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 beta-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 degrees C at pD 8.0, and the domain has a delta G degree folding = 2.8 +/- 0.8 kcal/mol at 40 degrees 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 beta-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. Study holds ProTherm entries: 8825, 8826, 8827 Extra Details: antiparallel beta-sheet; cooperative; two-state mechanism;,equilibrium thermal unfolding transition
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||5|
|Proteins||50S ribosomal protein L9 ; 50S ribosomal protein L9|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MKVIFLKDVKGKGKKGEIKNVADGYANNFLFKQGLAIEATPANLKALEAQKQKEQRQAAEELANAKKLKEQLEKLTVTIPAKAGEGGRLFGSITSKQIAESLQAQHGLKLDKRKIELADAIRALGYTNVPVKLHPEVTATLKVHVTEQK|