Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle

Z. Török, L. Vígh, Pierre Goloubinoff

Research output: Contribution to journalArticle

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Abstract

The GroEL14 chaperonin from Escherichia coli was labeled with 5- ((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein. Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES7-dependent changes in the fluorescence intensity of AEDANS-GroEL14, corresponding to the sequential binding of two GroES7 heptamers and the formation of two types of chaperonin heterooligomers, GroEL14GroES7 and GroEL14(GroES7)2. The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL14 that allowed the direct measurement in solution at equilibrium of ATP- and GroES7-dependent protein release from the chaperonin. Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL14GroES7 particles bind newly formed ATP. 2) MDH- GroEL14GroES7 particles bind a second GroES7. 3) MDH-GroEL14(GroES7)2 particles productively release MDH. 4) Released MDH completes folding. Therefore, the symmetrical GroEL14(GroES7)2 heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle.

Original languageEnglish
Pages (from-to)16180-16186
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number27
DOIs
Publication statusPublished - 1996

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Chaperonins
Malate Dehydrogenase
Fluorescence
Adenosine Triphosphate
Adenosine Diphosphate
Proteins
Adenylyl Imidodiphosphate
Protein folding
Sulfonic Acids
Protein Folding
Fluorescent Dyes
Escherichia coli
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle. / Török, Z.; Vígh, L.; Goloubinoff, Pierre.

In: Journal of Biological Chemistry, Vol. 271, No. 27, 1996, p. 16180-16186.

Research output: Contribution to journalArticle

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abstract = "The GroEL14 chaperonin from Escherichia coli was labeled with 5- ((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein. Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES7-dependent changes in the fluorescence intensity of AEDANS-GroEL14, corresponding to the sequential binding of two GroES7 heptamers and the formation of two types of chaperonin heterooligomers, GroEL14GroES7 and GroEL14(GroES7)2. The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL14 that allowed the direct measurement in solution at equilibrium of ATP- and GroES7-dependent protein release from the chaperonin. Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL14GroES7 particles bind newly formed ATP. 2) MDH- GroEL14GroES7 particles bind a second GroES7. 3) MDH-GroEL14(GroES7)2 particles productively release MDH. 4) Released MDH completes folding. Therefore, the symmetrical GroEL14(GroES7)2 heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle.",
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