Probing the functional mechanism of Escherichia coli GroEL using circular permutation.
BACKGROUND
The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit.


RESULTS
Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion.


CONCLUSIONS
The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan. mizobata@bio.tottori-u.ac.jp
Tomohiro
T
Mizobata
Tatsuya
T
Uemura
Kazuhiro
K
Isaji
Takuma
T
Hirayama
Kunihiro
K
Hongo
Yasushi
Y
Kawata
D000595:Amino Acid Sequence; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008854:Microscopy, Electron; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D021122:Protein Subunits; D013884:Thiosulfate Sulfurtransferase