Self-Association of the Molecular Chaperone HSC70

Abstract

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)

Hsc70 Protein Interaction with Soluble and Fibrillar α-Synuclein

“…The sedimentation coefficient measured for ␣-Syn (1 S) is compatible with the molecular mass of a natively unfolded 14-kDa polypeptide. The species we observe for Hsc70 have been previously described and correspond to monomeric, dimeric, and oligomeric Hsc70 (31). Determination of sedimentation coefficients gives valuable information on the hydrodynamic shape of protein complexes and conformational changes within these complexes.…”

Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

“…Initially, we found that Hsp90 was predominantly dimeric and FKBP52 monomeric, both were anticipated. Recently there has been some controversy over the oligomeric state of Hop reported as dimeric (6,20,34) or monomeric (22,35) and Hsp70 can form high oligomeric species under some conditions (36). We have shown that under the conditions used here both proteins are monomeric.…”

The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70

“…Protein Expression and Purification-Recombinant HSC70 was expressed and purified as described previously (30,34). The recombinant NH 2 -terminal domain of HSC70 was expressed and purified as described for HSC70 except that the first ion exchange chromatographic step was replaced by a 60% ammonium sulfate precipitation.…”

“…After centrifugation, the ammonium sulfate concentration of the supernatant was adjusted to 80%, and the sample was submitted to centrifugation. The resulting pellet was resuspended in 20 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM ␤-mercaptoethanol, and 3 mM MgCl 2 , dialyzed in the same buffer, and applied onto an ATP-agarose affinity column as described previously (30,34).…”