The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this ''random coil'' protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine N-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.The adaptation of certain higher organisms to harsh environments is enabled by the intracellular presence of small organic solutes (osmolytes) that protect proteins and other cell components from the denaturing environmental stresses (1). Arakawa and Timasheff (2) have shown that the osmolytes act by raising the chemical potential of the denatured state relative to the native state, thereby increasing the (positive) Gibbs energy difference (⌬G) between the native and denatured ensembles. A pictorial description of the results of Arakawa and Timasheff is presented in the Gibbs energy diagram below, where ⌬G 1 is the unfolding Gibbs energy difference between native (N aq ) and unfolded (U aq ) protein in aqueous buffer solution, and ⌬G 3 is the Gibbs energy change for the same reaction in the presence of osmolytes. Transfer of N aq or U aq from water to osmolyte solution (N os and U os respectively) raises the chemical potential of the unfolded ensemble (⌬G 2 ) much more than it does that of the native state ensemble (⌬G 4 ), resulting in a greater stability of the protein in the osmolyte solution than in buffer, i.e., ⌬G 3 Ͼ⌬G 1 . We have determined the transfer Gibbs energy changes of amino acid side chains and peptide backbone from water to solutions of the osmolytes trimethylamine N-oxide (TMAO), sarcosine, and sucrose (3, 4). From knowledge of these tran...