A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits. Five proteins isolated in this way were studied with high-resolution 'H NMR at 500 MHz. These are S21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found. Protein L33 appears to be arandom coil. Several resonancesin the 'H N M R spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and &-protons of lysines.During the last decade considerable progress has been made regarding our knowledge of the ribosome, in particular the ribosome of the Escherichia coli bacterium. A clear picture of the topology of the ribosomal subunits is beginning to emerge from the application of immunoelectronmicroscopy [I], from chemical and radiochemical cross-linking [2, 31 and from neutron scattering [4]. Moreover the primary structures of the ribosomal RNAs of E. coli and of its 53 ribosomal proteins have been established. Data available for the three-dimensional structure at an atomic level are still very limited. Some ribosomal proteins have been crystallized and in some cases diffraction patterns to about 0.3 nm resolution are available [5]. Studies of the solution structures of ribosomal proteins, making usc of high-resolution NMR, have bcen undertaken and have provided some insight as to whether individual proteins adopt tertiary structures in solution [6]. Most proteins of the E. coli ribosome have molecular weights smaller than 15000, making them attractive for high-resolution N M R studies. Apart from the intrinsic interest for an understanding of the conformations of free ribosomal proteins, such studies will be of significance for obtaining insight into the process of ribosome assembly and in the feedback regulation of ribosomal-protein gene expression [7-91.Ribosomal proteins are normally isolated under denaturing conditions; low pH and 6 M urea as solvent are currently used [lo, 111. It has been shown however, that such procedures do not lead to irreversible denaturation. The denatured material isolated in this way can still be used succesfully in, for example, reconstitution experiments [12] and affinity chromatography [13]. For NMR studies, where the aim is to obtain information about the three-dimensional structure of proteins, it is essential to use a procedure which yields material that has remained native or that can be renatured. An example of a method where denaturation is avoided throughout the isolation procedure is that of Littlechild and Dijk which is based on extraction of proteins from 3 0 4 and 50-S subunits by high LiCl concentrations [I 41. A different approach, capable of yielding Abbreviations. PAGE, polyacrylamide gel electrophoresis; WEFT, water elimination Fourier transform. ribosomal proteins with lertiary structure retained is dialysis of proteins in urea solutions against appropriate buffers upon wh...
