## 3 Results
### ANK and TPR Domains Work Together as a Molecular chaperone  
Because AtTPR10 showed molecular chaperone activity (Figure 6) and contains two domains, ANK and TPR (Figure 2), which are reported to be involved in protein-protein interactions and intracellular protein complex assembly [4; 5; 7; 21; 34], we analyzed the contribution of each domain to the chaperone function of AtTPR10. To do the experiment, two truncated versions of the AtTPR10 protein, ANK-D (1-300 aa) and TPR-D (300-456 aa), were constructed (Figure 7a). These truncated recombinant proteins were expressed in _E. coli_, homogeneously purified, and used for biochemical analysis (Figure 7b). Chaperone activity of the truncated proteins was measured using MDH and CS as substrates at a truncated protein:substrate molar ratio of 1:5. Both domains exhibited a similar level of chaperone activity (Figure 7c), but each truncated protein showed approximately 30%-40% lower chaperone activity than the native AtTPR10. This suggests that each domain contributes to the chaperone function of AtTPR10 in a synergistic manner. To investigate the structural changes in the truncated proteins, we analyzed the hydrophobicity of these proteins by incubation with bis-ANS. Consistent with the result of chaperone activity, the hydrophobicity of AtTPR10 was considerably higher than that of each truncated protein (Figure 7d). Figure 6: Chaperone activity analysis and heat shock-dependent activity regulation of AtTPR10. (**a**) and (**b**) Analysis of the chaperone function of AtTPR10 using MDH (**a**) and citrate synthase (CS) (**b**) as substrates. Thermal aggregation of MDH and CS was monitored at 340 nm after a 15 min incubation with AtTPR10. In (**a**), 1.5 \(\upmu\)M MDH was incubated at 43 \({}^{\circ}\)C, either alone (\(\oslash\); control) or with 7.5 \(\upmu\)M GST (**a**), 4.5 \(\upmu\)M 2-Cys Prx (\(\oslash\)), and 1.5 \(\upmu\)M (**a**), 4.5 \(\upmu\)M (\(\blacklozenge\)), and 7.5 \(\upmu\)M (**\(\blacktriangle\)**) AtTPR10 in 50 mM HEPES (pH 7.0). In (**b**), 1.2 \(\upmu\)M CS was incubated at 43 \({}^{\circ}\)C, either alone (\(\oslash\); control) or with 6 \(\upmu\)M GST (\(\bullet\)), 3.6 \(\upmu\)M 2-Cys Prx (\(\oslash\)) and 1.2 \(\upmu\)M (**\(\blacksquare\)**), 3.6 \(\upmu\)M (\(\blacklozenge\)), and 6 \(\upmu\)M (\(\blacktriangle\)**) AtTPR10 in 50 mM HEPES (pH 7.0). (**c**) Heat-shock dependent chaperone activity of AtTPR10. In this experiment, 1.5 \(\upmu\)M MDH was incubated either alone (\(\oslash\); control) or with 3 \(\upmu\)M AtTPR10 at 23 \({}^{\circ}\)C (\(\blacksquare\)), 50 \({}^{\circ}\)C (\(\blacklozenge\)), and 60 \({}^{\circ}\)C (\(\blacktriangle\)) for 15 min. Furthermore, because the chaperone activity of AtTPR10 increased upon heat shock treatment (Figure 6c), we investigated changes in the chaperone activity and hydrophobicity of the truncated mutants under different temperatures (Figure 8). Similar to AtTPR10 (Figure 6c), chaperone activity of ANK-D and TPR-D increased significantly upon increasing the temperature from 23 \({}^{\circ}\)C to 60 \({}^{\circ}\)C (Figure 8a). Additionally, exposure of the hydrophobic surfaces of the truncated proteins at different temperatures was verified by incubation with bis-ANS (Figure 8b,c). In contrast to the hydrophobicity of proteins under normal temperature (23 \({}^{\circ}\)C), the proportion of hydrophobic patches on the surface was greatly increased to allow the binding of denatured substrates under heat stress. In addition, chaperone activities of both truncated proteins were greatly enhanced by the heat shock treatment, suggesting that the structure of these proteins was transformed by thermal exposure. Because AtTPR10 polymerized and formed HMW complexes in response to high temperature (Figure 5a,b), we examined the heat stress-induced structural shift in the two truncated proteins by native gel electrophoresis and SEC. The results of native gel electrophoresis showed that the MW of both ANK-D and TPR-D increased significantly under high temperatures (Figure 9a,c), similar to AtTPR10.