The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4'-bis(1-anilino-8- naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to approximately 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at approximately 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at approximately 1 M urea. The same destabilization by ADP and stabilization by Mg2+ were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5'-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg2+ and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed. Study holds ProTherm entries: 5205, 5206 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:29 p.m.
|Number of data points||4|
|Proteins||60 kDa chaperonin ; 60 kDa chaperonin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MAAKDVKFGNDAGVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDKAVTVAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATAVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGGMGGMM|