Figure 3
Thermal denaturation of apoA-I WT, Milano and A164S proteins in HDL. (a) Lipid-bound (LB) WT, Milano and A164S proteins in HDL were scanned by SRCD spectroscopy (in the range of 190–260 nm) in the temperature range 25.1 °C to 95.1 °C, with increments of 1 °C followed by cooling to 25 °C (left panel). Spectra were analyzed to determine the temperature-dependent relative secondary structure distribution (alpha helix, beta strand, turns and unordered) of the proteins (right panel). (b) Native gel analysis was used to analyze the size distribution of the WT (W), Milano (M) and A164S (A) HDL particles before (start) and after (end) the thermal cycle procedure. Lipid-free proteins were included as controls (LF) in the analysis. 8 µg (Native PAGE) or 2.5 µg (SDS-PAGE) of protein was loaded per lane. The species obtained upon thermal denaturation were quantified and the amount of each species expressed as percentage with respect to the total signal (right panel). (c) The SRCD signal amplitude at 222 nm, which reflects the alpha helix content, was used to monitor the thermal unfolding process of the lipid-bound proteins (WT, Milano, A164S) as a function temperature. Data is shown as mean ± SD.
