In situ orientations of protein domains: Troponin C in skeletal muscle fibers

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca2+-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Call-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32degrees +/- 5degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.