P22 tailspike folding mutants revisited: effects on the thermodynamic stability of the isolated beta-helix domain

Abstract

The folding of the trimeric phage P22 tailspike protein is influenced by amino acid substitutions of two types, virtually all of which affect residues in the central domain, a large parallel beta-helix. Temperature sensitive folding (tsf) mutations lead to drastically decreased folding yields at elevated temperature. Their phenotype can be alleviated by global suppressor (su) mutations. Both types of mutations appeared to have no influence on the stability of the native protein at the time of their first isolation and were thus suggested to carry information needed for the folding pathway exclusively. The monomeric beta-helix of tailspike, expressed as an isolated domain, exhibits freely reversible unfolding and refolding transitions, allowing us to analyse the effects of two well-characterised tsf and all four known su mutations on its thermodynamic stability. We find a marked decrease in stability for the tsf mutants and a striking increase in stability for all su mutants. This leads to the conception that the isolated beta-helix domain, although active in receptor-binding and native-like in its spectroscopic properties, is close in conformation to a crucial monomeric folding intermediate whose thermolability is responsible for the kinetic partitioning between productive folding and irreversible aggregation during the maturation process of P22 tailspike protein.