FIGURE 4.

Hsp70-PP5 and Hsp90 interact with different stoichemetries. PP5 binds intact Hsp70 and Hsp90. A, SEC-MALS analysis of Hsp70:PP5 (blue) shows elution volume shift and molecular mass of 145 kDa, indicating stable 1:1 complex compared with Hsp70 (red) and PP5 (green) alone. B, Hsp90:PP5 (blue) is shifted in elution volume and determined to be a 2:1 complex at 232 kDa compared Hsp90 dimer alone (red). Molecular masses were determined from the Raleigh ratio, measured by static light scattering, and the protein concentration (right y axis), measured by a refractive index detector, are indicated. Molecular masses based on sequence are as follows (in kDa): 70 (Hsp70), 56 (PP5), and 170 (Hsp90 dimer). SEC columns WTC-030S5 and WTC-050S5 (Wyatt Technology) were used to optimally separate free Hsp70 and Hsp90 from PP5 complexes, respectively.