Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta-galactosidase.
We have studied the effect of the components of the GroE molecular chaperone machine on the refolding of the Escherichia coli enzyme beta-galactosidase, a tetrameric protein whose 116-kDa promoters should not completely fit within the central cavity of the GroEL toroid. In the absence of other additives, GroEL formed a weak complex with chemically denatured beta-galactosidase, reduced its propensity to aggregate, and increased the recovery yields of active enzyme twofold without altering its folding pathway. When present together with the chaperonin, ATP--and to a lesser extent AMP-PNP--reduced the recovery yields and led to the resumption of aggregation. The use of the complete chaperonin system (GroEL, GroES, and ATP) eliminated the GroEL-mediated increase in recovery and folding proceeded less efficiently than in buffer alone. This unusual behavior can be explained in terms of a chaperonin "buffering" effect and the different affinities of GroE complexes for denatured beta-galactosidase.
Ayling
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A
Department of Chemical Engineering, University of Washington, Seattle 98195-1750, USA.
Baneyx
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F
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D018835:Chaperonin 10 / Q000737:chemistry / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism; D018833:Chaperonins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000494:pharmacology*; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006146:Guanidines; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D018833:Chaperonins; D019791:Guanidine