Fig. 2: Stoichiometry of Brichos binding to Aβ42 fibrils is low, and is predictive of secondary nucleation inhibition.
From: Structural defects in amyloid-β fibrils drive secondary nucleation
a Illustration of MDS experiment. Mixtures of Alexa-488-labelled proSP-C Brichos chaperone and 24 μM Aβ42 fibrils are introduced to the centre of a microfluidic channel. Free Brichos diffuses rapidly toward the channel edges, while fibril-bound Brichos displays slower lateral diffusion and remains therefore localised in the centre of the channel. Top schematic reprinted ("Adapted” or “in part'') with permission from ref. 40. Copyright 2016 American Chemical Society. b The proportion of Brichos bound to fibrils, inferred by mathematical analysis of the diffusion profiles, decreases with increasing total concentration C of proSP-C Brichos. By C = 1.5 μM it is already below the reliable detection limit of 10%; measurements at higher C would therefore be uninformative. Sample sizes consisted of 2–3 repeats per condition. c Converting these proportions to free (x axis) and bound (y axis) Brichos concentrations enables global regression of a Langmuir adsorption isotherm to both datasets. This yields KD = 314 ± 85 nM and stoichiometry of 1 binding site per ca. 143 ± 16 Aβ42 monomers (R2 = 0.96). Sample sizes consisted of 2–3 repeats per condition. d Kinetic aggregation profiles of 3 μM Aβ42 with 1% seed and increasing total concentrations C of Brichos. Lines indicate global fits using a rate law for Aβ42 with an effective secondary nucleation rate constant k2(C) (see Methods “Kinetic modelling”; adjusted MSE = 0.00052). The sample size consisted of 1 repeat per condition. e Decrease in k2(C) with increasing C (red data, left axis) and fractional fibril coverage by Brichos (blue data, right axis). Superimposed are model projections using parameters determined in c and d selective binding to secondary nucleation sites, k2(C)/k2(0) = 1/(1 + C/KD) (red line; R2 = 0.99), and Langmuir isotherm (blue line; R2 = 0.97). Both closely match the data. Conditions: 20 mM sodium phosphate, 0.2 mM EDTA, pH 8.0 and 21 ∘C.
