May R P, Nowotny V, Nowotny P, Voss H, Nierhaus K H
Institut Laue-Langevin, Grenoble, France.
EMBO J. 1992 Jan;11(1):373-8. doi: 10.1002/j.1460-2075.1992.tb05060.x.
Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.
从大肠杆菌核糖体中选取成对的质子化核糖体蛋白，重组成氘代50S亚基。氘代核糖体基质的rRNA来自于在76% D2O中生长的细胞，氘代蛋白部分来自于在84% D2O中生长的细胞。该程序确保氘代蛋白和rRNA的相干中子散射几乎相同，且等于约90%的D2O溶液的相干中子散射。在含有约90% D2O的重构缓冲液中记录中子散射。结果是与传统方法相比，相干信号与噪声比有显著提高；由于可以使用这种稀溶液，从而避免了不利的粒子间效应。从衍射图中可以推导出两种质子化蛋白质心之间的距离。通过这种方式，已经确定了大亚基内蛋白质之间的50个距离，这为描述核糖体蛋白空间分布的大亚基未来模型提供了基础。本文给出了一个包含七种核糖体蛋白的模型。
