Refolding of barnase mutants and pro-barnase in the presence and absence of GroEL.
Three mutants of barnase and a pro-barnase variant, which have a variety of different physical properties but the same overall protein structure, were analysed for their folding in the presence of the molecular chaperone GroEL. Mutants were chosen on the basis that changes in their refolding rate constants in solution are not correlated with the changes in their stability. All barnase variants fold considerably more slowly when bound to GroEL. However, barnase refolding on GroEL parallels that in solution: there is a linear relationship between the refolding rate constants, obtained for wild-type and all mutants of barnase, in the presence and absence of GroEL. Barnase is synthesized in vivo with a 13 amino acid pro-sequence attached to the N-terminus. The pro-sequence of pro-barnase is shown by NMR spectroscopy to be devoid of defined structure. The presence of this pro-sequence has no effect on the overall refolding rate constant or the activity of barnase. In the presence of GroEL, the refolding of pro-barnase is retarded relatively more strongly than that of wild-type and the mutant barnase proteins, suggesting that the pro-sequence provides additional binding sites for the chaperone.
Gray
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TE
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, UK.
Eder
J
J
Bycroft
M
M
Day
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AG
Fersht
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AR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D018834:Chaperonin 60; D006360:Heat-Shock Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease