Nierhaus K H, Lietzke R, May R P, Nowotny V, Schulze H, Simpson K, Wurmbach P, Stuhrmann H B
Proc Natl Acad Sci U S A. 1983 May;80(10):2889-93. doi: 10.1073/pnas.80.10.2889.
Ribosomes are heterogeneous for neutrons because RNA and proteins have different neutron-scattering-length densities. This heterogeneity is an obstacle to the shape determination of single ribosomal components within the ribosome. Therefore, we homogenized (matched) the neutron-scattering-length densities of RNA and proteins. 23S and 5S RNA from the large ribosomal subunit were isolated from cells grown in a medium containing 76% 2H2O. The total protein fraction of the large ribosomal subunit was isolated from cells grown in a medium containing 84% 2H2O. When these constituents were used for total reconstitution of 50S subunits, neutron scattering measurements of the reconstituted particles revealed excellent matching near 100% 2H2O. A three-step reconstitution procedure was developed that allowed the reconstitution of 50S subunits from deuterated RNA, deuterated total (i.e., unfractionated) proteins, and single protonated proteins. The reconstituted particles contain one protonated protein or two in a matched ribosomal matrix and were used for shape determination or distance measurement of mass centers of gravity, respectively. The signal/noise ratio is high enough to allow measurement in solutions containing nearly 100% 2H2O at concentrations of only 300-500 A260 nm units/ml. Our experiments have proved the feasibility of our biochemical strategy. The shape determinations of ribosomal proteins in situ gave radii of gyration for L1, L3, L4, and L23 of 26 +/- 2, 22 +/- 2, 20 +/- 2, and 13 +/- 2 A, respectively.
核糖体对中子而言是异质的，因为RNA和蛋白质具有不同的中子散射长度密度。这种异质性阻碍了核糖体中单个核糖体组分的形状确定。因此，我们使RNA和蛋白质的中子散射长度密度均匀化（匹配）。从在含有76% 2H2O的培养基中生长的细胞中分离出大核糖体亚基的23S和5S RNA。从在含有84% 2H2O的培养基中生长的细胞中分离出大核糖体亚基的总蛋白组分。当这些组分用于50S亚基的完全重组时，对重组颗粒的中子散射测量显示在接近100% 2H2O时具有极佳的匹配。开发了一种三步重组程序，该程序允许从氘代RNA、氘代总蛋白（即未分级的）和单个质子化蛋白重组50S亚基。重组颗粒在匹配的核糖体基质中分别包含一种或两种质子化蛋白，并分别用于形状确定或质心距离测量。信噪比足够高，能够在仅300 - 500 A260 nm单位/ml的浓度下，在含有接近100% 2H2O的溶液中进行测量。我们的实验证明了我们生化策略的可行性。原位核糖体蛋白的形状确定给出L1、L3、L4和L23的回转半径分别为26 +/- 2、22 +/- 2、20 +/- 2和13 +/- 2 Å。
