Charge Regulation during Amyloid Formation of α-Synuclein

Electrostatic interactions play crucial roles in protein function. Measuring pK_a value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pK_a value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pK_a^ave value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pK_a^ave values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein.