Nucleotide and Mg2+ induced conformational changes in GroEL can be detected by sulfhydryl labeling.
The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the molecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg2+ and/or adenine nucleotides. Three peptides, each containing one of the cysteines of GroEL (C138, C458 and C519) were identified. GroEL labeled in 50 mM TrisHCl, pH 7.8, incorporated approximately 0.3 labels each on C138 and C458. With 10 mM MgCl2, the labeling increased to approximately 0.8 labels each on C138 and C458. The increase was partially due to a conformational change which occurred upon Mg2+ binding as well as to an increase in ionic strength. When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg2+, C138 incorporated approximately 0.8 labels, while C458 incorporated approximately 0.1 labels. These results suggest that the binding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible. GroEL slowly dissociated into monomers when it was extensively labeled at C458. GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was able to bind but not release active rhodanese. The observed variations in sulfhydryl accessibility are consistent with mechanisms that suggest binding of GroES to GroEL differs from the binding of substrate protein to GroEL, and that the binding of Mg2+ or Mg-adenine nucleotides results in conformational changes in GroEL.
Department of Biochemistry, University of Texas Health Sciences Center at San Antonio, 78240-7760, USA.
E A
EA
Jai
P M
PM
Horowitz
D000225:Adenine / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D005452:Fluoresceins / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D017510:Protein Folding; D017421:Sequence Analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation
D000345:Affinity Labels; D018835:Chaperonin 10; D018834:Chaperonin 60; D005452:Fluoresceins; D009711:Nucleotides; D013438:Sulfhydryl Compounds; C037041:fluorescein 5-maleimide; D013884:Thiosulfate Sulfurtransferase; D010450:Endopeptidases; D008274:Magnesium; D000225:Adenine; D003545:Cysteine