pH Dependence of Amyloid‐β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes

Abstract Central to Alzheimer's disease (AD) is the assembly of the amyloid‐beta peptide (Aβ) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aβ’s isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid‐point or pK a of 7.0, close to the pK a of Aβ histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aβ, in particular histidine protonation, has little impact on this stage of Aβ assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aβ into toxic oligomers and fibrils.


Supplemental Methods:
Aβ Peptides Synthetic Aβ40 and Aβ42 were purchased from EZBiolab Inc in a lyophilized form.Aβ peptides were synthesized by F-moc (N-(9-fluorenyl)methoxycarbonyl) chemistry, and were purified with reverse-phase high performance liquid chromatography.Sequence was verified by mass spectrometry.Unless otherwise stated all other solvents and reagents were purchased from Sigma-Aldrich.

Monomeric Aβ by size-exclusion chromatography (SEC)
The lyophilized Aβ peptides were solubilized in ultra-high quality (UHQ) water to 0.7 mg/ml at pH 10 and left at 4°C for 2 h.A solution was then centrifuged for 15 min at 20,000 g at 4°C, to remove high molecular weight aggregates.Monomeric Aβ was isolated using AKTA FPLC with a Superdex 75 10/300 GL column (GE Healthcare) at a flow rate of 0.5 mL•min −1 .The column was pre-equilibrated with 5 mM sodium phosphate buffer (pH 6.0).The Aβ peptides concentration were determined using ε280=1280•cm −1 mol −1 .Negative-stain TEM and thioflavin T fluorescence assay confirmed that SEC-purified Aβ peptides were seed-free.Monomeric samples were used directly after SEC.

Fibril growth Assay
The kinetics of amyloid fibril formation were monitored by a fibril-specific fluorescent dye, thioflavin T (ThT).Monomeric Aβ peptides (5 μM) and ThT (10 μM) were placed in a 96-well plate in NaCl (50 mM) and sodium phosphate (50 mM) buffer from pH 6.0 to pH 8.0.All fibril formation was performed under quiescent conditions.Experiments were recorded using a BMG-Omega FLUOstar microplate reader (BMG Labtech, Aylesbury, UK), with excitation and emission filters at 440 nm and 490 nm respectively.
In the seeded aggregation assay 10% fibrils, 0.5 μM monomer equivalent, were added.Aβ fibril seeds were obtained by incubating 5 μM Aβ peptides in NaCl (50 mM) and sodium phosphate (50 mM) buffer from pH 6.0 to pH 8.0 at 30 °C for 4 days.Samples also contained DMSO 0.5% (v/v).The formation of Aβ fibrils was confirmed by ThT fluorescent assay and TEM imaging.

Fitting fibril growth curves
The empirical kinetic values for t1/2 and tlag were extracted from the data by fitting the fibril growth curve to the equation below [14] Where Y is the ThT fluorescence intensity, x is the time and xo is the time at which the ThT fluorescence has reached half maximal intensity referred to as t1/2.The initial and final fluorescence signals, yi and γf, were used to determine the time at 10% and 90% ThT maximal for a measure of the growth time, tgrowth.

Analysis of Aβ aggregation kinetics
Global kinetic analysis of Aβ peptides aggregation were analyzed using the AmyloFit platform.
[10] The integrated rate law which based on Michaelis-Menten-Like kinetics for Aβ aggregation traces is: where the additional coefficients are functions of κ and λ: which are two combinations of the microscopic rate constants of: where m(0) is the initial monomer concentration, M(0) is initial fibril mass concentration, M(∞) is mass concentration of fibrils at equilibrium, P(0) is the initial aggregate concentration and P(∞) is the aggregates concentration of at equilibrium.The microscopic rate constants kn, k2, k+ are the rate constants for primary nucleation, secondary nucleation, and elongation respectively.The exponents nc and n2 are the reaction orders for primary and secondary nucleation respectively, and KM is the saturation constant for secondary nucleation.
Using predetermined values for KM; and initial k+kn and k+k2 values, the experimental macro kinetic traces were globally fitting to the integrated rate law over the range of pH's.The microscopic rate constants kn; k+; and k2 values were fitted to the fibril growth curves at pH 6.0, the other kinetic traces at increasing pH's, were then fitted in three scenarios in which only one of the rate constants were permitted to vary, while the other two remain constant.This approach has been used to investigate how increasing concentrations of an inhibitor of fibril formation effect individual microrate constants.[15]In a similar way raising the pH (concentration of hydroxide ions) also inhibits fibril formation.

Transmission electron microscopy
A fibril samples were generated with the same protocol for A fibril growth assay but without ThT addition.5 μL aliquot of sample were added onto glow discharged carbon-coated copper grids (Agar Scientific, Essex, UK) by the droplet method then blotted after 90 seconds and rinsed with ddH20 at room temperature.Glow discharge was carried out using the Pelco EasiGlow glow discharge unit.to negatively stain the assemblies a 5 μL uranyl acetate (2 % w/v) was used, then blotted and rinsed after 10 seconds.Images were recorded by a JEOL JEM-1230 electron microscope (JEOL, Ltd., Japan) at 80,000 magnifications, operated at 120 kV, paired with a 2k Morada CCD camera and corresponding Olympus iTEM software package (Olympus Europa, UK).Node-to-node fibril distance was measured using image-J software.

Figure S6 .Figure S7 .
Figure S6.pH effects primary nucleation process of Aβ40 aggregate.(A-C) Kinetics profiles of Aβ40 (5 μM) at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6 7.8 and 8.0, from left (black, pH 6.0) to right (purple, pH 8.0).The solid lines represent global fits of the kinetic traces when only primary nucleation (A), secondary nucleation (B) and fibril elongation (C) rate constants are altered to fit pH dependent traces.(D) Change in primary nucleation rate constants (kn) versus pH, derived from global fits in Figure 2A.(E) Schemes of the microscopic steps for primary nucleation, secondary nucleation and fibril elongation.Error bars represent standard errors of the mean (SEM) of four replicates.

Figure S8 .
Figure S8.TEM fibril images produced at pH 6.0, 7.0 and 8.0 for Aβ40.Scale bars: 500 nm, right panel: 50 nm.pH has negligible effect on the morphology of Aβ40.Typical node-to-node twist periodicity 141 nm.