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.
Subject(s)
Actins/metabolism , Fetal Proteins/chemistry , Microfilament Proteins/chemistry , Muscle, Skeletal/metabolism , Nuclear Proteins/chemistry , Actins/chemistry , Animals , Calorimetry, Differential Scanning , Electron Spin Resonance Spectroscopy , Formins , Hot Temperature , Protein Binding , Protein Multimerization , Protein Stability , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.
Subject(s)
Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Carrier Proteins/pharmacology , Actin Cytoskeleton/chemistry , Actins/metabolism , Animals , Carrier Proteins/metabolism , Electron Spin Resonance Spectroscopy , Formins , Mice , Motion , Movement/drug effects , Rotation , Spin Labels , Temperature , Time FactorsABSTRACT
The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains alpha-skeletal and alpha-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55 degrees C for the alpha-cardiac actin isoform and 58 degrees C for the alpha-skeletal one when the filaments were generated from ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5 degrees C). We concluded that phalloidin stabilized the actin filaments of alpha-skeletal and alpha-cardiac actin isoforms to the same extent when they were polymerized from ADP-actin monomers.
