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Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.

Publication ,  Journal Article
Zondlo, J; Fisher, KE; Lin, Z; Ducote, KR; Eisenstein, E
Published in: Biochemistry
August 1995

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.

Duke Scholars

Published In

Biochemistry

ISSN

0006-2960

Publication Date

August 1995

Volume

34

Issue

33

Start / End Page

10334 / 10339

Location

united states

Related Subject Headings

  • Biochemistry & Molecular Biology
  • 1101 Medical Biochemistry and Metabolomics
  • 0601 Biochemistry and Cell Biology
  • 0304 Medicinal and Biomolecular Chemistry
 

Citation

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Zondlo, J., Fisher, K. E., Lin, Z., Ducote, K. R., & Eisenstein, E. (1995). Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES. Biochemistry, 34(33), 10334–10339.
Zondlo, J., K. E. Fisher, Z. Lin, K. R. Ducote, and E. Eisenstein. “Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.Biochemistry 34, no. 33 (August 1995): 10334–39.
Zondlo J, Fisher KE, Lin Z, Ducote KR, Eisenstein E. Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES. Biochemistry. 1995 Aug;34(33):10334–9.
Zondlo, J., et al. “Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.Biochemistry, vol. 34, no. 33, Aug. 1995, pp. 10334–39.
Zondlo J, Fisher KE, Lin Z, Ducote KR, Eisenstein E. Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES. Biochemistry. 1995 Aug;34(33):10334–10339.
Journal cover image

Published In

Biochemistry

ISSN

0006-2960

Publication Date

August 1995

Volume

34

Issue

33

Start / End Page

10334 / 10339

Location

united states

Related Subject Headings

  • Biochemistry & Molecular Biology
  • 1101 Medical Biochemistry and Metabolomics
  • 0601 Biochemistry and Cell Biology
  • 0304 Medicinal and Biomolecular Chemistry