Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

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 the 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 this 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 exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO 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 are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

Spanning the scales of granular materials through microscopic force imaging.

If you walk on sand, it supports your weight. How do the disordered forces between particles in sand organize, to keep you from sinking? This simple question is surprisingly difficult to answer experimentally: measuring forces in three dimensions, between deeply buried grains, is challenging. Here we describe experiments in which we have succeeded in measuring forces inside a granular packing subject to controlled deformations. We connect the measured micro-scale forces to the macro-scale packing force response with an averaging, mean field calculation. This calculation explains how the combination of packing structure and contact deformations produce the observed nontrivial mechanical response of the packing, revealing a surprising microscopic particle deformation enhancement mechanism.