THE STABILITY AND DYNAMICS OF RIBOSOMAL-PROTEIN L9 - INVESTIGATIONS OF A MOLECULAR STRUT BY AMIDE PROTON-EXCHANGE AND CIRCULAR-DICHROISM

Nuclear magnetic resonance and circular dichroism experiments were use d to investigate the stability and dynamic aspects of ribosomal protei n L9 from Bacillus stearothermophilus in solution. This unusually shar ed protein, with its two widely spaced RNA-binding domains linked by a connecting helix, has been hypothesized to serve as a ''molecular str ut'', most likely playing a role in ribosome assembly and/or maintaini ng the catalytically active conformation of ribosomal RNA. Protection factors for amide proton exchange were quantitatively measured in an e xtensive series of NMR experiments, providing probes of the stability and dynamics of localized regions of the protein. Results show that ea ch of the two RNA-binding domains contains a highly stable care. The e xposed central helix that connects the two domains is helical in solut ion, albeit not rigid, a result that is supported by amide proton prot ection factors, circular dichroism measurements, and carbon-13 and pro ton chemical shift index values. A conserved glycine and lysine-rich l oop in the N-terminal domain is ordered and quite stable, a surprising result, since this loop had been presumed to be disordered in the ori ginal crystallographic analysis. Interestingly, the most dynamic parts of the protein are the regions that contain the likely RNA-binding re sidues in each of the two domains. The present results add further sup port to the notion that the L9 protein plays an architectural role wit hin the ribosome, with the central helix serving as a molecular strut, or perhaps a spring, Linking the two widely spaced RNA-binding domain s. (C) 1997 Academic Press Limited.