Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron-electron resonance) of force activation in muscle

The recent development of site specific spin labelling and advances in pulsed electron paramagnetic. resonance. (EPR) have established spin labelling as a viable structural biology technique. Specific protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined., from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of beta-strands (periodicity of 2 residues) or alpha-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60-70 angstrom of each other create a local dipolar field experienced by the other spin labels. The strength of this field is related to the interspin distance, alpha r(-3). The dipolar field can be measured by the broadening of the EPR lines for the short distances (8-20 angstrom) or for the longer distances (17-70 angstrom) by the pulsed EPR methods, double electron-electron resonance (DEER) and double quantum coherence (DQC). A brief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below.