Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of <i>m</i>‑Values

To quantify interactions of the osmolyte l-proline with protein functional groups and predict their effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dμ₂/d<i>m</i>₃ = μ₂₃, quantifying the preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and amide or cationic N surface. Solubility data yield μ₂₃ values for four less-soluble solutes. Values of μ₂₃ are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of μ₂₃ predicted from these α-values agree with the experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (<i>K</i>_p) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surfaces and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surfaces exposed in unfolding and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids