Comparison of the effect of fluorinated aliphatic and aromatic amino acids on stability at a solvent-exposed position of a globular protein

Abstract

ABSTRACT Rational protein engineering has often focused on increasing the thermal stability of proteins by incorporating stabilising mutations. Introducing fluorinated amino acids into proteins has been used to enhance protein stability. Free energy simulations of the immunoglobulin binding domain of streptococcal protein G in both the folded and unfolded states were performed to understand the detailed mechanism of the effect of fluorinated aromatic and aliphatic amino acid side chains on the stability of Leu → Hfl (hexafluoroleucine) and Phe → Pff (pentafluorophenylalanine) mutations at the solvent-exposed position of the globular protein. Our simulated free energy differences (ΔΔG) for the Leu → Hfl and Phe → Pff mutations are −0.43 ± 0.14 kcal/mol and −0.45 ± 0.20 kcal/mol, respectively. These calculated free energy differences are in excellent agreement with the corresponding reported experimental values. Surprisingly, a significant contribution to the increased stability of the Hfl-containing fluorinated protein comes from van der Waals interactions and, in the case of Pff, electrostatic interactions. The simulated quantitative agreement with experimental values validates the free energy simulation method to elucidate the effect of fluorination on protein stability. The implications of the simulation results for our understanding of the effect of fluorination on protein stability are discussed.