Abstract
The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation (EPSL) to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.