Mutations that stabilize folding intermediates of phage P22 tailspike protein: Folding in vivo and in vitro, stability, and structural context.

The folding of the trimeric phage P22 tailspike protein is affected by single amino acid substitutions designated temperature-sensitive folding (tsf) mutations. Their phenotypes are alleviated by two repeatedly isolated global suppressor (su) mutations (su V331A and su A334V) and by two additional substitutions (su V331G and su A334I), accessible through site-directed mutagenesis. We investigated the influence of the suppressor mutations on tailspike refolding in vitro, on its maturation at high expression levels' in vivo, and on the rates of thermal unfolding of the native protein. All su mutations improved the folding efficiency in vitro and in vivo, but the relative effects of substitutions at position 334 were more pronounced in vivo, whereas the 331 substitutions were more effective in vitro. V331G caused the strongest increase in refolding yields of any single mutation, and was as effective as the V331A/A334V double mutation, where the two single mutations exhibited an additive effect. Both V331A and V331G retarded thermal denaturation, while A334V did not affect, and A334I accelerated unfolding. A334I is the first mutation found to affect the folding of the tailspike and the thermal stability of the native protein in opposite directions. The observed effects can be rationalized on the basis of the recently determined crystal structure of an N-terminally shortened tailspike. As the backbone dihedral angles of Val331 (? = -119°, ψ = -142°) are unusual for non-glycine residues, V331G and V331A may remove steric strain and thereby stabilize folding intermediates and the native protein. The β-branched side-chains of Val and Ile substituted for Ala334 in the interior of the protein may improve a hydrophobic stack of residues in the large parallel β-helix. This is likely important in loosely structured early folding intermediates, but not in the very rigid native structure, where the side-chain of Ile can hardly be accommodated.ope