pH-Resolved SAXS Analysis of the Solution Assembly of Murine Amelogenin Variants

Amelogenin is an intrinsically disordered enamel matrix protein whose pH-dependent self-assembly is central to enamel biomineralization. Although AMELX-associated mutations and engineered variants are known to alter enamel structure, how these sequence changes affect amelogenin solution-state ensembles and assembly pathways remains unclear. Here, we combine small-angle X-ray scattering (SAXS) with monomeric replica exchange molecular dynamics (REMD) simulations to characterize nonphosphorylated murine amelogenin (rM179), Ser16-phosphorylated amelogenin (pS16), the amelogenin point mutants P41T and T21I, and the amelogenin deletion variants H3 (minus H67, H68, and H69) and Delta (minus A166 - D179 at C-terminal) at pH 3, 6, and 8. SAXS data shows that all variants remain disordered at pH 3 but differ in compaction and degree of self-association: H3 samples a more compact ensemble, Delta exhibits expansion or weak self-association, and T21I forms ribbon-like aggregates. REMD provides monomer-level context for these SAXS trends, predicting variant-dependent differences in conformational dimensions and transient structural propensities. At pH 6, pS16 and P41T favor smaller, more dynamic low-order assemblies, whereas T21I and Delta promote larger self-associated species; H3 occupies an intermediate regime. At pH 8, most variants form nanosphere-like assemblies with sequence-dependent size and heterogeneity, while the soluble Delta fraction lacks a well-defined nanosphere population. These results show that phosphorylation, point mutation, histidine deletion, and C-terminal truncation redistribute amelogenin among distinct pH-dependent assembly states, linking sequence variation to enamel-relevant self-assembly behavior.