Thermodynamic characterization of the osmolyte effect on protein stability and the effect of GdnHCl on the protein denatured state.

To understand the biomolecular interactions of osmolytes or guanidine hydrochloride (GdnHCl) with protein functional groups, we have determined the apparent transfer free energies (Delta'(tr)) of a homologous series of cyclic dipeptides (CDs) from water to aqueous solutions of osmolytes or GdnHCl through solubility measurements, as a function of osmolyte or GdnHCl concentration at 25 degrees C under atmospheric pressure. The materials investigated in the present study included the CDs of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val), the osmolytes of trimethylamine N-oxide (TMAO), sarcosine, betaine, proline, and sucrose, and the denaturant of GdnHCl. We observed positive values of (Delta'(tr)) for CDs from water to osmolyte, indicating that interactions between osmolytes and CDs are unfavorable. In contrast, negative (Delta'(tr)) contributions were observed for CDs from water to GdnHCl, revealing that favorable interactions are predominant. The experimental results were further used to estimate the transfer free energies (Delta'(tr)) of the peptide bond (-CONH-), the peptide backbone unit (-CH2C=ONH-), and various functional groups from water to aqueous solutions of osmolyte or GdnHCl.