Ribosome neutron scattering

To determine the shape of ribosomal proteins in solution, ultracentrifugation, digital densimetry, viscosity, gel filtration, quasi-elastic light scattering, and small-angle X-ray or neutron scattering have all been used. With each technique it is possible to obtain a physical characteristic of the protein. Combining these techniques should allow the size and shape of the protein to be characterized quite well. However, the values determined in various laboratories for the same ribosomal proteins differ considerably. To help understand some of the reasons we will initially discuss each method briefly as it relates to proteins and then review the size and shape of the ribosomal proteins that have been so characterized. 

The question can be raised as to whether the structure of the proteins within the ribosomal particle is the same as in the isolated state. The only direct evidence we have that the structures of proteins are not changed upon incorporation into the subunit is provided by the neutron-scattering studies of Nierhaus et al. (1983b). They showed that individual proteins in solution had radii of gyration indistinguishable from those obtained from their counterparts on the ribosomal subunits in the same buffers and under identical preparation conditions. 

One of the most powerful techniques by which protein-protein neighborhoods within the ribosomal particles can be elucidated is neutron scattering. When using this method to determine the relative positions of proteins in the 30 S subunit, the pardcle is reconstituted with two specific proteins that are deuterated whereas all other ribosomal components are in the protonated form (Moore, 1980). The subunits containing the two deuterated proteins give additional contributions to the scattering curves which provide information on the lengths of the vectors between the two deuterated proteins. 

Fig. 11. Map of proteins within the E. coli 30 S ribosomal subunit as determined by neutron scattering studies (Moore, 1980 Moore et al., 1984). Reproduced with permission from Wittmann (1983).

Finally, low-angle neutron scattering can provide information about the shape of these molecules within the ribosome, which may not be the same as their shape when free in solution. 

Engelman, D. M., Moore, P. B., and Schoenbom, B. P. (1975). Neutron-scattering measurements of separation and shape of proteins in 30s ribosomal-subunit of Escherichia coli—S2-S5, S5-S8, S3-S7. Proc. Natl Acad. Sci. USA 72, 3888-3892. 

Another important approach has been to isolate ribosomal proteins from bacteria grown in D2O and then to reconstitute ribosomal subunits with pairs of deuterated proteins. By studying neutron scattering the distances between the centers of mass of these pairs could be measured. By triangulation the three-dimensional relationship of the entire group of proteins could be determined. The results of such studies for the SOS subunit are shown in Fig. 

With sufficient information on the location of the subunits, more detailed models of the ribosomal quaternary structure can be tested by neutron scattering. Thus an early analysis of the 30S ribosomal particle was performed by comparing experimental neutron contrast variation curves with models based on Debye spheres set at two density levels to correspond to a V-shaped 16S RNA moiety, together with the putative locations of the 21 ribosomal proteins [441]. More recently, the triangula-tion of the 19 30S ribosomal proteins shows that they are not uniformly distributed about the RNA in the 30S subunit, as once believed. The use of deuterated RNA within the 30S ribosomal particle showed an asymmetry in the RNA and protein distribution to confirm this result, where a separation A of 2.5 nm between their centres was calculated [446]. 

Several aspects of neutron scattering studies are particularly elegant and unique in terms of the contrast variation method to examine internal structures as well as the external morphology. Thus the protein positions within the 30S ribosomal subunit can be determined by triangulation of deuterated subunits [489,490] the shell-like spherical structures of protein, lipid and nucleic acid within viruses [546,555,566] and lipoproteins can be analysed the distinct disposition of carbohydrates within glycoproteins [197,198,214], membranes within membrane proteins [39], or proteins within protein-nucleic acid complexes [385,387,392] can be elucidated. Likewise X-ray scattering has been of value in protein-lipid systems since these two components can be readily distinguished from one another [75,76]... 

The ribosome has been a favorite nucleo-protein for study by neutron scattering (References in (3)). We shall mention briefly a recent experiment which made use of the scattering curve I(Q) and [H2O] [ D2O ] variation in the solvent (3). Electron micrographs of 16S RNA had been obtained and showed it to have a Y shaped structure. Is this structure maintained within the 30 S ribosome particle The scattering curve of the model was calculated and compared with neutron data. In 42% D2O, the excess scattering mass of protein is negligible, so that data collected from the 30 S in this buffer should only reflect the RNA structure within the particle. The... 

Electron Microscopy and Neutron Scattering Studies of Ribosomal Quaternary Structure... 

The methods that have provided most information about the distribution of ribosomal components within the shape of the subunits are electron microscopy and neutron scattering. 

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