Complex additivity of the effects of suppressor mutations in differing protein environments.
In the alpha-complementation of beta-galactosidase, a defective beta-galactosidase protein interacts with an autologous peptide fragment (alpha-peptide) to restore enzymatic activity. Within a specific site of a defective alpha-peptide we have previously isolated a large number of mutations, many of which suppress the functional defect. The alpha-peptide was originally defective due to both insertional and substitutional sequence alterations near its N-terminus, which provided an increase in the sensitivity of detection of (suppressor) secondary mutations which conferred improved function. We have now studied the effects of the suppressor mutations when the primary deleterious mutations are sequentially reversed. This was done in intact beta-galactosidase, as we have shown that mutations in the alpha-peptide have related functional effects in the whole protein. Evidence was obtained showing that the effects of at least some suppressor mutations were not simply additive when the mutations are placed into the original wild-type protein environment. One suppressor appeared to function less effectively in the normal environment, while another when tested in the same manner functioned at a relatively increased level. This failure to show simple additivity may be attributable to the physical proximity of the original defective mutations and the introduced suppressors. Nevertheless, even in such cases it may be feasible to use a defective protein as a sensitive starting point for the identification of mutations which improve the wild-type protein.
Dunn
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CSIRO Division of Biomolecular Engineering, North Ryde, NSW, Australia.
Jennings
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PA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D013489:Suppression, Genetic / Q000187:drug effects / Q000235:genetics*; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D001616:beta-Galactosidase; D008274:Magnesium