Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca2+-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the −Ca2+ state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time τ = 1.9 ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca2+ state showed that Cys133 existed on a stable segment (τ = 4.8 ns) held by TnC. Spectra of reconstituted thin filaments (−Ca2+ state) revealed that slow mobility (τ = 45 ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI–actin and TnI–tropomyosin–actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca2+ concentrations.