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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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Upper 2 spectra: conventional EPR spectra of MSL–formin and its complex with F-actin. Lower 2 spectra: conventional EPR spectra of MSL–G-actin and MSL–F-actin. In contrast with MSL–F-actin, the spectra of MSL–formin contain two components with different rotational mobility. The peak heights of the low-field components are labeled I+1 and I+1m
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Fig2: Upper 2 spectra: conventional EPR spectra of MSL–formin and its complex with F-actin. Lower 2 spectra: conventional EPR spectra of MSL–G-actin and MSL–F-actin. In contrast with MSL–F-actin, the spectra of MSL–formin contain two components with different rotational mobility. The peak heights of the low-field components are labeled I+1 and I+1m

Mentions: EPR experiments were performed with MSL-labeled mDia1-FH2 formin fragments. Nitroxide maleimide reporter molecules usually react with the cysteine residues in proteins (Mossakowska et al. 1988; Thomas et al. 1975). Two EPR components were found and they were attributed to a shorter and a longer correlation time (Fig. 2). By successive subtractions of the two composite EPR spectra it was possible to estimate their relative contributions. At room temperature approximately 60 % of the signal was attributed to the component with the longer correlation time and the corresponding hyperfine splitting constant was 2= 6.538 ± 0.044 mT (n = 4).Fig. 2


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

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

Upper 2 spectra: conventional EPR spectra of MSL–formin and its complex with F-actin. Lower 2 spectra: conventional EPR spectra of MSL–G-actin and MSL–F-actin. In contrast with MSL–F-actin, the spectra of MSL–formin contain two components with different rotational mobility. The peak heights of the low-field components are labeled I+1 and I+1m
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824300&req=5

Fig2: Upper 2 spectra: conventional EPR spectra of MSL–formin and its complex with F-actin. Lower 2 spectra: conventional EPR spectra of MSL–G-actin and MSL–F-actin. In contrast with MSL–F-actin, the spectra of MSL–formin contain two components with different rotational mobility. The peak heights of the low-field components are labeled I+1 and I+1m
Mentions: EPR experiments were performed with MSL-labeled mDia1-FH2 formin fragments. Nitroxide maleimide reporter molecules usually react with the cysteine residues in proteins (Mossakowska et al. 1988; Thomas et al. 1975). Two EPR components were found and they were attributed to a shorter and a longer correlation time (Fig. 2). By successive subtractions of the two composite EPR spectra it was possible to estimate their relative contributions. At room temperature approximately 60 % of the signal was attributed to the component with the longer correlation time and the corresponding hyperfine splitting constant was 2= 6.538 ± 0.044 mT (n = 4).Fig. 2

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