The effect of regulatory Ca2+ on the in situ structures of troponin C and troponin I: a neutron scattering study

Abstract

The effects of regulatory amounts of Ca2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied ±Ca2+ in 41.6% 2H2O buffers in which protonated subunits were rendered “invisible”. We found that the radius of gyration (Rg) of TnI decreased by ≈10% upon addition of regulatory Ca2+ indicating that it was significantly more compact in the presence of Ca2+. The apparent cross-sectional radius of gyration (Rc) of TnI increased by about 9% when regulatory Ca2+ was bound to TnC. Modeling studies showed that the high-Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca2+ addition decreased the center of mass separation by about 1.5 nm. The Rg of TnI, its high-Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca2+) TnC·TnI complex.The Rg of TnC indicated that it was elongate in situ. The Rg of TnC was not sensitive to the Ca2+ occupancy of its regulatory sites. However, Rc increased upon Ca2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca2+-induced changes in TnC.