Complete 1H Chemical Shift Assignments Research Articles

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 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.

A two‐dimensional NMR strategy for the complete 1H chemical shift assignment of extended proton spin systems in triterpenoids

AbstractAn assignment strategy that utilizes one‐dimensional lanthanide‐induced methyl proton shifts, reported 13C NMR data of model compounds and two‐dimensional 1H and 13C shift correlated spectroscopy, homonuclear proton chemical shift correlated spectroscopy and nuclear Overhauser enhancement spectroscopy methods was demonstrated to be effective for the complete 1H chemical shift assignments of extended proton spin‐systems in a triterpenoid viz., 3‐keto‐urs‐12‐en‐24‐oic acid methyl ester. Complete 13C chemical shift assignments have also been accomplished.