Tailored solution environment for protein crystallization: tuning solubility, nucleation, and growth by urea and salt

Urea has recently been shown to modulate protein-protein interactions at sub-denaturing concentrations (M. Madani et al. Soft Matter 2025, 21, 1937-1948). Here, we investigate how urea and salt together influence protein crystallization by tuning both thermodynamic and kinetic parameters. Solubility, a key thermodynamic property, is affected differently by the two additives: urea increases lysozyme solubility, while sodium chloride decreases it without inducing a salting-in effect. When solubility is plotted as a function of the second virial coefficient (B₂), the data collapse onto a master curve. From these measurements, we calculate the chemical potential difference (Δμ) between solution and crystal, generating a Δμ map across the phase diagram. Crystallization kinetics at selected points were monitored using video microscopy. Salt reduces the induction time and accelerates crystal growth, whereas urea produces the opposite effect. The dependencies of the kinetic parameters on Δμ are described by classical nucleation theory and a birth-and-spread growth model, respectively. Strikingly, urea enables crystallization at lower supersaturation levels, and at a fixed Δμ, enhances both nucleation and growth compared to salt alone. Overall, our results reveal how urea and salt independently govern thermodynamic and kinetic factors. We propose that tuning the solution environment through salt and nonspecific additives such as urea provides a general strategy to optimize crystallization conditions for globular proteins.