Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.