PMID:7654686
| Citation |
Zondlo, J, Fisher, KE, Lin, Z, Ducote, KR and Eisenstein, E (1995) Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES. Biochemistry 34:10334-9 |
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| Abstract |
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. |
| Links | |
| Keywords |
Amino Acid Sequence; Chaperonin 10/chemistry; Chaperonin 10/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Escherichia coli/genetics; Histidine/chemistry; Kinetics; Light; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Fusion Proteins/chemistry; Scattering, Radiation; Spectrometry, Fluorescence; Thiocyanates/pharmacology; Ultracentrifugation |
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