Effect of a Concentrated “Inert” Macromolecular Cosolute on the Stability of a Globular Protein with Respect to Denaturation by Heat and by Chaotropes: A Statistical-Thermodynamic Model

Abstract

An equilibrium statistical-thermodynamic model for the effect of volume exclusion arising from high concentrations of stable macromolecules upon the stability of a trace globular protein with respect to denaturation by heat and by chaotropes is presented. The stable cosolute and the native form of the trace protein are modeled by effective hard spherical particles. The denatured state of the trace protein is represented as an ensemble of substates modeled by random coils having the same contour length but different rms end-to-end distances (i.e., different degrees of compaction). The excess or nonideal chemical potential of the native state and of each denatured substate is calculated as a function of the concentration of stable cosolute, leading to an estimate of the relative abundance of each state and substate, and the ensemble average free energy of the transition between native and denatured protein. The effect of the addition of stable cosolute upon the temperature of half-denaturation and upon the concentration of chaotrope required to half-denature the tracer at constant temperature is then estimated. At high cosolute concentration (Ͼ100 g/l) these effects are predicted to be large and readily measurable experimentally, provided that an experimental system exhibiting a fully reversible unfolding equilibrium at high total macromolecular concentration can be developed.

Effects of macromolecular crowding agents on protein folding in vitro and in silico

“…We have probed thermal and chemical stability changes due to the presence of synthetic macromolecular crowding agents using spectroscopic detection methods for several strategic proteins: i.e., α/β-structured Desulfovibrio desulfuricans apoflavodoxin (148 residues), α-helical single-domain Borrelia burgdorferi VlsE (341 residues), α-helical horse-heart cytochrome c (104 residues), the ring-shaped heptamer human mitochondrial co-chaperonin protein 10 (cpn10, 101 residues per monomer), and an α/β thermophilic ribosomal protein S16 (116 residues). In agreement with expectations based on excluded-volume effects (Minton 2000), all proteins are chemically and thermally stabilized by the presence of crowding agents, although to different degrees (Perham et al 2007;Homouz et al 2008Homouz et al , 2009bSamiotakis et al 2009;Christiansen et al 2010). In Table 1, we report measured thermal and chemical effects on our studied proteins (Perham et al 2007;Stagg et al 2007;Christiansen et al 2010), along with other such reports taken from the literature.…”

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

“…These stability changes approximate those made by excluded volume theory, which estimates the change 2 in stability of a protein on the basis of the difference between the compactness of the native and denatured states [23]. Minton predicts that crowding at around 300 g/L of cosolute should increase the equilibrium constant for folding by ten or a hundred times, or 1.4-2.8 kcal/mol for proteins at 310 K [23]. This prediction can be made because small, globular proteins undergo similar changes in compactness when unfolded, hence the small range of values obtained for a several different proteins.…”

“…Compaction of the denatured state is a prediction of macromolecular crowding theory [23] and compaction of denatured [57] and native [58] states has been observed experimentally. However, no collapse was observed for rapid transfer of unfolded ubiquitin to native conditions [59] by small-angle X-ray scattering in the absence of crowding agent, suggesting that glucose is required to promote partial collapse of the denatured state of ubiquitin.…”

Thermodynamics and kinetics of protein folding under confinement

“…Experimental and theoretical studies have shown that effects arising from excluded volume interactions either due to macromolecular crowding (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) or localization (confinement) of the protein in a small volume (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), such as the ribosome tunnel (32) or a chaperonin cavity (33)(34)(35), can indeed have significant effects on folding. In the limit where the crowding particles are much bigger and heavier than the protein, the macromolecular crowding effects can be approximated by confinement; the shape and dimensions of the cavity will depend on the crowding concentration.…”