Fig. 4: Disruption of the NT-IXI motif results in reduced chaperone activity in both native-like globular and fibril states.

a Chaperone assays against unfolding lysozyme client conducted with varying molar ratios of αB-AXA in the native-like globular state (blue traces), as monitored by light scattering (turbidity) at 360 nm at 37° C as a function of time (hours). Lysozyme-only (10 μM, light gray trace) and αB-wt (40 μM, dark gray trace) were run as positive and negative controls, respectively. αB-AXA prepared at 1:1, 2:1 and 4:1 (chaperone:client) molar ratios. Data are normalized to lysozyme-only conditions. Each condition contained 3 biological replicates, conducted with 1-3 technical replicates (n = 7–9). Standard error of the mean (s.e.m.) shown with semi-transparent shading. b Percent protection versus untreated lysozyme summarized in box plot representation and colored as in panel a. Statistical significance (p = 0.0079; **), calculated from all replicates using a two-tailed t-test. c, d Representative electron micrographs of end-state reactions for the 4:1(chaperone:client) ratios obtained for αB-AXA and αB-wt, respectively. Scale bar = 250 nm. αB-AXA chaperone/client complexes appear as more irregular and elongated particles (dotted outlines), as compared to αB-wt under these same conditions (see also, Supplemental Fig. 9). e Chaperone assay of αB-AXA in the fibril state conducted at 25° C (orange-yellow traces). Lysozyme-only (10  μM, light gray trace) and αB-wt (40 μM, dark gray trace) were run as positive and negative controls, respectively. αB-AXA prepared at 0.1:1, 1:1 and 10:1 (chaperone:client) molar ratios. Due to limited sample, each condition contained 1-3 biological replicates, conducted with 1-6 technical replicates (n = 6–9). Standard error of the mean (s.e.m.) shown with semi-transparent shading. f Percent protection summarized in box plot representation and colored as in panel (e). Statistical significance (p = 5.4 × 10⁻⁵; ***), calculated from all replicates using a two-tailed t-test. g, h. Representative electron micrographs of end-state reactions for the 10:1 and 1:1 (chaperone:client) ratios obtained for αB-AXA fibril state, respectively. Scale bar = 250 and 500 nm in panels (g and h), respectively. Increasing client ratios correlate with an increase tangling and/or co-aggregation of αB-AXA fibrils. Box plots show the central 50% of the data, spanning from the first quartile (Q1) to the third quartile (Q3), with a line at the median. Whiskers extend to the minimum and maximum values. Micrographs are representative of at least 3 independent observations. Source data are provided in a Source Data file.