The thermodynamics of solvent exchange

354

370

The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid-and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.denaturation ͉ hydrogen exchange ͉ osmolyte ͉ solvation B ecause of its fundamental importance, the study of protein molecular stability has engaged the attention of protein chemists for the last 100 years (1, 2). Experimental and theoretical investigations of protein stability have often focused on small-molecule osmolytes that can be used to modulate stability in vitro (3-6) and are used by virtually all organisms to counter biochemical stress in vivo (7). The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.It is a thermodynamic truism (20-22) that the concentration of destabilizing osmolytes must be enriched relative to water molecules in the vicinity of the protein surface that becomes newly exposed upon unfolding (preferential interaction). In a thermodynamic sense, denaturing osmolytes ''bind'' selectively to the increased surface of unfolded proteins and thus bias the folded/ unfolded equilibrium toward denaturation. Similarly, stabilizing osmolytes must be preferentially excluded from the protein surface. This is true independently of the physical mechanism of the ''binding'' or ''antibinding'' interaction. Thermodynamically based studies directed at the binding/antibinding problem have been designed to measure the transfer free energy of the various component groups of proteins between water and osmolyte solutions. Results show that the main-chain peptide group makes the largest contribution to the energetics, accounting for Ϸ80% of the effect of urea and of strong stabilizers such as trimethyl amine N-oxide (TMAO) (23). Similar determinations for guanidinium are not yet available, but it seems clear that guanidinium also favorably interacts with the peptide group (8,11,24).To search for the mechanistic basis of thermodynamic destabilization due to urea and guanidinium, we tested the possibility that they exert their denaturing effect through peptide group hydrogen bonding. This can be done experimentally by the straightforward measurement of acid-and base-catalyzed hydrogen exchange (HX) in a small-molecule peptide model. Osmolyte that is H-bonded to the peptide NH ...

Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

354

370

“…‡ The concept of exchange has been treated by Schellman in a set of classical papers (11)(12)(13)(14)(15)(16) in which he examined the interaction of proteins with weakly interacting ligands. Schellman (11)(12)(13) has treated the exchange reaction (Eq.…”

Section: Interaction Of Solvent Components With Protein Loci; Exchangementioning

confidence: 99%

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Timasheff

2002

627

663

Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water-protein-cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein-water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration. For the better part of a century, it has been common practice to modulate biochemical (biological) reactions by the addition to the aqueous solvent (dilute buffer) of compounds that were required at high concentration (ϳ0.5 M or higher) to exert their effect. For example, sucrose and glycerol were used to stabilize biological systems, whereas urea and guanidine hydrochloride were used to solubilize coagulated systems and to unfold (denature) proteins. The aim in the use of these additives, referred to as cosolvents, was to displace to the right or left the chemical equilibrium, Reactant º Product. Although this practice was widespread, a theoretical underpinning was lacking until the discovery by Wyman in 1948 of the phenomenon of linked functions (1) and his development of the linkage relationship equations (2, 3), which showed the necessary thermodynamic interdependence between the displacement of a chemical equilibrium and the change in binding of a ligand to the system during the course of the reaction. Complete understanding of the action of cosolvents in such modulation required the combination of the Wyman linkage relations with the multicomponent solution thermodynamics theory developed by Kirkwood and Goldberg (4), Stockmayer (5), and Scatchard (6).The basic Wyman linkage equation states that, at any ligand concentration, m L , the gradient of the equilibrium constant with respect to ligand activity is equal to the change in the binding of the ligand to the biological system during the course of the reaction (at constant temperature and pressure that will be maintained throughout):where K is the equilibrium constant of the reaction, a L is the activity of the ligand (a L ϭ m L ␥ L ), L Prod and L React are the bindings of the ligand to the two end states of the reaction, and m L and ␥ L are the molal concentration and activity coefficient of the ligand. [In our notation, the subscripts W, L, and P refer to water, ligand (cosolvent), and protein (macromolecule), respectively.] Tanford, in 1969 (7), showed that ...

“…(i) Does urea denature proteins by interacting with the peptide group (1)(2)(3) or by solubilizing nonpolar side chains, especially aromatic groups (4-6)? (ii) Can the urea dependence of the a-helix unfolding reaction be explained quantitatively by the popular models [binding-site model (7,8) and solventexchange model (9,10)] for the solvent denaturation of proteins? (iii) Can the urea-induced unfolding transition of the a-helix be used as a simple model system to discriminate between these thermodynamic models?…”

mentioning

confidence: 99%

Urea unfolding of peptide helices as a model for interpreting protein unfolding.

Scholtz

Barrick²,

York³

et al. 1995

171

173

To provide a model system for understanding how the unfolding of protein a-helices by urea contributes to protein denaturation, urea unfolding was measured for a homologous series of helical peptides with the repeating sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying from 14 to 50 residues. The dependence of the helix propagation parameter of the Zimm-Bragg model for helix-coil transition theory (s) on urea molarity ([urea]) was determined at 0°C with data for the entire set of peptides, and a linear dependence ofIn s on [urea] was found. The results were fitted by the binding-site model and by the solvent-exchange model for the interaction of urea with the peptides. Each of these thermodynamic models is able to describe the data quite well and we are not able to discern any difference between the ability of each model to fit the data. Thus a linear relation, In s = In so -(m/R7). [urea], fits the data for av-helix unfolding, just as others have found for protein unfolding. When the m value determined here for a-helix unfolding is multiplied by the number of helical residues in partly helical protein molecules, the resulting values agree within a factor of 2 with observed m values for these proteins. This result indicates that the interaction between urea and peptide groups accounts for a major part of the denaturing action of urea on proteins, as predicted earlier by some model studies with small molecules.We use helix-coil theory to examine how the a-helix to random coil transition depends on urea molarity for a homologous series of peptides. We address four specific questions. (i) Does urea denature proteins by interacting with the peptide group (1-3) or by solubilizing nonpolar side chains, especially aromatic groups (4-6)? (ii) Can the urea dependence of the a-helix unfolding reaction be explained quantitatively by the popular models [binding-site model (7, 8) and solventexchange model (9, 10)] for the solvent denaturation of proteins? (iii) Can the urea-induced unfolding transition of the a-helix be used as a simple model system to discriminate between these thermodynamic models? (iv) Can the dependence on urea molarity of native protein and molten globule unfolding be considered to result from the action of urea on protein a-helices and other elements of secondary structure?The observation that short, alanine-based peptides form monomeric helices in aqueous solution (11) has permitted the direct determination of the energetics of helix formation [see review by Scholtz and Baldwin (12)]. Thermally induced helix-coil transitions in these peptides have been shown by spectroscopy and calorimetry (13,14) to conform to helix-coil theory, which predicts that peptide helix unfolding is cooperative and multistate (15,16), and have provided estimates of Gibbs energies and enthalpies of helix unfolding (13, 14).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely t...