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 a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in the present study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine-N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations showed multiphasic kinetics. Equilibrium “cotitration” experiments were performed using both TMAO and urea during the titration to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are relatively slow compared to intrinsically folded proteins of similar size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.