The Denatured State Ensemble Contains Significant Local and Long-Range Structure under Native Conditions: Analysis of the N-Terminal Domain of Ribosomal Protein L9

The denatured state ensemble (DSE) represents the starting state for protein folding and the reference state for protein stability studies. Residual structure in the DSE influences the kinetics of protein folding, the propensity to aggregate, and protein stability. The DSE that is most relevant for folding is the ensemble populated under native conditions, but the stability of proteins and the cooperativity of their folding normally prevent direct characterization of this ensemble. Indirect experiments have been used to infer residual structure in the DSE under nondenaturing conditions, but direct characterization is rare. The N-terminal domain of ribosomal protein L9 (NTL9) is a small mixed α–β domain that folds cooperatively on the millisecond time scale. A destabilized double mutant of NTL9, V3A/I4A-NTL9, populates the DSE in the absence of denaturant and is in slow exchange with the native state on the nuclear magnetic resonance time scale. The DSE populated in buffer was compared to the urea-induced ...