Enthalpy of helix-coil transition: missing link in rationalizing the thermodynamics of helix-forming propensities of the amino acid residues

Abstract

It is known that different amino acid residues have effects on the thermodynamic stability of an alpha-helix. The underlying mechanism for the thermodynamic helical propensity is not well understood. The major accepted hypothesis is the difference in the side-chain configurational entropy loss upon helix formation. However, the changes in the side-chain configurational entropy explain only part of the thermodynamic helical propensity, thus implying that there must be a difference in the enthalpy of helix-coil transition for different residues. This work provides an experimental test to this hypothesis. Direct calorimetric measurements of folding of a model host peptide in which the helix formation is induced by metal binding is applied to a wide range of residue types, both naturally occurring and nonnatural, at the guest site. Based on the calorimetric results for 12 peptides, it was found that indeed there is a difference in the enthalpy of helix-coil transition for different amino acid residues, and simple empirical rules that define these differences are presented. The obtained difference in the enthalpies of helix-coil transition complement the differences in configurational entropies and provide the complete thermodynamic characterization of the helix-forming tendencies.

Fig. 1.

Structure of the model peptide and of the residues incorporated at the guest position. (A) Cartoon representation of the structure of the P2A peptide in the presence of La³⁺ [Protein Data Bank (PDB) ID code 1NKF, ref. 24]. The side chain of Ala-14 is shown as a green sphere. (B) Ball-and-stick models of the amino acid residues that were used in this study.

Fig. 2.

Comparison of the ellipticities of the P2X peptides in the absence of La³⁺ with the ellipticities of peptide models used previously by Baldwin and coworkers (29, 30) and Kallenbach and coworkers (12, 31). Correlation coefficients are 0.88 and 0.76, respectively.

Fig. 3.

Temperature dependence of the fraction helix of selected P2X peptides in the absence of La³⁺ ions. □, P2G; ○, P2A; ⋄, P2N; ▵, P2V; ▿, P2B; , P2T. The lines through the points are shown only to guide the eye.

Fig. 4.

Comparison of helix propensity scales. (A) Comparison of the apparent thermodynamic helix propensity, ΔG_o(P2X), calculated by using experimentally defined parameters according to Eq. 5, , or using (○) and (▵) computed by agadir (see Materials and Methods for details). The dashed lines are the linear fits with the slopes of 1.02 and 1.05, and correlation coefficients of 0.990 and 0.991, respectively. (B) Comparison of the thermodynamic helix propensity scale obtained from La³⁺ binding to P2X peptides (Eq. 6), ΔΔG_o(P2X), with the unified helix propensity scale of Pace and Scholtz (35), ΔΔG_P&S (○). The ΔΔG data for nonnatural amino acids (□) are from ref. . The correlation coefficient is 0.92 and the slope is 0.9. Solid lines on A and B have a slope of 1.

Fig. 5.

Enthalpies obtained from ITC experiments. (A) The temperature dependence of the experimental enthalpies, ΔH_cal, that accompany La³⁺ binding to P1 and selected P2X peptides. (B) The temperature dependence of the enthalpies of helix–coil transition, Δhα, for the selected peptides. □, P2G; ○, P2A; ⋄, P2N; ▵, P2V; ▿, P2B; , P1.

Fig. 6.

Comparison of the enthalpy of helix formation Δhα obtained from different peptides by using Eq. 3. Horizontal lines are drawn at -0.9 kcal/mol (type I), -0.6 kcal/mol (type II), and -0.4 kcal/mol (type III). For comparison, the values of Δhα calculated by using (○) and (▵) computed by agadir (see Materials and Methods for details) are also shown.

Fig. 7.

Comparison of two different helical propensity scales, ΔΔG_P&S (gray bars) and ΔΔG_o(P2X) (black bars), with the change in configurational entropy upon helix–coil transition. T·ΔΔS only (blue bars), or with the sum of T·ΔΔS and the enthalpy of helix formation ΔΔhα (red bars). The configurational entropy changes upon helix–coil transition are taken from Blaber et al. (16), with the exception of those for nonnatural amino acids B and J, which were taken from Creamer and Rose (14). All parameters are calculated relative to A.