Characterization of the Unfolded State Under Native Conditions: A Missing Piece of the Protein Folding Puzzle

Abstract

The nature of the denatured state ensemble is controversial owing in large part to the difficulty of characterizing the structure and energetics of denatured state interactions. Denatured states can be populated under a variety of extreme conditions but the state which is most relevant for protein folding and engineering is the denatured state ensemble which is populated in the absence of denaturant under native conditions. Unfortunately this state is usually experimentally inaccessible. We reported detailed characterized of the denatured state populated under native conditions for two α-β proteins, the N-terminal domain of the ribosomal protein L9 (NTL9) and the C-terminal domain of the same protein (CTL9) , as well as for a rapid folding all helical structure the villin headpiece helical subdomain, HP-36. Conditions have been found where the native and denatured states of CTL9 are both populated in the absence of denaturant and 1H, 15N and 13C NMR was used to define the conformation propensities of the denatured state. For NTL9 the thermodynamic linkage between proton binding and protein stability was used to characterize denatured state electrostatic interactions. Peptide models were exploited to characterize the denatured state of HP-36. In all three cases, the denatured states contains significant structure. The impact of this preformed structure on the kinetics and mechanism of protein folding is discussed.