Figure 2

pCH1 characterization. (A) – Analytical SEC profiles of the monomeric mutant of 14-3-3ζ and the 14-3-3σ fusion with HSPB6 phosphopeptide expressed in the absence (CH1) or in the presence (pCH1) of PKA, obtained using a calibrated Superdex 200 10/300 Increase column (GE Healthcare). Elution profiles were followed at 280 nm and normalized to absorbance at the peak maxima. Average hydrodynamic radii corresponding to peak maxima obtained from column calibration are indicated. Peaks I and II of the CH1 profile are marked. Inset shows the migration of CH1 (1), CH1 co-expressed (2) or in vitro phosphorylated (3) by PKA, or pCH1 in vitro dephosphorylated by alkaline phosphatase (4) during native gel-electrophoresis. (B) – Heating of 14-3-3σ∆C (1.5 µM), unphosphorylated CH1 (5 µM) or phosphorylated CH1 (1–5 µM) samples from 10 to 80 °C at a constant rate of 1 °C/min followed by intrinsic tryptophan fluorescence (direction is shown by arrow) and analyzed by plotting fraction of unfolded protein versus temperature (See Methods).