Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30.

Small heat shock proteins (shsps) act as molecular chaperones by preventing heat-induced aggregation and unfolding of cellular proteins by a mechanism that is still unclear. Previously we found that the C-terminal end of Xenopus shsp, hsp30C (30C), was essential for optimal chaperone activity. Examination of the C-terminal tail of 30C revealed that it had a net negative charge. Involvement of this negative charge in chaperone activity was assessed by the creation of two mutants, D209G (Asp converted to the more neutrally charged and less polar Gly at position 209) and D209/213G (Asp to Gly at position 209 and 213). Compared to 30C and D209G, D209/213G was impaired in inhibiting heat-induced citrate synthase aggregation. In rabbit reticulocyte lysate and Xenopus oocyte microinjection refolding assays the mutants were not as efficient as 30C in maintaining heat-treated luciferase in a folding competent state. Circular dichroism analysis revealed that D209G was similar in secondary structure to 30C whereas D209/213G displayed a loss of alpha-helical-like and beta-sheet structure. Also, C-terminal truncation of 30C or 30D (an hsp30 isoform) resulted in a loss of secondary structure and function. This study clearly shows that mutation of aspartic acid residues in the C-terminal end of hsp30 or its truncation disrupts secondary structure and impairs its chaperone activity.