Intragenic Suppression of a Capsid Assembly-Defective P22 Tailspike Mutation

AUTOR(ES)

Maurides, P. A.

RESUMO

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to ``partially unfold.'' Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein. To test this hypothesis we have generated suppressors of the hmH3034 mutation by site-directed, random mutagenesis of codon 13. From the broad spectrum of amino acids at position 13 which function as suppressors of hmH3034 we have concluded that elimination of Arg at position 13 is sufficient in most cases to restore capsid assembly activity to the hmH3034 protein.

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