Structure and stability of the N-terminal domain of the ribosomal protein L9: Evidence for rapid two-state folding

The N-terminal domain, residues 1-56, of the ribosomal protein L9 has been chemically synthesized. The isolated domain is monomeric as judged by analytical ultracentrifugation and concentration-dependent CD. Complete ¹H chemical shift assignments were obtained using standard methods. 2D-NMR experiments show that the isolated domain adopts the same structure as seen in the full-length protein. It consists of a three-stranded antiparallel β- sheet sandwiched between two helixes. Thermal and urea unfolding transitions are cooperative, and the unfolding curves generated from different experimental techniques, 1D-NMR, far-UV CD, near-UV CD, and fluorescence, are superimposable. These results suggest that the protein folds by a two-state mechanism. The thermal midpoint of folding is 77± 2 °C at pD 8.0, and the domain has a ΔG°(folding) = 2.8 ± 0.8 kcal/mol at 40 °C, pH 7.0. Near the thermal midpoint of the unfolding transition, the 1D-NMR peaks are significantly broadened, indicating that folding is occurring on the intermediate exchange time scale. The rate of folding was determined by fitting the NMR spectra to a two-state chemical exchange model. Similar folding rates were measured for Phe 5, located in the first β-strand, and for Tyr 25, located in the short helix between strands two and three. The domain folds extremely rapidly with a folding rate constant of 2000 s^-1 near the midpoint of the equilibrium thermal unfolding transition.