Addition of αA-Crystallin Sequence 164-173 to a Mini-Chaperone DFVIFLDVKHFSPEDLT Alters the Conformation but Not the Chaperone-like Activity

It has been shown that alpha A-mini-chaperone, a peptide representing the chaperone binding site in alpha A-crystallin, prevents destabilized protein aggregation. alpha A-Mini-chaperone has been shown to form amyloid fibrils. This study was undertaken to improve the stability of alpha A-mini-chaperone while preserving its anti-aggregation activity by fusing the flexible and solvent-exposed C-terminal 164-173 region of alpha A-crystallin to the mini-chaperone sequence DFVIFLDVKHFSPEDLT. The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTS-APSS (designated CP1), was characterized. Circular dichroism studies showed that unlike alpha A-mini-chaperone with its beta-sheet structure, the CP1 peptide exhibited a random structure. Transmission electron microscopy (TEM) examination of the CP1 peptide incubated in a shaker at 37 degrees C for 72 h did not reveal amyloid fibrils, whereas alpha A-mini-chaperone showed distinct fibrils. Consistent with TEM observation, the thioflavin T binding assay showed an increased level of dye binding in the mini-chaperone incubated at 37 degrees C and subjected to shaking but not of the CP1 peptide incubated under similar conditions. The chaperone activity of the CP1 peptide was comparable to that of alpha A-minichaperone against denaturing alcohol dehydrogenase, citrate synthase, and alpha-lactalbumin. Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects. The antioxidation assay involving the H2O2 treatment of COS-7 cells revealed that alpha A-mini-chaperone and the CP1 peptide have comparable cytoprotective properties against H2O2-induced oxidative damage in COS-7 cells. This study therefore shows that the addition of C-terminal sequence 164-173 of alpha A-crystallin to alpha A-mini-chaperone influences the conformation of alpha A-mini-chaperone without affecting its chaperone function or cytoprotective activity.