Journal: bioRxiv

Article Title: Evolutionarily Conserved Amyloid Aggregation in the PACAP Peptide Family Is Controlled by Heparin-Sensitive Lys/Arg Gatekeeper Residues

doi: 10.64898/2026.05.04.722589

Figure Lengend Snippet: (A) General overview of the experimental setup used to monitor amyloid formation. Peptides were applied at a final concentration of 0.35 mM. (B) CD spectra were recorded for the initial peptide states prior to incubation. The samples were then divided into two groups and incubated for 72 hours under Thioflavin-T fluorescence monitoring, either in the presence of enoxaparin sodium (2.125 mg mL⁻¹) or in the absence of heparin. The end states were subsequently analysed again by CD spectroscopy. For the rest of screening results see the supporting material. (PACAP-38 – S ; VIP – S ; PRP – S ; PHI – S ; GHRH – S ; SCT – S ) (C) The deconvolution of the collected CD curves yields 7 components: two slightly different unfolded components; one helical component and two-two typical and atypical β-sheeted components. For the in-depth review of the CD deconvolution see S . (D) Overview of the measured biophysical characteristics of the end states extended with the overall charge of the peptides across the examined pH range. ThT values represent the fold increase in fluorescence relative to the corresponding blank, whereas CD values indicate the relative proportions of unfolded, helical, and β-sheet components determined by spectral deconvolution. ThT values and component triplets are highlighted in bold in cases where amyloid fibrils were confirmed by AFM. Overall charges: heparin carries a negative charge under all investigated pH values. The sulfonate groups of polyanionic heparin remain fully deprotonated between pH 2 and 10. In contrast, its carboxyl groups are predominantly protonated at low pH, present in roughly equal protonated and deprotonated proportions near their pK a and become fully deprotonated above pH 7. Approximate net charges of the peptides were calculated using generalized pKₐ values. (E) AFM micrographs recorded from the end states of the incubated samples after 20-fold dilution reveal nanoscale amyloid filaments with varying morphologies ( S -6 /G shows additional micrographs ) , often accompanied by amorphous aggregates. PACAP (pH 6 – /C ) and GHRH (pH 4 – S /G ) display clear polymorphism within the same sample. In the case of SCT incubated at pH 5, the observed structures are not typical amyloid fibrils; however, they are too structured to be considered mere precipitation.

Article Snippet: Thioflavin T (ThT) stock solutions (Acros Organics, Thermo Fisher Scientific) were prepared in distilled water, with the pH adjusted between 2 and 10 in increments of one pH unit.

Techniques: Concentration Assay, Circular Dichroism, Incubation, Fluorescence