Figure 4
Structural transition of apoA-I WT, Milano and A164S proteins in lipid-binding and HDL formation. (a) Lipid Clearance Assay. Lipid-free apoA-I WT, Milano and A164S proteins were incubated with multilamellar DMPC vesicles with a 1:100 lipid to protein molar ratio and the decrease of the absorbance at 325 nm was measured every 10 s for 10 min. Experimental points were fitted to one-way decay of non-linear regression and the rates of lipid binding (t1/2) were calculated. Data shown are the mean ± SEM of 3 independent experiments carried out in triplicate. Significance is calculated according to one-way ANOVA (*p < 0.05). (b) Lipid-free apoA-I WT, Milano and A164S proteins were mixed with DMPC phospholipids (1:100 molar ratio) and scanned by SRCD spectroscopy (in the range of 190–260 nm) at the indicated times of incubation. Secondary structure distribution (alpha helix, beta strand, turns and unordered) of the proteins was estimated by deconvolution of the obtained spectra and plotted as a function of time (top panel). Native gel analysis was used to analyze formation and size distribution of the WT, Milano and A164S HDL particles during the time-dependent lipidation process (bottom panel). Lipid-free (LF) proteins were included for comparison. Red arrows indicate delayed increases in alpha helix secondary structure content. 1.5 µg of protein was loaded per lane. Data is shown as mean ± SD.
