ABSTRACT
Donna
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.
Salmonella
typhimurium
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.