Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea.

Many proteins fold in apparent two-state behavior, as partially folded intermediates only transiently accumulate and easily escape detection. Besides a native form and a mainly unfolded form, we captured a partially unfolded form of an acyl carrier protein from Micromonospora echinospora (meACP) in the folding/unfolding equilibrium using chemical exchange saturation transfer NMR experiments. The C-terminal region of the partially unfolded form is mainly folded and the N-terminal is unfolded. Furthermore, to understand how the folding process of meACP is influenced by solvent environments, we compared the folding dynamics of meACP in D2 O, H2 O and low concentration of urea. As the environment becomes more denaturing from D2 O to H2 O and then to urea, the unfolded state becomes increasingly populated, and the folding rate decreases. Adding a small amount of urea, which does not change solvent viscosity, has little effects on the unfolding rates, while changing H2 O to D2 O reduces the unfolding rates possibly due to the increase of solvent viscosity. The quantified solvent effects on the protein folding Gibbs energy and activation energy suggest that the transition state of folding may have a similar structure to the native state of the protein.