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Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies.

Kupi T, Gróf P, Nyitrai M, Belágyi J - Eur. Biophys. J. (2013)

Bottom Line: EPR results suggested that the MSL was attached to a single SH group in the FH2.The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments.In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs, 7624, Hungary.

ABSTRACT
Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.

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(a) Temperature dependence of 2 of MSL–formin as a function of temperature. At approximately 41 °C a breakpoint is apparent. (b) Temperature dependence of the hyperfine splitting constants of: MSL–formin complex with F-actin (1:5 mol/mol) (filled squares), MSL–F-actin complex with formin (5:1 mol/mol) (asterisks), and MSL–F-actin without formin (filled triangles)
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Fig3: (a) Temperature dependence of 2 of MSL–formin as a function of temperature. At approximately 41 °C a breakpoint is apparent. (b) Temperature dependence of the hyperfine splitting constants of: MSL–formin complex with F-actin (1:5 mol/mol) (filled squares), MSL–F-actin complex with formin (5:1 mol/mol) (asterisks), and MSL–F-actin without formin (filled triangles)

Mentions: EPR spectra were recorded as a function of temperature between 0 and 60 °C. First, the temperature dependence of the hyperfine splitting of MSL–formin was determined in the absence of actin filaments (Fig. 3a). At 40.4 °C a breakpoint appeared in the temperature dependence of 2 (Fig. 3a). Statistical analysis showed that the difference between the slopes of the two straight lines was significant by t-test at the P = 0.05 level and F-test declared that the linear fit with two straight lines is significantly better than a simple linear fit.Fig. 3


Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies.

Kupi T, Gróf P, Nyitrai M, Belágyi J - Eur. Biophys. J. (2013)

(a) Temperature dependence of 2 of MSL–formin as a function of temperature. At approximately 41 °C a breakpoint is apparent. (b) Temperature dependence of the hyperfine splitting constants of: MSL–formin complex with F-actin (1:5 mol/mol) (filled squares), MSL–F-actin complex with formin (5:1 mol/mol) (asterisks), and MSL–F-actin without formin (filled triangles)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824300&req=5

Fig3: (a) Temperature dependence of 2 of MSL–formin as a function of temperature. At approximately 41 °C a breakpoint is apparent. (b) Temperature dependence of the hyperfine splitting constants of: MSL–formin complex with F-actin (1:5 mol/mol) (filled squares), MSL–F-actin complex with formin (5:1 mol/mol) (asterisks), and MSL–F-actin without formin (filled triangles)
Mentions: EPR spectra were recorded as a function of temperature between 0 and 60 °C. First, the temperature dependence of the hyperfine splitting of MSL–formin was determined in the absence of actin filaments (Fig. 3a). At 40.4 °C a breakpoint appeared in the temperature dependence of 2 (Fig. 3a). Statistical analysis showed that the difference between the slopes of the two straight lines was significant by t-test at the P = 0.05 level and F-test declared that the linear fit with two straight lines is significantly better than a simple linear fit.Fig. 3

Bottom Line: EPR results suggested that the MSL was attached to a single SH group in the FH2.The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments.In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Medical School, University of Pécs, Szigeti str. 12, Pécs, 7624, Hungary.

ABSTRACT
Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.

Show MeSH
Related in: MedlinePlus