H. Smith and Jonathan King (1981) J. Mol. Biol., 145, 653-676.
Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tailspike protein. III. Inactive polypeptide chains synthesized at 39 degrees.
cells infected by temperature-sensitive mutants in gene 9 of bacteriophage
P22 at the restrictive temperature (39°C) fail to accumulate functional
tailspike protein. We report here studies of the inactive mutant tailspike polypeptide
chains synthesized at 39°C by temperature-sensitive mutants at 15 different
sites of gene 9. For all 15 mutants, the gene 9 polypeptide chains
were synthesized at 39°C at rates similar to wild type. The mutant polypeptide
chains were stable within the infected cells.
The inactive polypeptide chains were tested for three functions displayed by the native tailspike protein: irreversible binding to phage heads, endorhamnosidase activity, and reaction with anti-tail antibody. The 15 mutant proteins that accumulated at 39°C lacked all three functions. Since the amino acid substitutions do not affect these functions of the mature protein, the mutant polypeptide chains synthesized at 39°C have a conformation very different from the wild type, and different from the same proteins when matured at 30°C. The fact that amino acid substitutions throughout the 76,000 Mr polypeptide chain prevent all three functions suggests that the mutations prevent the correct folding of the gene 9 polypeptide chain at restrictive temperature. Thus, these mutations indentify sites in the polypeptide chain critical for protein maturation.
Many of the mutant proteins could be activated in the absence of new protein synthesis by shifting infected cells from restrictive to permissive temperature before cell lysis. For these mutants, the immature chains accumulating at high temperature must be reversibly related to intermediates in protein folding or subunit assembly.
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