Serial femtosecond crystallography reveals the pH-driven allosteric mechanism of hexamer glargine

Insulin glargine is formulated at acidic pH but acts after transferring to near-neutral tissue, where its prolonged effect is commonly attributed to isoelectric depot formation. However, the structural pathway linking precipitation to delayed release has remained unresolved. Here we combine ambient-temperature serial femtosecond crystallography, solution biophysics, and multiscale network analyses to define the pH-dependent conformational landscape of hexameric glargine across pH 8.4, 7.3, 6.4, and 5.1. We resolve full hexameric glargine structures and identify a previously unreported, pH-coupled lattice transition from P12 1 1 (near-neutral) to R3:H (acidic), accompanied by redistribution from compact phenolic R f 6 -state assemblies to more plastic yet structurally coherent TR f /T 3 R f 3 states. This transition is accompanied by B-chain N-terminal unpeeling, phenol-pocket collapse, hydration loss, and electrostatic rewiring, and is mirrored in solution by oligomeric heterogeneity, Raman amide-I broadening, reduced thermal stability, and a blue-shifted intrinsic fluorescence maximum. Multiscale analyses further indicate that acidification does not create a new dynamical regime but reweighs pre-existing collective modes along a continuous free-energy landscape. These results support a revised mechanism in which isoelectric precipitation and delayed dissociation are mechanistically coupled through structurally organized molten-like intermediate states, linking glargine pharmacology to intrinsic allosteric redistribution within the hexamer. These findings establish a structural blueprint for benchmarking biosimilar glargine and for engineering next-generation basal insulins by tuning allosteric plasticity and intermediate-state stability.