Fig 2: Deuterium uptake of selected ApoA-I peptides. Individual deuterium uptake plots for ApoA-I were the amino acidic substitutions led to a decreased structural stability compared with WT rHDL state (blue trace). Deuterium uptake for peptides from the lipid-free WT protein (black trace) is also reported as comparison. Amino acidic substitutions are indicated with a red X in the sequence of relevant plots. Data shown are the mean ± SD. N = 2. Mass envelopes, mass, and retention time difference for point mutations can be found in the supplementary information file (supplemental Fig. S5).

Fig 3: Conformation and stability of ApoA-I amyloidogenic variants in 8.4-nm rHDLs. (A) SRCD spectroscopy analysis of ApoA-I variants in 8.4-nm rHDL particles, in the presence or absence of FC. SRCD spectra (upper panels) were deconvoluted with the CONTINLL algorithm for the estimation of the ApoA-I proteins' secondary structure (lower panels). (B) Thermal stability of ApoA-I variants in 8.4-nm rHDL particles, in the presence or absence of FC. The unfolding curves (left panels) were obtained by monitoring the CD signal amplitude at 222 nm as a function of temperature, and Tm (right panels) were calculated by fitting the experimental data with a biphasic nonlinear regression. Data shown are the mean ± SEM, and significance is calculated according to (A) two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ##P < 0.01, ###P < 0.001 ####P < 0.0001 for L75P and L174S rHDL, respectively, as compared with WT rHDL, or (B) 1-way ANOVA (*P < 0.05, **P < 0.01). Data shown are the mean ± SEM. N = 3.

Fig 4: Hydrogen-deuterium exchange mass spectrometry reveals regions with high flexibility in 8.4-nm rHDLs. Differential HDX heatmap of (A) lipid-bound vs lipid-free states for WT ApoA-I, (B) L75P versus WT in 8.4-nm POPC particles, and (C) L174S versus WT in 8.4-nm POPC particles. HDX peptide coverage is shown by the bars above each heatmap. The heatmaps show the deuterium uptake at the different timepoints (30 s, 300 s, 3,000 s, and 9,000 s). Cold color = slower exchange; warm color = faster exchange. The red dashed box (top) surrounding 115–159 sequence denotes the region where bimodal HDX kinetics (EX1) could be observed for the rHDL particles. Amino acidic substitutions are indicated with a black X.

Fig 5: Cholesterol efflux ability of reconstituted HDLs of different sizes. 9.6- and 8.4-nm reconstituted HDLs (rHDLs) were produced by incubating recombinant ApoA-I amyloidogenic variants with POPC, in the presence or absence of cholesterol, and tested for their ability to mediate cholesterol efflux from J744 macrophages, in dose-response experiments. The experimental data (upper panel) were fitted, and Kd (middle panel) and Bmax (lower panel) were calculated according to Michaelis-Menten equation. Data shown are the mean ± SEM, and significance is calculated according to two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ##P < 0.01, ###P < 0.001 for L75P and L174S rHDL, respectively, as compared with WT rHDL). N = 4–5.