Probing Conformational Change in the First Actin-Binding Domain of Dystrophin

Abstract

We have used time-resolved EPR and fluorescence to resolve structural transitions of dystrophin upon actin binding. Dystrophin (Dys) is a muscle cytoskeletal protein that binds to filamentous actin (F-actin) and the dystroglycan complex in the sarcolemmal membrane. Dys acts to dissipate mechanical forces generated during the contraction and relaxation of muscle thereby maintaining sarcolemmal membrane integrity and protecting from tears. The protein-protein interactions and allostery underlying this function of Dys have not been well studied in the context of conformational change and thermodynamics, partly because acquisition of structural and thermodynamic detail on large and flexible proteins is difficult. Two techniques capable of measuring large-scale conformational changes are dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET). Using a combination of DEER and TR-FRET, we placed a single label (nitroxide or fluorescent) in each CH domain Dys ABD1 and subsequently measured the interprobe distance to assess conformational change upon association with F-actin. To probe the allosteric network of Dys ABD1, we also subjected the protein to differential scanning calorimetry.