Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of DSC traces of formin samples: formin, G-actin, formin plus F-actin (1:3 mol/mol), and F-actin. The heat flows are plotted in arbitrary units to demonstrate the differences between the transition temperatures and the peak width at half maximum of the protein samples
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3824300&req=5

Fig4: Comparison of DSC traces of formin samples: formin, G-actin, formin plus F-actin (1:3 mol/mol), and F-actin. The heat flows are plotted in arbitrary units to demonstrate the differences between the transition temperatures and the peak width at half maximum of the protein samples

Mentions: To further characterize the thermodynamic properties of formin, DSC experiments were also conducted. The DSC transitions of formin and formin–actin complexes were irreversible. The DSC results for formin (96 μM) are shown in Fig. 4. The transition temperature (Tm) was found to be 43.1 °C. Thermodynamic data for the transition were determined and are summarized in Table 1. The calorimetric enthalpy change (ΔH) was 104 kJ/mol, the entropy change (ΔS) was 0.33 kJ/mol K at Tm, and the Gibbs free energy change (ΔG) was 7.7 kJ/mol at 20 °C.Fig. 4


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

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

Comparison of DSC traces of formin samples: formin, G-actin, formin plus F-actin (1:3 mol/mol), and F-actin. The heat flows are plotted in arbitrary units to demonstrate the differences between the transition temperatures and the peak width at half maximum of the protein samples
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Comparison of DSC traces of formin samples: formin, G-actin, formin plus F-actin (1:3 mol/mol), and F-actin. The heat flows are plotted in arbitrary units to demonstrate the differences between the transition temperatures and the peak width at half maximum of the protein samples
Mentions: To further characterize the thermodynamic properties of formin, DSC experiments were also conducted. The DSC transitions of formin and formin–actin complexes were irreversible. The DSC results for formin (96 μM) are shown in Fig. 4. The transition temperature (Tm) was found to be 43.1 °C. Thermodynamic data for the transition were determined and are summarized in Table 1. The calorimetric enthalpy change (ΔH) was 104 kJ/mol, the entropy change (ΔS) was 0.33 kJ/mol K at Tm, and the Gibbs free energy change (ΔG) was 7.7 kJ/mol at 20 °C.Fig. 4

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