Stone D B, Timmins P A, Schneider D K, Krylova I, Ramos C H, Reinach F C, Mendelson R A
Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0130, USA.
J Mol Biol. 1998 Aug 28;281(4):689-704. doi: 10.1006/jmbi.1998.1965.
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 approximately 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.
通过中子散射研究了调节量的Ca2+对完整肌钙蛋白中肌钙蛋白C（TnC）和肌钙蛋白I（TnI）原位结构的影响。在单独的差异实验中，研究了完整肌钙蛋白中97%氘代的TnC和TnI在41.6% 2H2O缓冲液中±Ca2+的情况，其中质子化亚基变得“不可见”。我们发现，添加调节性Ca2+后，TnI的回转半径（Rg）降低了约10%，这表明在Ca2+存在下它明显更紧凑。当调节性Ca2+与TnC结合时，TnI的表观截面回转半径（Rc）增加了约9%。模型研究表明，TnI的高Q散射模式可以由一个由两个子域组成的TnI拟合：一个是包含约65%质量的高度扁长的旋转椭球体，另一个是由约35%质量组成的高度细长的旋转椭球体。用这类模型找不到其他拟合。当这些椭球体的旋转轴彼此陡峭倾斜时，可实现最佳拟合。添加Ca2+使质心间距降低了约1.5 nm。TnI的Rg、其高Q散射模式以及所得结构与之前关于（+Ca2+）TnC.TnI复合物中TnI的中子散射结果不同。TnC的Rg表明它在原位是细长的。TnC的Rg对其调节位点的Ca2+占有率不敏感。然而，随着Ca2+的添加，Rc增加，这与分离的TnC的NMR和晶体学预期一致。目前的观察结果表明，TnI的作用类似于一个分子开关，由TnC中较小的Ca2+诱导变化控制。
