Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

King, Jonathan; Hall, Cecil E.; Casjens, Sherwood

doi:10.1016/0092-8674(78)90023-5

Cited by 47 publications

(60 citation statements)

References 35 publications

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“…Only four strains of independent origin defective in scaffolding protein function have been isolated over a period spanning two decades (11,12). This represents a property of the protein, rather than the gene, because nonsense mutations generating stop codons occur at expected frequencies (10,13).…”

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confidence: 99%

Folding and Stability of Mutant Scaffolding Proteins Defective in P22 Capsid Assembly

Greene

King

1999

Journal of Biological Chemistry

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Bacteriophage P22 scaffolding subunits are elongated molecules that interact through their C termini with coat subunits to direct icosahedral capsid assembly. The soluble state of the subunit exhibits a partially folded intermediate during equilibrium unfolding experiments, whose C-terminal domain is unfolded (Greene, B., and King, J. (1999) J. Biol. Chem. 274, 16135-16140). Four mutant scaffolding proteins exhibiting temperature-sensitive defects in different stages of particle assembly were purified. The purified mutant proteins adopted a similar conformation to wild type, but all were destabilized with respect to wild type. Analysis of the thermal melting transitions showed that the mutants S242F and Y214W further destabilized the C-terminal domain, whereas substitutions near the N terminus either destabilized a different domain or affected interactions between domains. Two mutant proteins carried an additional cysteine residue, which formed disulfide cross-links but did not affect the denaturation transition. These mutants differed both from temperaturesensitive folding mutants found in other P22 structural proteins and from the thermolabile temperature-sensitive mutants described for T4 lysozyme. The results suggest that the defects in these mutants are due to destabilization of domains affecting the weak subunitsubunit interactions important in the assembly and function of the virus precursor shell.

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Folding and Stability of Mutant Scaffolding Proteins Defective in P22 Capsid Assembly

Greene

King

1999

Journal of Biological Chemistry

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“…In the absence of the gene 13 product, infected cells do not lyse and phage protein synthesis continues past the normal lysis time. Late after infection by the 5-113-phage, the tail spike polypeptide chain is one of the major proteins synthesized (18).…”

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Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Goldenberg¹,

King²

1982

Proc. Natl. Acad. Sci. U.S.A.

113

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Newly synthesized tail spike polypeptide chains mature from trypsin-and NaDodSO4-sensitive unfolded chains to trypsin-and NaDodSO4-resistant native trimers with a t1,2 of 5 min at 300C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.

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“…This mutation prevents the packaging of phage DNA into newly formed procapsids, thereby preventing addition of the neck protein and prolonging lysis. As a result, all newly synthesized tailspike chains remain in the cytoplasm (30). In addition, unpackaged DNA also is involved in upregulating the total amount of tailspike synthesized.…”

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confidence: 99%

Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium

Pope

Haase-Pettingell

King

2004

Appl Environ Microbiol

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The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.

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Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

Cited by 47 publications

References 35 publications

Folding and Stability of Mutant Scaffolding Proteins Defective in P22 Capsid Assembly

Folding and Stability of Mutant Scaffolding Proteins Defective in P22 Capsid Assembly

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium

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