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

Abstract

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...

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

“…In this method, the parameters AGH*O and An are somewhat sensitive to the choice of k (Pace, 1986). Binding constants for the denaturation of proteins and peptide helices and from the study of denaturant interaction with model compounds are not in exact agreement, so the proper binding constants to use for urea and Gdn HCI are not clear (Pace, 1986;Makhatadze & Privalov, 1992;Scholtz et al, 1995, and references therein). Evidence for specific binding of denaturant molecules to protein is not very strong in any case.…”

Kosmotropes and chaotropes: modelling preferential exclusion, binding and aggregate stability

“…I). We stress at this point that the chaotropic action of urea is by no means universal: in a number of systems, such as the ultra-small solute methane [20,54], and proteins with variable hydrophobicity [49] or significant interactions between chain segments and the cosolvent molecules [52,53], its effect may in fact be inverted. However, because of its ubiquity as a solubilising agent for most binary systems, we continue to consider urea as a generic example of a strong chaotrope.…”

Denaturant-induced helix–coil transition of oligopeptides: theoretical and equilibrium studies of short oligopeptides C17 and AK16

“…For AK16, s 0 was 2.6-2.8, Z was 0.09-0.26 and y H0 was À22 000 (deg cm À2 dmol À1 ) when p¼1 and p¼2. In both cases, these s 0 values were greater than that (s 0 ¼1.39) reported by Scholtz et al 12 As shown in Figure 10, f H was calculated from the fitted curve, and the experimental data shown in Figure 8 were plotted against C for C17 and AK16. At a GuHCl concentration of 0 M, the proportion of the helix form of the peptide was 485%, which indicates that all of the residues in the peptide formed a helix, except for the residues located at the ends of the peptide.…”

“…According to Scholtz et al, 12 the value of y H0 for peptides is approximately À42 500 (deg cm À2 dmol À1 ). Initially, we attempted to fit the data to a y H0 value of À40 000 (deg cm À2 dmol À1 ); however, the simulation did not provide satisfactory results.…”

“…Using Schellman's theory, the effects of denaturants on helix-coil transitions have been studied by Scholtz et al, 12 who connected the thermodynamic solvation model derived by Schellman 10,11 to the Zimm and Bragg model for the analysis of the unfolding of peptide helices by urea. However, the method was phenomenological and was not consistently based on statistical physics.…”