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

Abstract

The recent development of site specific spin labelling and advances in pulsedelectron paramagnetic resonance (EPR) have established spin labelling as a viablestructural biology technique. Specific protein sites or whole domains can beselectively targeted for spin labelling by cysteine mutagenesis. The secondarystructure of the proteins is determined from the trends in EPR signals of labelsattached to consecutive residues. Solvent accessibility or label mobility displayperiodicities along the labelled polypeptide chain that are characteristic ofβ-strands (periodicityof 2 residues) or α-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from thedistance restraints. Two spin labels placed within 60–70 Å of each other create a local dipolarfield experienced by the other spin labels. The strength of this field is related to theinterspin distance, . The dipolar field can be measured by the broadening of the EPR lines for the shortdistances (8–20 Å) or for the longer distances (17–70 Å) by the pulsed EPR methods, doubleelectron–electron resonance (DEER) and double quantum coherence (DQC). A brief reviewof the methodology and its applications to the multisubunit muscle protein troponin ispresented below.