Interactions between Hsp90 and Hsp70 were not observed when incubating the proteins alone. A control experiment revealed no interactions between Hop and FKBP52 alone, confirming that Hop and FKBP52 compete for binding sites on Hsp90. The (Hsp90) 2(Hop) 1 complex was also observed and was more prevelant than its analogue (Hsp90) 2(FKBP52) 1. We next challenged the Hsp90/Hop complexes with different amounts of FKBP52 one Hop could readily be exchanged by FKBP52. Likewise, incubation with the immunophilin FKBP52 led to the formation of (Hsp90) 2(FKBP52) 1 and (Hsp90) 2(FKBP52) 2 complexes. By incubating equimolar amounts of Hsp90 and Hop we found that (Hsp90) 2(Hop) 1 is the predominant complex although binding of a second Hop was also observed albeit at low intensities. Hop is a crucial interaction partner of Hsp90 facilitating client transfer from Hsp70 to Hsp90. We first explored the heterogeneity of the Hsp90 complexes formed in the presence of the co-chaperones Hop, FKBP52 and Hsp70. We propose that the Hsp70 dimer forms within the stable intermediate complex, as the two chaperone cycles meet to facilitate handover of client proteins from Hsp70 to Hsp90. Addition of the immunophilin p23 to this client-transfer complex induced the transfer of the client from Hsp70 to Hsp90 preparing it for its further action and eventual transfer to the nucleus. This final client-transfer complex not only contained the anticipated Hsp90 dimer but also contained an unexpected Hsp70 dimer. Prevalent intermediates in both cycles were identified and a final client-transfer complex containing the glucocorticoid receptor (GR) was defined. We applied MS to study the dynamic complexes of the Hsp70/Hsp90-chaperone machinery. Significantly for this research it enables the analysis of dynamic equilibria and heterogeneous protein assemblies, such as chaperone cycles. MS allows the determination of subunit stoichiometries, interaction modules and the topology of protein complexes. Recent developments have made mass spectrometry (MS) a powerful tool in structural biology. It contains a C-terminal dimerisation domain, a middle domain and an N-terminal nucleotide-binding domain, connected by a charged linker. Its main function is to stabilise client proteins and to regulate their activation with the help of numerous co-chaperones. It interacts with misfolded proteins to prevent their aggregation, however, Hsp90 alone cannot refold these proteins to their native state. Hsp90 plays a role at the later stages of the Hsp70/Hsp90 cycle. We previously characterised Hsp70 as being predominantly monomeric, although Hsp70 dimers have been reported in solution and crystal structures. Hsp70 contains nucleotide- and substrate-binding domains which move independently prompting proposals of allosteric control mechanisms between the two domains leading to an elongated ADP conformation and a docked/compact ATP state. Hsp40 is required to form complexes with Hsp70 and act as a catalyst to bind partially folded substrates or clients. p23) then lead to formation of the mature complexes which keep the client in an activatable state. A general model that has emerged over the last few decades includes binding of a client protein to the Hsp70/40 chaperones followed by transfer from Hsp70 to Hsp90 via Hop (the Hsp70-Hsp90 organising protein). Client proteins include steroid hormone receptors, transcription factors or kinases. It requires a cohort of co-chaperones that interact at different stages of the Hsp70/Hsp90 cycles and consequently regulate specificity for the high number of substrates. The Hsp70/Hsp90-based cellular machinery stabilises proteins for correct folding or re-folding in response to stress.
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