Structural and Functional Impacts of Extended N-Terminal End of the Small Heat Shock Protein Tpv HSP 14.3

Abstract

Small heat shock proteins (sHSPs) are composed of the α-crystallin domain, which is highly conserved, and variable N-terminal and C-terminal domains. In contrast to the α-crystallin domain, structures of the flanking N- and C-terminal domains are poorly defined. The N-terminal domain is the most divergent region in sequence and length among small heat shock proteins. In this study, to provide further insight into the importance of N-terminal tags in the chaperone function of small heat shock proteins, two variants of Tpv HSP 14.3 containing polyhistidine tags (11-aa and 26-aa in length) in the proximal part of their N-termini were used. These variants were generated by expressing the cloned Tpv HSP 14.3 gene in Escherichia coli using the expression vectors pQE-31 and TAGZyme pQE-2. The His-tagged recombinant proteins were purified by affinity chromatography. The effects of poly-His tags on chaperone activity of the Tpv HSP 14.3 were evaluated using pig heart citrate synthase as the model substrate. The results showed that Tpv HSP 14.3 variants with N-terminal tags were more effective chaperones than the one without tag. In addition, the alterations in intrinsically disordered states of N-termini were analyzed by means of the PONDR predictor. The results indicated that the disordered nature of the fused tags and additional hydrophobic residues they contributed to the N terminus may increase the capacity of Tpv HSP 14.3 to interact with its substrate protein and thereby improve its chaperone activity.