Inductive effects on the energetics of prolyl peptide bond isomerization: Implications for collagen folding and stability

The hydroxylation of proline residues in collagen increases 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-[beta,gamma-C-13]D,L-proline methyl ester (1); N-acetyl-4(R)-hydroxy-L-proline [C-13]methyl ester (2); and N-acetyl-4(R)-fluoro-L-proline methyl ester (3). The pK(a)'s of the nitrogen atom in the parent amino acids decrease in the following 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 following order 1 > 2 > 3. At 37 degrees 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 degrees C in water or dioxane equilibrium concentrations of the trans isomer increase in the order: 1 < 2 < 3. Inductive effects may therefore accelerate the;folding and enhance the stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.