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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\n                    <jats:italic>ortho<\/jats:italic>\n                    position of an invariant phenylalanine (2\u2010F\u2010Phe\n                    <jats:sup>B24<\/jats:sup>\n                    , 2\u2010Cl\u2010Phe\n                    <jats:sup>B24<\/jats:sup>\n                    , and 2\u2010Br\u2010Phe\n                    <jats:sup>B24<\/jats:sup>\n                    ; \u0394\u0394\n                    <jats:italic>G<\/jats:italic>\n                    <jats:sub>u<\/jats:sub>\n                    \u2009=\u2009\u22120.5 to \u22121.0\u2009kcal\/mol) located at the edge of a protein crevice; corresponding\n                    <jats:italic>meta<\/jats:italic>\n                    and\n                    <jats:italic>para<\/jats:italic>\n                    substitutions had negligible effects. Although receptor\u2010binding affinities were generally decreased (in accordance with packing of the native Phe at the hormone\u2010receptor interface), the\n                    <jats:italic>ortho<\/jats:italic>\n                    \u2010analogs retained biological activity in mammalian cells and in a rat model of diabetes mellitus. Further, the\n                    <jats:italic>ortho<\/jats:italic>\n                    \u2010modified analogs exhibited enhanced resistance to fibrillation above room temperature in two distinct assays of physical stability. Regiospecific halo\u2010aromatic stabilization may thus augment the shelf life of pharmaceutical insulin formulations under real\u2010world 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.\n                  <\/jats:p>","DOI":"10.1002\/pro.70064","type":"journal-article","created":{"date-parts":[[2025,2,19]],"date-time":"2025-02-19T08:04:26Z","timestamp":1739952266000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Stabilization of a protein by a single halogen\u2010based aromatic amplifier"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4837-3888","authenticated-orcid":false,"given":"Krystel","family":"El Hage","sequence":"first","affiliation":[{"name":"Department of Chemistry University of Basel  Basel Switzerland"},{"name":"Qubit Pharmaceuticals  Paris 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