THE PEPTIDE BACKBONE PLAYS A DOMINANT ROLE IN PROTEIN STABILIZATION BY NATURALLY-OCCURRING OSMOLYTES

Transfer free energy measurements of amino acids from water to the osm olytes, sucrose and sarcosine, were made as a function of osmolyte con centration. From these data, transfer free energies of the amino acid side chains were obtained, and the transfer free energy of the peptide backbone was determined from solubility measurements of diketopiperaz ine (DKP). Using static accessible surface evaluations of the native a nd unfolded states of ribonuclease A, solvent exposed side chain and p eptide backbone areas were multiplied by their transfer free energies and summed in order to evaluate the transfer free energy of the native and unfolded states of the protein from water to the osmolyte solutio ns. The results reproduced the main features of the free energy profil e determined for denaturation of proteins in the presence of osmolytes . The side chains were found collectively to favor exposure to the osm olyte in comparison to exposure in water, and in this sense the side c hains favor protein unfolding. The major factor which opposes and over rides the side chain preference for denaturation and results in the st abilization of proteins observed in osmolytes is the highly unfavorabl e exposure of polypeptide backbone on unfolding. Except for urea and g uanidine hydrochloride solutions, it is shown that all organic solvent s (e.g., dioxane, ethanol, ethylene glycol) and solutes (osmolytes) fo r which transfer free energy measurements have been determined exhibit unfavorable transfer free energy of the peptide backbone. The prevale nce of the unfavorable transfer free energy of the peptide backbone, a nd the fact that the peptide backbone unit is the most numerous group in a protein, makes it an extremely important factor in influencing th e character of protein conformations existing in both stabilizing and destabilizing solvents.