Thermodynamic Consequences of Incorporating 4-Substituted Proline Derivatives into a Small Helical Protein

Abstract

Although proline residues are incompatible with an alpha-helix conformation, they fit well into the N-terminal end of alpha-helices. Proline can form either a C(gamma)-exo ring pucker or a C(gamma)-endo ring pucker. An electron-withdrawing substituent on the 4R position of proline favors an exo ring pucker while an endo ring pucker is preferred if the substituent is on the 4S position due to stereoelectronic effects. The villin headpiece subdomain (HP36) is a small helical protein composed of three alpha-helices and contains a proline residue (Pro62) at the N-terminus of its C-terminal alpha-helix. Pro62 has a C(gamma)-exo ring pucker and forms an aromatic-proline interaction, with Trp64 in the native structure. This work reports the use of 4-substituted proline derivatives, including (2S,4R)-4-hydroxyproline (Hyp), (2S,4R)-4-fluoroproline (Flp), (2S,4R)-4-methoxyproline (Mop), (2S,4S)-4-hydroxyproline (hyp), (2S,4S)-4-fluoroproline (flp), and (2S,4S)-4-methoxyproline (mop), to replace Pro62 and study how the pucker conformation affects the proline-aromatic interaction and the stability of HP36. CD and NMR measurements indicate that all of the HP36 variants incorporated with proline derivatives maintain a structure similar to that of the wild type. Thermal unfolding and urea-induced denaturation measurements have shown that all of the mutants with the exception of the one with the flp substitution are less stable than the wild type. Our results reveal that, upon the replacement of Pro62 to proline derivatives, not only do stereoelectronic effects influence the aromatic-proline interaction but the steric and hydrophobic effects induced by the substituents also play an important role in modulating the stability of HP36.