Structures of alpha-tropomyosin magnesium paracrystals. II. Stimulation of staining patterns from the sequence and some observations on the mechanism of positive staining.
Structures of alpha-tropomyosin magnesium paracrystals. II. Stimulation of staining patterns from the sequence and some observations on the mechanism of positive staining.
复制标题
α-原肌球蛋白镁副晶体的结构。
DOI：
10.1016/0022-2836(81)90184-4
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发表时间：
1981
影响因子：
5.6
通讯作者：
Stewart,M
中科院分区：
文献类型：
--
作者：
Stewart,M
用醋酸双氧铀染色的α-原肌球蛋白的镁类晶体的电子显微镜照片显示出14条暗带的重复模式。先前的研究（Casparet等人，1969; Ohtsuki，1974）已经表明，在次晶中的分子是反平行的，它们的末端靠近两个明显的白色带。这些白色线将图案分为两个区域，分别含有九条和五条暗带，较长的区域对应于C末端之间的重叠。本研究表明，用羧肽酶A消化原肌球蛋白后，显著的白色线的强度降低。这意味着，即使在假定阳性染色的材料中，白色线也是由于与连续平行分子末端重叠（NC重叠）相关的局部增厚排除了残余阴性染色而产生的。计算机图像处理和最小二乘分析已被用于将在消化和未消化材料中观察到的阳性染色图案与分子位置和氨基酸序列相关联。在一系列不同的染色标准中，显示当C末端重叠176 ± 5个残基并且连续平行分子的末端重叠11 ± 5个残基时，图案拟合最佳。铀酰离子似乎与结构中的羧基结合，除非它们与碱性残基形成盐桥，或者它们位于原肌球蛋白卷曲螺旋的最内部位置。在分子末端附近预测和观察到的模式之间的系统差异表明NC重叠的构象可能不是完全的α-螺旋。具有球状N末端和延伸C末端的模型与观察到的染色模式更一致，并且还为一些其他观察结果提供了解释。
Electron micrographs of magnesium paracrystals of α-tropomyosin stained with uranyl acetate show a repeating pattern of 14 dark bands. Previous studies (Casparet al., 1969; Ohtsuki, 1974) have shown that the molecules in the paracrystal lie antiparallel with their ends near two prominent white bands. These white lines divide the pattern into two zones containing nine and five dark bands, respectively, with the longer zone corresponding to the overlap between C termini. The present study shows that the intensity of the prominent white lines is reduced after digesting tropomyosin with carboxypeptidase A. This implies that, even in supposedly positively stained material, the white lines result from the exclusion of residual negative stain by the local thickening associated with the overlap of the ends of consecutive parallel molecules (NC overlap). Computer image processing and least-squares analysis have been employed to relate the positively stained patterns observed in both digested and undigested material to molecular positions and the amino acid sequence. Over a range of different staining criteria, it is shown that the pattern is fitted best when the C termini overlap by 176 ± 5 residues and the ends of consecutive parallel molecules overlap by 11 ± 5 residues. Uranyl ions appear to bind to carboxyl groups in the structure unless they form salt bridges with basic residues or they lie in the innermost positions of the tropomyosin coiled coil. Systematic differences between predicted and observed patterns near the molecular ends suggest that the conformation of the NC overlap may not be completely α-helical. A model with a globular N terminus and an extended C terminus is more consistent with the observed staining patterns and also offers an explanation for some other observations.