%0 Generic
%A Graf, Christian
%D 2008
%F heidok:8696
%K H/D-Austausch , Hsp70 , Hsp90 , Co-Chaperonhydrogen exchange , mass spectrometry , Hsp70 , Hsp90 , fluorescence spectroscopy
%R 10.11588/heidok.00008696
%T Analysis of the Conformational Dynamics of Hsp70 and Hsp90 Chaperones
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8696/
%X Molecular chaperones of the Hsp70 and Hsp90 family are central components in protein folding processes in the cell. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the control of stability and activity of some 200 natively folded clients including receptors, protein kinases and transcription factors, many of which are key regulators in essential signal transduction pathways. Both chaperones are multi-domain proteins that use ATP-controlled cycles for substrate binding and release which are regulated by co-chaperones. The aim of this thesis was to elucidate the allosteric mechanism of Hsp70 and Hsp90 chaperones and their interactions with the co-chaperone CHIP by investigating their conformational changes and dynamics in solution. These conformational studies were performed primarily using amide hydrogen exchange (HX) combined with high-resolution mass spectrometry (MS) and fluorescence spectroscopy. HX-MS experiments with the E. coli Hsp70 homologue DnaK revealed specific ATP-induced conformational changes. Upon ATP binding, the nucleotide-binding domain was more compact, while the substrate binding domain was more flexible. The exposed interdomain linker became completely protected from solvent upon ATP binding. Comparison of the dynamics of the full-length protein with the dynamics of the isolated domains demonstrated a mutual stabilization of both domains. Substrate binding to DnaK in the ATP-bound state reverses the ATP-induced conformational changes in the linker and selected parts of the NBD. The HX-MS data outline a pathway for allosteric interdomain communication which mediates ATP-induced opening of the substrate binding pocket and the substrate-induced stimulation of ATP hydrolysis. Continuous-labeling and pulse-labeling HX-MS experiments with the dimeric E. coli Hsp90 homologue HtpG demonstrate drastic ATP-induced conformational changes throughout the protein that do not occur simultaneously but progress slowly like a wave from the nucleotide binding site towards the N-terminus and the middle domain. A conformation-sensitive fluorescent probe allowed the elucidation of the kinetics of the ATPase cycle of HtpG. Conversion into a compact conformational state was shown to be rate-limiting for ATP hydrolysis and the nucleotide-coordinating residue Glu34 was important for the rate of conversion.Analysis of the conformational dynamics of the eukaryotic Hsp90 homologues from yeast and human, Hsc82 and Hsp90b, by HX-MS revealed a significant higher flexibility as compared to the prokaryotic HtpG. Segments with higher dynamics were located predominantly in the middle and the dimerization domains. Nucleotide binding had long-range effects on the conformation of all domains, but these were more subtle compared to HtpG. Consistent with the hypothesis that conformational dynamics of Hsp90 is linked to regulation by co-chaperones, it could be shown by fluorescence resonance energy transfer experiments with fluorescent Hsp90b; that the binding of the co-chaperone p23 in presence of ATP leads to movement of the N-terminal Hsp90 domains towards each other. Two Hsp90 inhibitors competitive for nucleotide affected the conformational dynamics of Hsp90b differently from nucleotides and from each other. The human E3-ubiquitin ligase CHIP exhibited an extraordinary flexibility with an almost completely unfolded N-terminal TPR domain as measured by HX-MS. Complex formation with intact Hsp70 and Hsp90 or their respective C-terminal octapeptides induced folding of the TPR domain to a defined, stabilized structure. Interaction of CHIP with two different E2 ubiquitin-conjugating enzymes, UbcH5a and Ubc13, resulted in distinct effects on the conformational dynamics of CHIP suggesting different roles of the CHIP-E2 interaction for the ubiquitination of substrates.