May R P, Stuhrmann H B, Nierhaus K H
Basic Life Sci. 1984;27:25-45. doi: 10.1007/978-1-4899-0375-4_2.
The large (50 S) subunit from E. coli ribosomes consists of 32 different proteins and two RNA molecules of different length. In an attempt to determine the three-dimensional arrangement of the proteins in the subunit, we are also interested in obtaining direct information on the shape of the proteins within the subunit. This is possible only with ribosomal subunits which, unlike natural protonated subunits, are homogeneous for neutrons. These homogeneous particles are produced by reconstituting 50 S particles from RNA and proteins isolated from bacteria grown at different levels of D2O in the culture medium, 76% D2O for RNA and 84% D2O for proteins. Model calculations and test experiments reveal that the pursued strategy allows direct determination of radii of gyration of 50 S components within the particle with reasonable precision. Data evaluation and interpretation are significantly facilitated by contrast variation of the reconstituted particles. The determination of protein shape parameters is only one aspect of the new strategy. The pair distance measurements are completely independent of its success. Data on radii of gyration of five ribosomal proteins in situ are reported: L1 (26 +/- 2 A), L2 (22 +/- 2 A), L3 (22 +/- 2 A), L4 (20 +/- 2 A), and L23 (13 +/- 2 A).
大肠杆菌核糖体的大亚基（50 S）由32种不同的蛋白质和两个长度不同的RNA分子组成。为了确定该亚基中蛋白质的三维排列，我们还希望获得有关亚基内蛋白质形状的直接信息。只有核糖体亚基才能做到这一点，与天然质子化亚基不同，它们对中子是均匀的。这些均匀颗粒是通过用从在培养基中不同D2O水平下生长的细菌中分离的RNA和蛋白质重构50 S颗粒而产生的，RNA用76% D2O，蛋白质用84% D2O。模型计算和测试实验表明，所采用的策略能够以合理的精度直接测定颗粒内50 S组分的回转半径。重构颗粒的对比度变化极大地促进了数据评估和解释。蛋白质形状参数的测定只是新策略的一个方面。对距离的测量与该策略的成功与否完全无关。本文报道了五种核糖体蛋白质原位回转半径的数据：L1（26±2 Å）、L2（22±2 Å）、L3（22±2 Å）、L4（20±2 Å）和L23（13±2 Å）。
