Folding properties of the nucleotide exchange factor GrpE from Thermus thermophilus: GrpE is a thermosensor that mediates heat shock response

Hsp70 proteins like DnaK bind unfolded polypeptides in a nucleotide-depende nt manner. The switch from high-affinity ADP-state to low-affinity ATP-stat e with concomitant substrate release is accelerated significantly by GrpE p roteins. GrpE thus fulfils an important role in regulation of the chaperone cycle. Here, we analysed the thermal stability of GrpE from Thermus thermo philus using differential scanning calorimetry and CD-spectroscopy. The pro tein exhibits unusual unfolding characteristics with two observable thermal transitions. The first transition is CD-spectroscopically silent with a tr ansition midpoint at 90 degreesC. The second transition, mainly constitutin g the CD-signal, ranges between 100 and 105 degreesC depending on the GrpE( Tth) concentration, according to the model N-2 reversible arrow I-2 reversi ble arrow 2U. Using a C-terminally truncated version of GrpE(Tth) it was po ssible to assign the second thermal transition to the dimerisation of GrpET th, while the first transition represents the completely reversible unfoldi ng of the globular C-terminal domain. The unfolding of this domain is accom panied by a distinct decrease in nucleotide exchange rates and impaired bin ding to DnaK(Tth). Under heat shock conditions, the DnaK(.)ADP(.)protein-su bstrate complex is thus stabilised by a reversibly inactivated GrpE-protein that refolds under permissive conditions. In combination with studies on G rpE from Escherichia coli presented recently by Christen and co-workers, it thus appears that the general role of GrpE is to function as a thermosenso r that modulates nucleotide exchange rates in a temperature-dependent manne r to prevent substrate dissociation at non-permissive conditions. (C) 2001 Academic Press.