Hsp70 proteins like DnaK bind unfolded polypeptides in a nucleotide-dependent manner. The switch from high-affinity ADP-state to low- affinity ATP-state with concomitant substrate release is accelerated significantly by GrpE proteins. GrpE thus fulfils an important role in regulation of the chaperone cycle. Here, we analysed the thermal stability of GrpE from Thermus thermophilus using differential scanning calorimetry and CD-spectroscopy. The protein exhibits unusual unfolding characteristics with two observable thermal transitions. The first transition is CD-spectroscopically silent with a transition midpoint at 90 degrees C. The second transition, mainly constituting the CD-signal, ranges between 100 and 105 degrees C depending on the GrpE(Tth) concentration, according to the model N(2) <==> I(2) <==> 2U. Using a C-terminally truncated version of GrpE(Tth) it was possible to assign the second thermal transition to the dimerisation of GrpE(Tth), while the first transition represents the completely reversible unfolding of the globular C-terminal domain. The unfolding of this domain is accompanied by a distinct decrease in nucleotide exchange rates and impaired binding to DnaK(Tth). Under heat shock conditions, the DnaK-ADP-protein-substrate complex is thus stabilised by a reversibly inactivated GrpE-protein that refolds under permissive conditions. In combination with studies on GrpE from Escherichia coli presented recently by Christen and co-workers, it thus appears that the general role of GrpE is to function as a thermosensor that modulates nucleotide exchange rates in a temperature-dependent manner to prevent substrate dissociation at non-permissive conditions. Study holds ProTherm entries: 12765, 12766 Extra Details: chaperones; DnaK; GrpE; nucleotide exchange
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||2|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MEERNHENTLEKDLEAVGQEAQALEERLKAAEEELKGLKDKYLRLLADFDNYRKRMEEELKAREREGVLKALRALLPVLDDLDRALEFAEASPESIRQGVRAIRDGFFRILAGLGVEEVPGEGEAFDPRYHEAVGLLPGEPGKVAKVFQRGFRMGEALVRPARVAVGEEKREEADLE|