New real-time Hsp90 high throughput screening method using fluorescence resonance energy transfer (FRET)

The molecular chaperone heat shock protein 90 (Hsp90) couples ATP hydrolysis

to conformational changes driving a reaction cycle that is required for substrate activation. Recent structural analysis provided snapshots of the open and closed states of Hsp90, which mark the starting and end points of these changes. To directly track structural rearrangements in Hsp90, fluorophores were attached to engineered cysteine residues in the N or M domains of Hsp90. The chosen amino acid positions are surface-exposed and not directly involved in Hsp90 function. In addition, a double-cysteine variant was created (Figure). The structural changes in Hsp90 can be tightly regulated by co-chaperones that are completely inhibited by Sti1 or accelerated by Aha1. Aha1 induces Hsp90 rearrangements that speed up the conformational cycle even in the absence of nucleotide. The comprehensive analysis of the Hsp90 cycle defines a controlled progression through distinct intermediates that can be modulated by conformation-sensitive co-chaperones. This new assay system allows high-sensitivity real-time detection and kinetic analysis of motions of Hsp90 in response to nucleotides or co-chaperones by fluorescence resonance energy transfer (FRET).

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