Protein Science, Vol 6, Issue 1 211-221, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Mechanism of the stabilization of ribonuclease A by sorbitol: Preferential hydration is greater for the denatured than for the native protein

G. XIE and S. N. TIMASHEFF
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, T(m), was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, {delta}{Delta}G{deg} = {Delta}G{deg}(sorbitol) - {Delta}G{deg}(water). Measurements of preferential interactions at 48{deg}C at pH 5.5, where protein is native, and pH 2.0, where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, {Delta}{mu}(2)(D) > {Delta}{mu}(2)(N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, {Delta}G{deg}(W) + {Delta}{mu}(2)(D) = {Delta}G{deg}(S) + {Delta}{mu}(2)(D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water {complex} co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Noronha, J. C. Lima, E. Paci, H. Santos, and A. L. Macanita Tracking Local Conformational Changes of Ribonuclease A Using Picosecond Time-Resolved Fluorescence of the Six Tyrosine Residues Biophys. J., June 15, 2007; 92(12): 4401 - 4414. [Abstract] [Full Text] [PDF]

				T. Fujii, T. Ohkuri, R. Onodera, and T. Ueda Stable Supply of Large Amounts of Human Fab from the Inclusion Bodies in E. coli J. Biochem., May 1, 2007; 141(5): 699 - 707. [Abstract] [Full Text] [PDF]

				N. El Kadi, N. Taulier, J. Y. Le Huerou, M. Gindre, W. Urbach, I. Nwigwe, P. C. Kahn, and M. Waks Unfolding and Refolding of Bovine Serum Albumin at Acid pH: Ultrasound and Structural Studies Biophys. J., November 1, 2006; 91(9): 3397 - 3404. [Abstract] [Full Text] [PDF]

				R. Mishra, R. Seckler, and R. Bhat Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes: HOW WELL ARE PROTEIN FOLDING AND STABILITY ASPECTS COUPLED? J. Biol. Chem., April 22, 2005; 280(16): 15553 - 15560. [Abstract] [Full Text] [PDF]

				R. Singh, I. Haque, and F. Ahmad Counteracting Osmolyte Trimethylamine N-Oxide Destabilizes Proteins at pH below Its pKa: MEASUREMENTS OF THERMODYNAMIC PARAMETERS OF PROTEINS IN THE PRESENCE AND ABSENCE OF TRIMETHYLAMINE N-OXIDE J. Biol. Chem., March 25, 2005; 280(12): 11035 - 11042. [Abstract] [Full Text] [PDF]

				B. J. Bennion and V. Daggett Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: A chemical chaperone at atomic resolution PNAS, April 27, 2004; 101(17): 6433 - 6438. [Abstract] [Full Text] [PDF]

				J. K. Kaushik and R. Bhat Why Is Trehalose an Exceptional Protein Stabilizer?: AN ANALYSIS OF THE THERMAL STABILITY OF PROTEINS IN THE PRESENCE OF THE COMPATIBLE OSMOLYTE TREHALOSE J. Biol. Chem., July 11, 2003; 278(29): 26458 - 26465. [Abstract] [Full Text] [PDF]

				A. A. Moosavi-Movahedi, J. Chamani, Y. Goto, and G. H. Hakimelahi Formation of the Molten Globule-Like State of Cytochrome c Induced by n-Alkyl Sulfates at Low Concentrations J. Biochem., January 1, 2003; 133(1): 93 - 102. [Abstract] [Full Text] [PDF]

				P. Lamosa, A. Burke, R. Peist, R. Huber, M.-Y. Liu, G. Silva, C. Rodrigues-Pousada, J. LeGall, C. Maycock, and H. Santos Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus Appl. Envir. Microbiol., May 1, 2000; 66(5): 1974 - 1979. [Abstract] [Full Text]

				Y.-S. Kim, S. P. Cape, E. Chi, R. Raffen, P. Wilkins-Stevens, F. J. Stevens, M. C. Manning, T. W. Randolph, A. Solomon, and J. F. Carpenter Counteracting Effects of Renal Solutes on Amyloid Fibril Formation by Immunoglobulin Light Chains J. Biol. Chem., January 5, 2001; 276(2): 1626 - 1633. [Abstract] [Full Text] [PDF]

				G. S. Ratnaparkhi and R. Varadarajan Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States J. Biol. Chem., July 27, 2001; 276(31): 28789 - 28798. [Abstract] [Full Text] [PDF]