Anatomy of energetic changes accompanying urea-induced protein denaturation

Abstract

Because of its protein-denaturing ability, urea has played a pivotal role in the experimental and conceptual understanding of protein folding and unfolding. The measure of urea's ability to force a protein to unfold is given by the m value, an experimental quantity giving the free energy change for unfolding per molar urea. With the aid of Tanford's transfer model [Tanford C (1964) J Am Chem Soc 86:2050 -2059], we use newly obtained group transfer free energies (GTFEs) of protein side-chain and backbone units from water to 1 M urea to account for the m value of urea, and the method reveals the anatomy of protein denaturation in terms of residue-level free energy contributions of groups newly exposed on denaturation. The GTFEs were obtained by accounting for solubility and activity coefficient ratios accompanying the transfer of glycine from water to 1 M urea. Contrary to the opinions of some researchers, the GTFEs show that urea does not denature proteins through favorable interactions with nonpolar side chains; what drives urea-induced protein unfolding is the large favorable interaction of urea with the peptide backbone. Although the m value is said to be proportional to surface area newly exposed on denaturation, only Ϸ25% of the area favorably contributes to unfolding (because of newly exposed backbone units), with Ϸ75% modestly opposing urea-induced denaturation (originating from side-chain exposure). Use of the transfer model and newly determined GTFEs achieves the long-sought goal of predicting urea-dependent cooperative protein unfolding energetics at the level of individual amino acid residues. m value ͉ transfer free energy ͉ transfer model ͉ activity coefficient ͉ self-avoiding random coil U nderstanding the energetics of protein-solute interactions is one of the most elusive goals of protein science, with urea-induced denaturation serving as a long-standing reminder of the inability to experimentally account for such fundamental interactions in a detailed manner. More than 40 years ago, Tanford set out to identify the sites and free energies of urea interaction with protein groups in enough detail to account for the energetics of urea-induced denaturation (1-3). In principle, the transfer model he developed provides a means of dissecting which groups (side chain and backbone) are involved in denaturation and how much they contribute to the free energy of denaturation by urea. For the transfer model (see Scheme 1) to be successful, two requirements must be met: (i) accurate transfer free energy changes for side-chain and backbone groups must be known, and (ii) the free energy of transfer of a native or denatured state of a protein from water to the urea solution must be equal to the sum of the transfer free energy contributions of its solvent-exposed parts. Of these two requirements, the ability to obtain accurate transfer free energies of side chains and backbone groups has been a particularly difficult impediment to quantifying the energetics of urea-induced denaturation. Here, we present results fo...

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

“…We assigned the urea dependence entirely to the intrinsic, rather than interfacial, folding energies. This partitioning is consistent with a recent study indicating that the magnitude of m-value changes are dominated by peptide backbone exposure, 47 since the majority of the Notch ankyrin domain backbone units are involved in hydrogen bonds within (not between) repeats.…”

Assessing the Interaction of Urea and Protein-Stabilizing Osmolytes with the Nonpolar Surface of Hydroxypropylcellulose

“…The exclusion energies of the polar protein stabilizing osmolytes from the hydrophobic HPC chain, however, are comparable to the energies that have been ascribed to exclusion from the peptide backbone (8,11,26). This indicates that the exclusion of these osmolytes from nonpolar peptide side chains should significantly contribute to the stabilization of native protein structure.…”

“…This, however, does not explain the chemical specificity of the interaction. Bolen and coworkers, for example, have concluded (8,11,12) that the inclusion of urea and the exclusion of stabilizing osmolytes from proteins are dominated by the interaction of these small molecules with the peptide backbone with little contribution from the exclusion of these polar solutes from hydrophobic side chains. One method for elucidating the physics of the interaction is to measure the distance dependence of the force through either a radial distribution function of solutes †…”

Backbone additivity in the transfer model of protein solvation

“…Recent work corrected the previous measurements for the activities of the model compounds in solution and showed that the strength of the hydrophobic interactions do not change substantially on transfer to urea, and that urea's favorable interaction with the protein backbone is responsible for its denaturing ability. 18 Thus, the free energy dependence of protein stability as a function of osmolyte concentration can be predicted if one assumes that the transfer free energies of solvent exposed sidechain and backbone groups on the native and denatured states are additive. [18][19][20] Here, using computational methods, we assess the transfer free energies of the peptide backbone from water to two osmolyte solutions, 2M urea and 2M TMAO.…”

“…18 Thus, the free energy dependence of protein stability as a function of osmolyte concentration can be predicted if one assumes that the transfer free energies of solvent exposed sidechain and backbone groups on the native and denatured states are additive. [18][19][20] Here, using computational methods, we assess the transfer free energies of the peptide backbone from water to two osmolyte solutions, 2M urea and 2M TMAO. The goal is to evaluate the peptide backbone transfer free energies as a function of chain length of oligoglycine peptides with capped end groups, and determine the extent to which the transfer free energies change in an additive manner with respect to chain length.…”