Kinetic Folding of Haloferax volcanii and Escherichia coli Dihydrofolate Reductases: Haloadaptation by Unfolded State Destabilization at High Ionic Strength

Salts affect protein stability by multiple mechanisms (e.g., the Hofmeister effect, preferential hydration, electrostatic effects and weak ion binding). These mechanisms can affect the stability of both the native state and the unfolded state. Previous equilibrium stability studies demonstrated that KCl stabilizes dihydrofolate reductases (DHFRs) from Escherichia coli (ecDHFR, E. coli DHFR) and Haloferax volcanii (hvDHFR1, H. volcanii DHFR encoded by the hdrA gene) with similar efficacies, despite adaptation to disparate physiological ionic strengths (0.2 M versus 2 M). Kinetic studies can provide insights on whether equilibrium effects reflect native state stabilization or unfolded state destabilization. Similar kinetic mechanisms describe the folding of urea-denatured ecDHFR and hvDHFR1: a 5-ms stopped-flow burst-phase species that folds to the native state through two sequential intermediates with relaxation times of 0.1–3 s and 25–100 s. The latter kinetic step is very similar to that observed for the refolding of hvDHFR1 from low ionic strength. The unfolding of hvDHFR1 at low ionic strength is relatively slow, suggesting kinetic stabilization as observed for some thermophilic enzymes. Increased KCl concentrations slow the urea-induced unfolding of ecDHFR and hvDHFR1, but much less than expected from equilibrium studies. Unfolding rates extrapolated to 0 M denaturant, k unf(H 2O), are relatively independent of ionic strength, demonstrating that the KCl-induced stabilization of ecDHFR and hvDHFR1 results predominantly from destabilization of the unfolded state. This supports the hypothesis from previous equilibrium studies that haloadaptation harnesses the effects of elevated salt concentrations on the properties of the aqueous solvent to enhance protein stability.