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 use ¹⁹F 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.

Figure 1

Amide proton temperature coefficients. Buffer, black; 100 g/L urea, red; 200 g/L sucrose, green; 300 g/L Ficoll, blue; 100 g/L lysozyme, orange; no bar means no data. RT, reverse turn; DT, diverging turn; n-Src loop; DF, distal loop. Bars ending in the blue box have a ≥85% probability of participating in an intramolecular hydrogen bond. Bars ending in a pink box have a ≤20% probability of participating in an intramolecular hydrogen bond. Values were determined using a linear least-squares fit of ¹H–N chemical shifts from 288 to 308 K in 5 K increments at pH 7.2. Uncertainties represent one standard deviation.

Figure 2

Temperature dependence of folding rates (black) and unfolding rates (red) for SH3 in buffer. Data were fit with eq 3, incorporating $\mathrm{\Delta }{C}_{p,\mathrm{F},\mathrm{U}\to \mathrm{U}\mathrm{‡}}^{{\circ }^{\prime }\mathrm{‡}}$. The uncertainties (one standard deviation) are smaller than the points, and are listed in the Supporting Information (Table S4).

Figure 3

Temperature dependence of folding (A) and unfolding (B) of SH3 in buffer and cosolutes. Buffer, black; 100 g/L urea, red; 50 g/L TMAO, blue; 200 g/L sucrose, cyan; 200 g/L Ficoll, green; 100 g/L lysozyme, magenta. Folding rates in panel A are fit with $\mathrm{\Delta }{C}_{p,\mathrm{U}\to \mathrm{U}\mathrm{‡}}^{{\circ }^{\prime }\mathrm{‡}}=-0.59$ kcal/mol K, while unfolding rates in panel B are fit with $\mathrm{\Delta }{C}_{p,\mathrm{F}\to \mathrm{U}\mathrm{‡}}^{{\circ }^{\prime }\mathrm{‡}}=0$. The uncertainties are smaller than the points, and are listed in Table S4.

Figure 4

Changes in the activation free energy (A), enthalpy (B), and entropic component (C) in cosolutes. Folding, black; unfolding, red. Changes in the activation free energy were determined at 303 K. Changes in activation enthalpy and entropy were determined at 303 K for folding, and without a reference temperature for unfolding, as described in the text. Uncertainties (one standard deviation) in panel A are smaller than the points. Changes in entropy were multiplied by 303 K. All values and uncertainties are listed in Table S5.