Nature and distribution of sites of temperature-sensitive folding mutations in the gene for the P22 tailspike polypeptide chain

Abstract

Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.