Conformational changes of troponin C within the thin filaments detected by neutron scattering

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered invisible to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 Angstrom (99% confidence limits between 22 Angstrom and 23 Angstrom) and 24 Angstrom (99% confidence limits between 23 Angstrom and 25 Angstrom) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 Angstrom (99% confidence limits between 49 Angstrom and 57 Angstrom) and 49 Angstrom (99% confidence limits between 44 Angstrom and 53 Angstrom) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4 Angstrom is associated with large conformational changes of the entire Tn molecule by binding of Ca2+. (C) 2004 Elsevier Ltd. All rights reserved.