We have examined the structure of the E. coli and B. stearothermophilus ribosomal S15 proteins. S15 is a primary binding ribosomal protein, and we have recently identified the minimal RNA binding site for S15 on 16S rRNA. As a prelude to determining the structure of the RNA-protein complex, we are studying the structure of the free protein. S15 is an 88 residue basic protein, predicted to form four alpha helical regions. Initial experiments on the E. coli protein confirm that S15 is a helical protein. Partial resonance assignments were possible using NOESY, COSY and TOCSY, and 15N-HMQC and NOESY-HMQC on labeled samples. Three helical segments were identified, however there was no sign of significant tertiary structure. No interresidue NOEs were observed for the aromatic side chains, consistent with the lack of formation of a hydrophobic core. Since initial experiments with the E. coli protein were of limited success, we prepared the homologous protein from B. stearothermophilus. We hoped that this protein would be more stable. However, a similar battery of experiments revealed a similar picture as for the E. coli protein. The focus of this project has now shifted to determining the structure of the RNA-protein complex.
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