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
Van’t Hoff plot for the double integral ratio of the first two components in the spectrum of MSL–formin. From the slope of the straight line the free energy change (ΔG) was determined to be 6.7 kJ mol−1 K−1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3824300&req=5

Fig5: Van’t Hoff plot for the double integral ratio of the first two components in the spectrum of MSL–formin. From the slope of the straight line the free energy change (ΔG) was determined to be 6.7 kJ mol−1 K−1

Mentions: In this work maleimide spin labels were attached to mDia1-FH2 formin fragments. Similar labeling of formin has not been reported previously. In the EPR experiments we observed two components for mDia1-FH2 (Fig. 2). To understand the origin of the two components we analyzed their contributions to the spectra obtained from temperature-dependent experiments (Fig. 3). It was possible to approximate the ratio of the double integrals of the two fractions at the m = +1 EPR transition as a function of temperature by computer manipulations (discussed in the supplementary material). The double integrals determined from the experimental spectra were temperature-independent and the natural logarithm of the ratio––double integral of the two components––showed linear dependence against reciprocal temperature (Fig. 5). This observation showed that the spin labels bound to a single cysteine residue only in formin.Fig. 5


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

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

Van’t Hoff plot for the double integral ratio of the first two components in the spectrum of MSL–formin. From the slope of the straight line the free energy change (ΔG) was determined to be 6.7 kJ mol−1 K−1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Van’t Hoff plot for the double integral ratio of the first two components in the spectrum of MSL–formin. From the slope of the straight line the free energy change (ΔG) was determined to be 6.7 kJ mol−1 K−1
Mentions: In this work maleimide spin labels were attached to mDia1-FH2 formin fragments. Similar labeling of formin has not been reported previously. In the EPR experiments we observed two components for mDia1-FH2 (Fig. 2). To understand the origin of the two components we analyzed their contributions to the spectra obtained from temperature-dependent experiments (Fig. 3). It was possible to approximate the ratio of the double integrals of the two fractions at the m = +1 EPR transition as a function of temperature by computer manipulations (discussed in the supplementary material). The double integrals determined from the experimental spectra were temperature-independent and the natural logarithm of the ratio––double integral of the two components––showed linear dependence against reciprocal temperature (Fig. 5). This observation showed that the spin labels bound to a single cysteine residue only in formin.Fig. 5

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