Cosolutes, Crowding, and Protein Folding Kinetics

Abstract

Long accepted as the most important interaction, recent work shows that steric repulsions alone cannot explain the effects of macromolecular cosolutes on the equilibrium thermodynamics of protein stability. Instead, chemical interactions have been shown to modulate, and even dominate, crowding-induced steric repulsions. Here, we use19F NMR to examine the effects of small and large cosolutes on the kinetics of protein folding and unfolding using the metastable 7 kDa N-terminal SH3 domain of the Drosophila signaling protein drk (SH3), which folds by a two-state mechanism. The small cosolutes consist of trimethylamine N-oxide and sucrose, which increase equilibrium protein stability, and urea, which destabilizes proteins. The macromolecules comprise the stabilizing sucrose polymer, Ficoll, and the destabilizing globular protein, lysozyme. We assessed the effects of these cosolutes on the differences in free energy between the folded state and the transition state and between the unfolded ensemble and the transition state. We then examined the temperature dependence to assess changes in activation enthalpy and entropy. The enthalpically mediated effects are more complicated than suggested by equilibrium measurements. We also observed enthalpic effects with the supposedly inert sucrose polymer, Ficoll, that arise from its macromolecular nature. Assessment of activation entropies shows important contributions from solvent and cosolute, in addition to the configurational entropy of the protein that, again, cannot be gleaned from equilibrium data. Comparing the effects of Ficoll to those of the more physiologically relevant cosolute lysozyme reveals that synthetic polymers are not appropriate models for understanding the kinetics of protein folding in cells.

Crowding and Confinement Can Oppositely Affect Protein Stability

“…To quantify the enthalpic and entropic components, we assessed Δ and −TΔ at 308 K and 298 K (Table ). Urea is enthalpically destabilizing but entropically stabilizing whereas TMAO is enthalpically stabilizing but entropically destabilizing as has been observed in other studies …”

“…Urea is enthalpically destabilizing but entropically stabilizing whereas TMAO is enthalpically stabilizing but entropically destabilizing as has been observed in other studies. [42][43][44][45] For Ficoll and dextran, the DH 0 0 u values in solutions of their monomers are smaller than those in polymers, showing that KH1 is enthalpically destabilized in monomers compared to polymers. It appears that the monomers have stronger attractive interactions with the unfolded ensembles than do the polymers, consistent with conclusions from Record's group.…”

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

“… is expected to be reduced by the excluded volume effect, if the transition state is more compact than the reactant state, as in the case of a protein folding reaction. A very recent study of the Pielak group could dissect the impact of crowding on the activation free energy into enthalpic and entropic contributions and reported on different thermodynamic mechanisms for Ficoll and lysozyme as crowding agents . However, if large‐scale motions between protein domains and thus the intrachain diffusion play a significant role, crowding may reduce the value of k UF,0 .…”

“…Av ery recent study of the Pielak group could dissect the impact of crowding on the activation free energy into enthalpic and entropic contributions and reported on different thermodynamic mechanisms for Ficoll and lysozyme as crowding agents. [241] However, if large-scale motions between protein domains and thus the intrachain diffusion play as ignificantr ole, crowdingm ay reduce the value of k UF,0 .T hereby,b oth the internal dynamics and the solventm icroviscosity can contribute to the effective diffusion constant along the folding path. [242] Them icroscopic spatialh eterogeneity of ac rowdeds olution can cause solvent microviscosities that might be much smaller than the bulk viscosity.…”

Molecular simulations of cellular processes

“…A cellular model should also include non-equilibrium processes, for their importance in regulating protein stability (Samiotakis et al 2009;Gershenson and Gierasch 2011;Wang et al 2015;Bui and Hoang 2016;Gorensek-Benitez et al 2017). Toward this goal, recent simulations of cotranslational folding showed that translation rates may affect the structure and stability of misfolding-prone proteins (Trovato and O'Brien 2017), as well as of multidomain proteins (Tanaka et al 2015).…”