RESUMO
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.
Assuntos
Actinas/metabolismo , Proteínas Fetais/química , Proteínas dos Microfilamentos/química , Músculo Esquelético/metabolismo , Proteínas Nucleares/química , Actinas/química , Animais , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância de Spin Eletrônica , Forminas , Temperatura Alta , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/farmacologia , Citoesqueleto de Actina/química , Actinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Forminas , Camundongos , Movimento (Física) , Movimento/efeitos dos fármacos , Rotação , Marcadores de Spin , Temperatura , Fatores de TempoRESUMO
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.
