Abstract
The 70-kDa heat-shock proteins (Hsp70), including its cognate Hsc70, are implicated as molecular chaperones, which facilitate the correct assembly or disassembly of proteins. These chaperones not only recognize a wide range of nascent polypeptides that share no apparent sequence homology, but also possess the ability to distinguish between properly folded and unfolded proteins. In this dissertation, a hybrid peptide (H2N-CNCKAPETALCAFYCLAKT-CONH2) was used to investigate the structural / chemical requirement essential for the Hsc70 recognition. The conformation of the peptide, both in the reduced and oxidized form, was examined by circular dichroism spectroscopy and 2D-NMR spectroscopy. Since the hybrid peptide took apamin as a structure template, its behavior was like a chameleon - the reduced form was a random coil, but the oxidized form was a a-helix. The ability of hybrid peptides to inhibit the formation of complexes between Hsc70 and denatured lactalbumin (CMLA) was measured by nondenaturing PAGE. The result indicated that the reduced form could inhibit the formation of Hsc70-CMLA complex, while the oxidized form could not. Fluorescence measurements of the dansylated peptide also indicated that the reduced form bound to Hsc70 much stronger than the oxidized form.Ｔhe conclusion based on above data was that Hsc70 possess the ability to distinguish between properly folded and unfolded peptides, and only bind to the unfolded one. The interaction was further investigated by monitoring the oxidation of the reduced form peptide in the presence of Hsc70. The results showed that Hsc70 slowed down the oxidation, and this had the meaning that Hsc70 protected the reduced form peptide from misfolding.