Fluorescence detection of conformational changes in GroEL induced by thermal switching and nucleotide binding.
GroEL assists other proteins to fold in vivo, usually requiring Mg2+ and ATP to function. Electron micrographs reveal that ATP binding induces a conformational change in this protein. Previously we have suggested that a thermally induced conformational change in GroEL results in the switching between enhanced and arrested reactivation of bacterial glucose-6-phosphate dehydrogenase. This thermal switching occurs over a narrow temperature range (25-30 degrees C). Recently it has aslo been reported that the binding affinity of the P22 tail spike polypeptide to GroEL changes abruptly over this same temperature range. To determine whether these conformational changes occur in GroEL in solution in absence of folding intermediates, the protein was stoichiometrically labeled with AEDANS (N-(acetylaminoacyl)-5-naphthalene-1-sulfonic acid), a fluorescent label whose emission is environment-sensitive. By measuring changes in the fluorescence Stokes shift we have detected two thermally induced conformational changes between 25 and 30 degrees C. The first change occurs between 25 and 26 degrees C, resulting in less than a 1-nm shift in the fluorescence. The second conformational change occurs between 27 and 30 degrees C, resulting in a 3-nm fluorescence shift. The conformational change induced by ATP binding also results in a 3-nm shift in fluorescence. Our data provide a correlation between functional change and structural change in GroEL.
Hansen
J E
JE
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-2007.
Gafni
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D018834:Chaperonin 60; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry*; D008027:Light; D008274:Magnesium / Q000378:metabolism; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D001426:Bacterial Proteins; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C006421:1,5-I-AEDANS; D000255:Adenosine Triphosphate; D008274:Magnesium