Abstract

The utility of halogenation in protein design is investigated by a combination of quantitative atomistic simulations and experiment. The approach is applied to insulin, a small, therapeutically relevant domain amenable to simulation and semi-synthesis. In a singly halogenated aromatic ring, the simulations predicted regiospecific inductive effects to modulate multiple surrounding electrostatic (weakly polar) interactions, thereby amplifying changes in thermodynamic stability. In accordance with the simulations, stabilization of insulin is demonstrated by single halogen atoms at the ortho position of an invariant phenylalanine (2-F-PheB24, 2-Cl-PheB24 and 2-Br-PheB24; {Delta}{Delta}Gu = -0.5 to -1.0 kcal/mol) located at the edge of a protein crevice. Corresponding meta and para substitutions have negligible effects. The ortho-modified insulin analogs exhibit enhanced resistance to fibrillation above room temperature and retain biological activity in mammalian cells and in a rat model of diabetes mellitus. Consequently, halogen-based stabilization of insulin and other therapeutic proteins may provide a biophysical strategy to circumvent the requirement for a distribution "cold chain" in the developing world and enhance the shelf life of pharmaceutical formulations.