Wagenknecht T, Berkowitz J, Grassucci R, Timerman A P, Fleischer S
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Biophys J. 1994 Dec;67(6):2286-95. doi: 10.1016/S0006-3495(94)80714-3.
Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.
钙调蛋白（CaM）是骨骼肌和心肌肌浆网钙释放通道（兰尼碱受体）的调节剂。通过电子显微镜确定了CaM在骨骼肌兰尼碱受体表面的结合位置。用直径为1.4纳米的金簇的马来酰亚胺衍生物对小麦胚芽CaM的半胱氨酸-27进行特异性标记，然后将金簇标记的CaM与纯化的兰尼碱受体结合。在不存在染色剂或固定剂的情况下，通过冷冻电子显微镜对复合物在冷冻水合状态下成像。在显微照片中，经常在兰尼碱受体方形图像的角附近观察到金簇。在一些图像中，受体的四个角都被金簇占据。图像平均法使得能够在二维上确定CaM的结合位点，估计精度为4纳米。在达到的分辨率（约3纳米）下，结合CaM后兰尼碱受体的四级结构没有明显变化。偶尔会观察到兰尼碱受体的侧视图，其中受体的取向大致垂直于更为常见的四重对称视图，结果表明CaM结合位点最有可能位于受体面向细胞质的表面。我们得出结论，CaM结合位点距离受体的跨膜通道至少10纳米，因此，CaM对受体钙通道活性的调节涉及长程构象变化。
