SELF-ASSOCIATION OF THE MOLECULAR CHAPERONE HSC70

The self association properties of the molecular chaperone HSC70 have been analyzed by a wide range of biochemical and biophysical technique s. Nondenaturing gel electrophoresis and crosslinking studies show the presence of multiple species going from monomer to at least trimer. S ize-exclusion chromatography gives two overlapping peaks, a major one corresponding to species having the molecular mass of monomer (70 kDa) and a minor broad one corresponding to species with a molecular mass range of 150-300 kDa. Progressive dilution of the protein leads to an increase in the size of the monomer peak at the expense of that of the oligomeric peak, thus indicating a concentration-dependent chemical e quilibrium. Sedimentation velocity reveals the presence of three speci es, whose proportions were dependent on concentration, but whose sedim entation coefficients, S-20,S-W, of 4.3, 6.6, and 8.5 S did not vary w ith concentration, indicative of a slowly equilibrating system. Sedime ntation equilibrium studies confirmed these results and showed a disso ciation into monomers at low concentrations and an association into di mers and trimers at high concentrations. The multiple sedimentation eq uilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equ ilibrium constants by a monomer-dimer-trimer association model in whic h the association constants for the monomer-dimer and dimer-trimer equ ilibrium were respectively K-1-2 = 1.1 X 10(5) M(-1) and K-2-3 = 0.9 X 10(5) M(-1). Interestingly, an isodesmic, indefinite type of associat ion describes the data almost equally well with a single constant of 1 .2 x 10(5) M(-1). Altogether, these results indicate that HSC70 slowly and reversibly self-associates in solution, likely in an unlimited fa shion, and might have implications for the chaperone function of-this protein.