Abstract
The 70 kilodalton heat shock cognate protein (Hsc70) is a constitutively expressed member of the molecular chaperones which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Hsc70 cooperates with cochaperone Hsp40, and functions by binding and releasing the exposed extended polypeptide of nascent and nonnative proteins, in the ATP-dependent manner. Binding specificities of Hsc70s to their substrates are quite different among bacteria, yeast, vertebrate and plant. To investigate the interactions and specificity between Hsc70 and its substrates, the carboxyl-terminal 30 kDa of three mung bean (Vigna radiata) Hsc70s were expressed and used as target proteins to select Hsc70-binding heptapeptides using phage display screening. Three heptapeptides of high frequencies were selected with the target proteins: KVWVLPI for VrHsc70-1, KLWVIPQ for VrHsc70-2, KLWVIPQ and YAPLSRL for VrHsc70-3. The target binding was confirmed by ELISA analysis. These three heptapeptides were subject to search for the homologous proteins. Our results suggested that VrHsc70-1 may assist the nonnative or newly synthesized proteins to fold correctly, and VrHsc70-2 and 3 may bind substrates during de novo synthesis stages. To study the binding patterns of three significant heptapeptides to VrHsc70s, InsightII program was used to docking each heptapeptide into its target VrHsc70. The lowest-energy binding models were inspected and a similar binding mode of VrHsc70s to KVWVLPI, KLWVIPQ and YAPLSRL was obtained.