The hexameric form of the KaiC protein is a core of the cyanobacterial biological clock, and its enzymatic activities exhibit circadian periodicity. The instability of the monomeric form of ...nucleotide-free KaiC has precluded its storage and detailed analyses of the activities of the reassembled hexamer. Here, we provide a protocol for preparing nucleotidefree KaiC monomer that is stable in solution and for triggering its reassembly into intact KaiC hexamer by the addition of ATP. A phosphate buffer containing glutamic acid and arginine enhanced the stability of KaiC monomer considerably. In addition, we found that reassembled KaiC hexamer was functionally active as the intact hexamer. This protocol provides a methodological basis for further analyses of first-turnover events of the ATPase/autokinase/autophosphatase activities of the KaiC hexamer.
The σ
E
pathway of extracytoplasmic stress responses in
Escherichia coli
is activated through sequential cleavages of the
anti-σ
E
protein, RseA, by membrane proteases DegS and RseP.
Without the ...first cleavage by DegS, RseP is unable to cleave full-length RseA.
We previously showed that a PDZ-like domain in the RseP periplasmic region is
essential for this negative regulation of RseP. We now isolated additional
deregulated RseP mutants. Many of the mutations affected a periplasmic region
that is N-terminal to the previously defined PDZ domain. We expressed these
regions and determined their crystal structures. Consistent with a recent
prediction, our results indicate that RseP has tandem, circularly permutated
PDZ domains (PDZ-N and PDZ-C). Strikingly, almost all the strong mutations
have been mapped around the ligand binding cleft region in PDZ-N. These
results together with those of an
in vitro
reaction reproducing the
two-step RseA cleavage suggest that the proteolytic function of RseP is
controlled by ligand binding to PDZ-N.
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