Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle.
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.
Török
Z
Z
Institute of Biochemistry, Biological Research Center, Szeged, P.O. Box 521, Hungary 6701.
Vigh
L
L
Goloubinoff
P
P
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D018835:Chaperonin 10 / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000378:metabolism; D008961:Models, Structural; D017510:Protein Folding; D055503:Protein Multimerization; D013050:Spectrometry, Fluorescence / Q000379:methods
D018835:Chaperonin 10; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; D008274:Magnesium