Browsing by Subject "Free‐energy simulation"

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    Stabilization of a protein by a single halogen‐based aromatic amplifier
    (Wiley, 2025) El Hage, Krystel; Dhayalan, Balamurugan; Chen, Yen-Shan; Phillips, Nelson B.; Whittaker, Jonathan; Carr, Kelley; Whittaker, Linda; Phillips, Manijeh H.; Ismail-Beigi, Faramarz; Meuwly, Markus; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    The utility of halogenation in protein design is investigated by a combination of quantitative atomistic simulations and experiment. Application to insulin is of complementary basic and translational interest. In a singly halogenated aromatic ring, regiospecific inductive effects were predicted to modulate multiple surrounding electrostatic (weakly polar) interactions, thereby amplifying changes in thermodynamic stability. In accordance with the simulations, we demonstrated stabilization of insulin by single halogen atoms at the ortho position of an invariant phenylalanine (2-F-PheB24, 2-Cl-PheB24, and 2-Br-PheB24; ΔΔGu = -0.5 to -1.0 kcal/mol) located at the edge of a protein crevice; corresponding meta and para substitutions had negligible effects. Although receptor-binding affinities were generally decreased (in accordance with packing of the native Phe at the hormone-receptor interface), the ortho-analogs retained biological activity in mammalian cells and in a rat model of diabetes mellitus. Further, the ortho-modified analogs exhibited enhanced resistance to fibrillation above room temperature in two distinct assays of physical stability. Regiospecific halo-aromatic stabilization may thus augment the shelf life of pharmaceutical insulin formulations under real-world conditions. This approach, extending principles of medicinal chemistry, promises to apply to a broad range of therapeutic proteins and vaccines whose biophysical stabilization would enhance accessibility in the developing world.