Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability

Abstract

The hydroxylation of proline residues in collagen enhances the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res.1994, 44, 262-269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[β,γ-(13)C]D,L-proline methylester (1); N-acetyl-4(R)-hydroxy-L-proline [(13)C]methylester (2); and N-acetyl-4(R)-fluoro-L-proline methylester (3). The pK(a)'s of the nitrogen atom in the parent amino acids decrease in the order: proline (10.8) > 4(R)-hydroxy-L-proline (9.68) > 4(R)-fluoro-L-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the order: 1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater for 3 than 1. Each of these results is consistent with the traditional picture of amide resonance coupled with an inductive effect that results in a higher bond order in the amide C=O bond and a lower bond order in the amide C-N bond. Further, at 37 °C in water or dioxane equilibrium concentrations of the trans isomer increase in the order: 1 < 2 < 3. Inductive effects may therefore have a significant impact on the folding and stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.

Figure 1

(a) Amide I vibrational modes of 1 (υ_max = 1658.99 cm⁻¹), 2 (1660.92 cm⁻¹), and 3 (1664.78 cm⁻¹) in dioxane. (b) Amide I vibrational mode of 1 (υ_max = 1608.10 cm⁻¹), 2 (1613.08 cm⁻¹), and 3 (1616.02 cm⁻¹) in D₂O.

Figure 2

(a) Eyring plots of the cis-to-trans isomerization of 1 (○) and 3 (◇), and the trans-to-cis isomerization of 1 (●) and 3 (◆) in dioxane. (b) Eyring plots of the cis-to-trans isomerization of 1 (○) and 2 (□), and the trans-to-cis isomerization of 1 (●) and 2 (■) in 0.10 M sodium phosphate buffer, pH 7.2.

Figure 3

(a) Van’t Hoff plots of the cis-to-trans isomerization of 1 (○), 2 (□), and 3 (◇) in dioxane. (b) Van’t Hoff plots of the cis-to-trans isomerization of 1 (○) and 2 (□) in 0.10 M sodium phosphate buffer, pH 7.2.

Chart 1