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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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Binding of smTM to native F-actin and evaluation of the association constant Ko, cooperativity ω, and binding stoichiometry n, using McGhee and von Hippel approach. Solid line represents the binding function (2) computed with the optimal parameters obtained from non-linear regression analysis. v is the number of mol of tropomyosin bound per mol of actin, c is the concentration of free tropomyosin. For details see Materials and methods.
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fig2: Binding of smTM to native F-actin and evaluation of the association constant Ko, cooperativity ω, and binding stoichiometry n, using McGhee and von Hippel approach. Solid line represents the binding function (2) computed with the optimal parameters obtained from non-linear regression analysis. v is the number of mol of tropomyosin bound per mol of actin, c is the concentration of free tropomyosin. For details see Materials and methods.

Mentions: R = {[1 − (n + 1) v]2 + 4 ωv (1 − nv)}1/2; v = the number of mol of TM bound per mol of actin, n = stoichiometry of TM binding to actin, and c = the concentration of free TM. The binding data were analyzed by fitting the experimental points to the Eq. (2) using non-linear regression software Scientists from MicroMath Scientific Software Inc., (Saint Louis, Mo) in which χ2-function between experimental and theoretical values of a mathematical model is minimized. The character of the curve generated by the Eq. (2) is shown in Fig. 2.


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)

Binding of smTM to native F-actin and evaluation of the association constant Ko, cooperativity ω, and binding stoichiometry n, using McGhee and von Hippel approach. Solid line represents the binding function (2) computed with the optimal parameters obtained from non-linear regression analysis. v is the number of mol of tropomyosin bound per mol of actin, c is the concentration of free tropomyosin. For details see Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Binding of smTM to native F-actin and evaluation of the association constant Ko, cooperativity ω, and binding stoichiometry n, using McGhee and von Hippel approach. Solid line represents the binding function (2) computed with the optimal parameters obtained from non-linear regression analysis. v is the number of mol of tropomyosin bound per mol of actin, c is the concentration of free tropomyosin. For details see Materials and methods.
Mentions: R = {[1 − (n + 1) v]2 + 4 ωv (1 − nv)}1/2; v = the number of mol of TM bound per mol of actin, n = stoichiometry of TM binding to actin, and c = the concentration of free TM. The binding data were analyzed by fitting the experimental points to the Eq. (2) using non-linear regression software Scientists from MicroMath Scientific Software Inc., (Saint Louis, Mo) in which χ2-function between experimental and theoretical values of a mathematical model is minimized. The character of the curve generated by the Eq. (2) is shown in Fig. 2.

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
Related in: MedlinePlus