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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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Plots of I+1/I+1m against reciprocal absolute temperature. Filled triangles, MSL–formin; squares, MSL–formin complex with F-actin (1:10 mol/mol); circles, MSL–formin complex with F-actin (1:5 mol/mol)
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Fig7: Plots of I+1/I+1m against reciprocal absolute temperature. Filled triangles, MSL–formin; squares, MSL–formin complex with F-actin (1:10 mol/mol); circles, MSL–formin complex with F-actin (1:5 mol/mol)

Mentions: The line shape of the EPR spectra of MSL–formin depended on temperature. At approximately 41 °C a characteristic heat-induced conformational change was detected, which indicated loosening of the formin structure (Fig. 3a). Addition of F-actin to MSL–formin increased the hyperfine splitting constant 2 (Fig. 3b). The rotational correlation time (τ2) characteristic of the slower rotating component was calculated from the temperature-dependent EPR spectra obtained in the presence of actin. The logarithm of this rotational correlation time is proportional to the reciprocal of the absolute temperature (Kupi et al. 2009) and this enables calculation of the activation energy by use of an Arrhenius-type relationship. A breakpoint appeared in the function of lnτ2 vs. 1,000/T at 42.8 °C (Fig. 6). The calculated activation energies were 23.6 and 14.6 kJ/mol before and after the breakpoint, respectively (Fig. 6; Table 1). These values are connected to the rotational diffusion as Gibb’s free energies. To better understand the origin of the two subpopulations and how they behave on interaction with actin we analyzed their contributions to the spectra (discussed in the supplementary material). Changes in the relative contributions of the two subpopulations can approximately be characterized by calculating the ratio of I+1/I+1m. Here I+1m is the peak height of the low-field maximum in the spectrum of the MSL–formin, and I+1 is the peak-to-peak height of the first component of the spectrum characterizing the faster component of the EPR spectrum (Fig. 2). The analyses revealed that actin binding tends to increase the contribution of the slower component (Fig. 7). Integrated intensities of the separated components were used to determine the relative contributions and basic thermodynamic data for MSL–formin. The corresponding thermodynamic data (ΔH, ΔS, and ΔG) were determined from the temperature dependence of the contribution of the two populations (Fig. 5 and supplementary material). The values are summarized in Table 1 and agree with those found in DSC experiments (Table 1). The relatively small differences can be accounted for the fact that the DSC technique provides information about the global changes in protein structure, whereas the EPR technique also reports local changes around the labeled site at different temperatures. Privalov and Potekhin (1986) and Sanchez-Ruiz (1992) assumed that the process responsible for the irreversible step has a much lower enthalpy change than the unfolding process. This assumption would explain the near agreement of the EPR results with the DSC measurements.Fig. 6


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

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

Plots of I+1/I+1m against reciprocal absolute temperature. Filled triangles, MSL–formin; squares, MSL–formin complex with F-actin (1:10 mol/mol); circles, MSL–formin complex with F-actin (1:5 mol/mol)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824300&req=5

Fig7: Plots of I+1/I+1m against reciprocal absolute temperature. Filled triangles, MSL–formin; squares, MSL–formin complex with F-actin (1:10 mol/mol); circles, MSL–formin complex with F-actin (1:5 mol/mol)
Mentions: The line shape of the EPR spectra of MSL–formin depended on temperature. At approximately 41 °C a characteristic heat-induced conformational change was detected, which indicated loosening of the formin structure (Fig. 3a). Addition of F-actin to MSL–formin increased the hyperfine splitting constant 2 (Fig. 3b). The rotational correlation time (τ2) characteristic of the slower rotating component was calculated from the temperature-dependent EPR spectra obtained in the presence of actin. The logarithm of this rotational correlation time is proportional to the reciprocal of the absolute temperature (Kupi et al. 2009) and this enables calculation of the activation energy by use of an Arrhenius-type relationship. A breakpoint appeared in the function of lnτ2 vs. 1,000/T at 42.8 °C (Fig. 6). The calculated activation energies were 23.6 and 14.6 kJ/mol before and after the breakpoint, respectively (Fig. 6; Table 1). These values are connected to the rotational diffusion as Gibb’s free energies. To better understand the origin of the two subpopulations and how they behave on interaction with actin we analyzed their contributions to the spectra (discussed in the supplementary material). Changes in the relative contributions of the two subpopulations can approximately be characterized by calculating the ratio of I+1/I+1m. Here I+1m is the peak height of the low-field maximum in the spectrum of the MSL–formin, and I+1 is the peak-to-peak height of the first component of the spectrum characterizing the faster component of the EPR spectrum (Fig. 2). The analyses revealed that actin binding tends to increase the contribution of the slower component (Fig. 7). Integrated intensities of the separated components were used to determine the relative contributions and basic thermodynamic data for MSL–formin. The corresponding thermodynamic data (ΔH, ΔS, and ΔG) were determined from the temperature dependence of the contribution of the two populations (Fig. 5 and supplementary material). The values are summarized in Table 1 and agree with those found in DSC experiments (Table 1). The relatively small differences can be accounted for the fact that the DSC technique provides information about the global changes in protein structure, whereas the EPR technique also reports local changes around the labeled site at different temperatures. Privalov and Potekhin (1986) and Sanchez-Ruiz (1992) assumed that the process responsible for the irreversible step has a much lower enthalpy change than the unfolding process. This assumption would explain the near agreement of the EPR results with the DSC measurements.Fig. 6

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