Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.
In an effort to clarify the role of GroES in chaperonin-facilitated protein folding, a plasmid-encoding expression system for GroES incorporating a histidine-tagged, thrombin-cleavable, N-terminal sequence was constructed. This approach facilitated the rapid purification of native-like, histidine-cleaved GroES (HC-GroES). The addition of NaSCN to purification buffers to mildly promote subunit dissociation enabled the complete separation of chromosomally encoded, wild-type GroES chains from recombinant chains, allowing the production of homogeneous mutant variants of GroES. A substitution of histidine-7 to tryptophan in GroES was used to demonstrate the concentration-dependent modulation of the heptameric quaternary structure of the chaperonin. Fluorescence and light scattering studies of this mutant suggest that GroES heptamers dissociate to monomers upon dilution with half-times of 2-4 min. Sedimentation equilibrium experiments using either wild-type or HC-GroES can best be described by a monomer--heptamer equilibrium, yielding dissociation constants of 1 x 10(-38) M6 for native GroES and 2 x 10(-32) M6 for HC-GroES. These results are supported by subunit exchange experiments using mixtures of native or HC-GroES and GroES containing the complete N-terminal histidine tail. Native polyacrylamide gel electrophoresis demonstrates that these mixtures form an eight-membered hybrid set within minutes. The studies described here suggest a dynamic equilibrium for the quaternary structure of GroES, which may be an important feature for its role in GroEL-mediated protein folding reactions.
Zondlo
J
J
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Fisher
K E
KE
Lin
Z
Z
Ducote
K R
KR
Eisenstein
E
E
D000595:Amino Acid Sequence; D018835:Chaperonin 10 / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates / Q000494:pharmacology; D014461:Ultracentrifugation
D018835:Chaperonin 10; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D013861:Thiocyanates; D006639:Histidine; C024553:sodium thiocyanate