Osmolyte-driven contraction of a random coil protein

Abstract

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.

Figure 1

Upper bound limits of Gibbs energies for transfer of side chains and peptide backbone of RCAM RNase from water to 1 M concentrations of urea and protecting osmolytes. DKP/2 represents the transfer of the peptide backbone unit summed over the entire chain length of RCAM RNase, and the values for the transfer of specific amino acid side chains represent the sum of all such side chains in the protein. Data for transfer Gibbs energies of side chains and backbone from water to 1 M concentration of urea and TMAO are from Wang and Bolen (3), sucrose and sarcosine are from Liu and Bolen (4), and proline are from the present work.

Figure 2

Upper bound transfer Gibbs energies of RCAM RNase from water to 1 M concentration of each solute indicated. Filled bars represent the sum total contribution of the peptide backbone, whereas unfilled bars show the sum total contribution of side chains to the transfer.

Figure 3

Side chain contributions to the transfer Gibbs energy (upper bound) of RCAM RNase from water to 1 M concentrations of urea, proline, sucrose, sarcosine, and TMAO. The transfer Gibbs energy changes are segregated into two classes of side chains, the hydrophobic class (W, F, Y, L, I, P, V, M, A; unfilled bars) and the polar/charged class (T, S, Q, N, K, R, H, E, D, G; filled bars).

Figure 4

K_d ratio of elution behavior for RCAM RNase:RNase A, and upper and lower bounds of Gibbs energies of transfer of RCAM RNase from water to 1 M of the solutes indicated. The K_d ratio of RCAM RNase:RNase A is proportional to the Stokes radius ratio of RNase A:RCAM RNase. Filled bars represent the K_d ratio data and unfilled bars are the transfer Gibbs energies of RCAM RNase (see text) representing the upper and lower bounds of transfer Gibbs Energies, ▵G_{tr ub} and ▵G_{tr lb} from Eqs. 2 and 3. Errors in K_d ratios estimated from elution volume data are less than 1%.