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 techniques. Nondenaturing gel electrophoresis and cross-linking studies show the presence of multiple species going from monomer to at least trimer. Size-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 equilibrium. Sedimentation velocity reveals the presence of three species, whose proportions were dependent on concentration, but whose sedimentation coefficients, s20,w, of 4.3, 6.6, and 8.5 S did not vary with concentration, indicative of a slowly equilibrating system. Sedimentation equilibrium studies confirmed these results and showed a dissociation into monomers at low concentrations and an association into dimers and trimers at high concentrations. The multiple sedimentation equilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equilibrium constants by a monomer-dimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium were respectively K1-2 = 1.1 x 10(5) M-1 and K2-3 = 0.9 x 10(5) M-1. Interestingly, an isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2 x 10(5) M-1. (ABSTRACT TRUNCATED AT 250 WORDS)
Benaroudj
N
N
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Batelier
G
G
Triniolles
F
F
Ladjimi
M M
MM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D014461:Ultracentrifugation
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate