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

Abstract

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 DSE. Analysis of (1)H and (13)C chemical shifts reveals residual secondary structure in the DSE in buffer, which is stabilized by both local and long-range interactions. (15)N R2 relaxation rates deviate from random coil models, suggesting hydrophobic clustering in the DSE. Paramagnetic relaxation enhancement experiments show that there are transient long-range contacts in the DSE in buffer. In contrast, the urea-induced DSE has significantly less residual secondary structure and markedly fewer long-range contacts; however, the urea-induced DSE deviates from a random coil.

19F Nuclear Magnetic Resonance and Crystallographic Studies of 5-Fluorotryptophan-Labeled Anthrax Protective Antigen and Effects of the Receptor on Stability

“…37 The 19 F NMR spectrum of MTSL-labeled PA, in the absence and presence of TCEP, is shown in Figure 5. The resonance at −49.5 ppm exhibited the largest increase in amplitude in the presence of TCEP, and thus, we assigned this resonance to Trp346.…”

A quantitative measure for protein conformational heterogeneity

“…[5][6][7][8] Studies of disorder in protein folding are focused on characterizing the ensemble of non-native conformations under denaturing as well as native conditions. [9][10][11] Of interest are questions pertaining to the degree of conformational heterogeneity, 12,13 the balance between intrachain and chainsolvent interactions that define polymeric properties, [14][15][16] effects of macromolecular crowding, 17,18 intermolecular interactions that lead to protein aggregation, [19][20][21] and the timescales for conversion between distinct conformations that contribute to internal friction. 22 Recent interest has also focused on the topic of IDPs.…”

Comparing protein folding in vitro and in vivo: foldability meets the fitness challenge

“…Another research topic in in-vitro folding that has seen impressive progress recently is the nature of the denatured or unfolded state ensemble [21,22] and under what conditions the chains collapse [23]. A subject of long standing debate is how collapsed the unfolded state ensemble is under differing denaturant concentrations, and a recent study shows that the apparent results depend on the method of observation [24].…”