Site-specifically Phosphorylated Hsp90C-terminal Domain Variants Provide Access to Deciphering the Chaperone Code

Area of Science:

• Molecular biology
• Protein biochemistry
• Cellular stress response

Background:

• Heat shock protein 90 (Hsp90) is a crucial molecular chaperone involved in protein folding.
• Posttranslational modifications (PTMs) on Hsp90, known as the 'chaperone code,' and their effects on Hsp90 function are not fully understood.
• The Hsp90 C-terminal domain (CTD) is vital for forming the active Hsp90 dimer and interacts with co-chaperones and client proteins.

Purpose of the Study:

• To investigate the impact of site-specific phosphorylations in the Hsp90 CTD on its structure, stability, and chaperone activity.
• To develop an efficient method for generating site-specifically modified Hsp90 variants to study the 'chaperone code.'

Main Methods:

• Expressed protein selenoester ligation (EPSL) was optimized and utilized to introduce site-selective phosphorylations into the Hsp90 CTD.
• Characterization of modified Hsp90 CTD variants, including assessment of secondary structure, thermal stability, and anti-aggregation activity using model client proteins.

Main Results:

• Site-specific phosphorylations were successfully introduced into the Hsp90 CTD without altering the native amino acid sequence or overall secondary structure.
• Combined phosphorylations led to a slight decrease in the thermal stability of the Hsp90 CTD.
• The introduced phosphorylations did not significantly affect the chaperone activity of the Hsp90 CTD in preventing the aggregation of model client proteins.

Conclusions:

• Optimized EPSL provides an efficient method for generating site-specifically PTM-modified Hsp90 CTD variants.
• While C-terminal phosphorylations impact Hsp90 CTD stability, their direct effect on chaperone activity against model clients is minimal.
• Further research is needed to fully elucidate the role of the 'chaperone code' in Hsp90 function.