Lesniewski_2026_Chem.Biol.Interact__112082

Structural characterization of proteins is essential for understanding their function, stability, and role in disease. Butyrylcholinesterase (BChE), expressed in the brain and localized within amyloid-beta plaques, may generate an extended 69-amino acid, cysteine-rich signal peptide capable of self-aggregation and iron binding. In this study, we investigated the structure and aggregation of the BChE signal peptide using far-UV circular dichroism (CD) spectroscopy combined with in silico modelling via AlphaFold and coarse-grained replica-exchange molecular dynamics simulations with the UNRES force field. CD spectra revealed a predominance of beta-sheet structure, with additional alpha-helices and statistical coil contributions. These experimental results were qualitatively consistent with AlphaFold and UNRES predictions, which revealed mixed alpha and beta secondary structure in oligomeric assemblies. UNRES also predicted a substantial statistical coil content. Thermal CD analyses revealed marked differences in stability between variants. The p.C31Y signal peptide showed no significant spectral changes after heating, indicating exceptional resistance to thermal unfolding. In contrast, the wild-type peptide exhibited partial structural change under identical conditions. To refine structural interpretation, three short fragments of the BChE signal sequence (P1-P3) were analyzed by far-UV CD. P1 and P3 were largely disordered, whereas P2 displayed mixed secondary structure. An equimolar mixture of all three fragments produced an approximately additive CD spectrum, suggesting minimal conformational interference. Notably, P1 and P2 improved the solubility of the hydrophobic P3 fragment. Overall, these findings support a stable, aggregation-prone architecture for the extended BChE signal peptide and provide molecular insights into its potential role in promoting Alzheimer's pathology.