Figure 2.
Thermodynamic stability and binding activity of TPR domain of PP5. (A) Far-UV CD spectrum of the TPR domain of PP5 (residues 24–177) at 12 μM protein concentration in a 0.1-cm pathlength cuvette. The CD spectrum was recorded with a bandwidth of 1 nm at 1-nm increments and 10-sec average time. All CD experiments were performed using an AVIV Model 215 CD spectrophotometer (AVIV Instruments) in 150 mM NaCl, 50 mM phosphate buffer (pH 6.5) at 25°C. (B) Thermal denaturation of PP5 TPR domain. Thermal denaturation was monitored at 6 μM protein concentration following the ellipticity at 222 nm from 15°C to 95°C and in the reverse direction from 95°C to 15°C in a 0.1-cm path-length cuvette. The temperature ramp was performed in 1°C steps with an equilibration time at each temperature of 1 min. (C) Interaction of the TPR domain of PP5 with the 24-mer C-terminal peptides of Hsp90 (•) and Hsp70 (○) measured by surface plasmon resonance (SPR) in HBS-EP buffer (150 mM NaCl, 3 mM EDTA, 0.005% [v/v] polysorbate 20, 10 mM Hepes at pH 7.5). SPR measurements were performed using a BIACORE 3000 (BIACORE AB) as described previously (Cortajarena et al. 2004). Equilibrium response units were plotted vs. the protein concentration. The data were fit to a 1:1 binding model to calculate the dissociation constant (KD TPR-PP5/Hsp90 = 660 nM).