A probe for chaperone/multiprotein complexes and inhibitors in biological matrices

Objective Hsp90α was immobilized onto the surface of magnetic beads and used to extract small ligands and client proteins from chemical mixtures and biological matrices. Methods Hsp90α was immobilized through the N‐terminus, Hsp90α‐NT‐MB or C‐terminus, Hsp90α‐CT‐MB. Ligand fishing experiments were carried out using C‐terminus binders (CA1, NOVO), N‐terminus binders (17‐AAG, GM) and non‐binders (nicotine, propanolol). Protein fishing experiments were performed with recombinant eNOS, p60 Hop and Hsp70. The supernatants and eluates were analyzed using lc‐ms (small ligands) or Western blot analysis (proteins). The effect of ATP and Hsp90α thiol modifications on multiprotein complex formation was also studied. Results Data indicated that Hsp90α‐CT‐MB selectively bound N‐terminus ligands and Hsp90a‐NT‐MB selectively bound C‐terminus ligands. With isolated proteins, Hsp90α‐NT‐MB formed complexes with eNOS and p60 HOP but not Hsp70. All three proteins were extracted when Hsp90α‐NT‐MB was incubated with a mixture of proteins and p60 Hop was isolated from KU‐812 cellular extracts using Hsp90α‐NT‐MB. Hsp90α‐protein complex formation was reversibly reduced by ATP/MgCl 2 and thiol modification. Conclusions The Hsp90α‐MBs can be used to isolate small molecule ligands and client proteins from complex chemical and biological matrices and to study Hsp90α multiple conformations.