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 co...

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Bibliographic Details
Published in:The EMBO journal 1993-11, Vol.12 (11), p.4145-4150
Main Authors: Gray, T.E., Eder, J., Bycroft, M., Day, A.G., Fersht, A.R.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacillus amyloliquefaciens
Bacterial Proteins - metabolism
Bacteriology
Base Sequence
Biological and medical sciences
Chaperonin 60
Conformational dynamics in molecular biology
Fundamental and applied biological sciences. Psychology
Heat-Shock Proteins - metabolism
Magnetic Resonance Spectroscopy
Metabolism. Enzymes
Microbiology
Molecular biophysics
Molecular Sequence Data
Mutation
Protein Conformation
Protein Folding
Protein Precursors - genetics
Protein Precursors - metabolism
Recombinant Proteins - metabolism
Ribonucleases - genetics
Ribonucleases - metabolism
Sequence Homology, Amino Acid
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Summary: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.
ISSN:0261-4189
1460-2075
DOI:10.1002/j.1460-2075.1993.tb06098.x