Osmolyte-Induced Folding of an Intrinsically Disordered Protein: Folding Mechanism in the Absence of Ligand

摘要

Understanding the interconversion between thermodynamically distinguishable states present in anprotein folding pathway provides not only the kinetics and energetics of protein folding but also insights intonthe functional roles of these states in biological systems. The protein component of the bacterial Rnase Pnholoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of itsncognate RNA or other anionic ligands. P protein was used in this study as a model system to explore generalnfeatures of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxiden(TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study thenfolding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at variousnfinal TMAO concentrations exhibited multiphasic kinetics. Equilibrium “cotitration” experiments werenperformed using both TMAO and urea during the titration to produce a urea-TMAO titration surfacenof P protein. Both kinetic and equilibriumstudies show evidence of a previously undetected intermediate statenin the P protein folding process. The intermediate state is significantly populated, and the folding ratenconstants are relatively slow compared to those of intrinsically folded proteins similar in size and topology.nThe experiments and analysis described serve as a useful example for mechanistic folding studies of othernIDPs.