in Materials and methods. Conditions: 5 mM imidazole, 1 mM DTT, 150 mM NaCl, 2 mM MgCl2, with pH adjusted to 7.0, in the case of D) concentrations of NaCl and MgCl2 were respectively 30 and 0.5 mM. Data are from 3 to 5 independent experiments. Binding curves were drawn by fitting the experimental points to Hill equation (Eq. 1); (solid line) native F-actin; (dashed line) F-actin−3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2628472&req=5

fig3: Effect of actin truncation on its affinity for tropomyosin isoforms. Binding of smTM (A), TM2 (B), TM1b9a (C), and TM5a (D) to native F-actin (closed symbols) or F-actin−3 (open symbols) was assayed as described in Materials and methods. Conditions: 5 mM imidazole, 1 mM DTT, 150 mM NaCl, 2 mM MgCl2, with pH adjusted to 7.0, in the case of D) concentrations of NaCl and MgCl2 were respectively 30 and 0.5 mM. Data are from 3 to 5 independent experiments. Binding curves were drawn by fitting the experimental points to Hill equation (Eq. 1); (solid line) native F-actin; (dashed line) F-actin−3.
Mentions: Removal of three residues from actin C-teminus had a small effect on the binding of each of the TM isoforms used (Fig. 3). The equilibrium binding constants (Kapp) obtained by fitting the experimental points to the Hill equation (Eq. 1) are summarized in Table 1 and show that actin truncation reduced the isoforms' affinity by a factor ranging between 1.2 and 1.5. The cooperativity coefficient (αH) of TMs binding to F-actin−3 was slightly greater than that for the binding to F-actin. The only TM isoform which bound to F-actin−3 with lower cooperativity was TM2 (Table 1). This might suggest that actin modification affects the affinity of TM either through changing direct TM–actin contacts or through distortion of binding cooperativity.

Bottom Line: Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms.Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state.We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

View Article: PubMed Central - PubMed

Affiliation: Kazimierz Wielki University in Bydgoszcz, Department of Experimental Biology, Chodkiewicza 30, 85-064 Bydgoszcz, Poland.

ABSTRACT
Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

Show MeSH