In vivo folding efficiencies for mutants of the P22 tailspike β-helix protein correlate with predicted stability changes Original Research Article

Parallel β-helices are among the simplest repetitive structural elements in proteins. The folding behavior of β-helix proteins has been studied intensively, also to gain insight on the formation of amyloid fibrils, which share the parallel β-helix as a central structural motif. An important system for investigating β-helix folding is the tailspike protein from the Salmonella bacteriophage P22. The central domain of this protein is a right-handed parallel β-helix with 13 windings. Extensive mutational analyses of the P22 tailspike protein have revealed two main phenotypes: temperature-sensitive-folding (tsf) mutations that reduce the folding efficiency at elevated temperatures, and global suppressor (su) mutations that increase the tailspike folding efficiency. A central question is whether these phenotypes can be understood from changes in the protein stability induced by the mutations. Experimental determination of the protein stability is complicated by the nearly irreversible trimerization of the folded tailspike protein. Here, we present calculations of stability changes with the program FoldX, focusing on a recently published extensive data set of 145 singe-residue alanine mutants. We find that the calculated stability changes are correlated with the experimentally measured in vivo folding efficiencies. In addition, we determine the free-energy landscape of the P22 tailspike protein in a nucleation–propagation model to explore the folding mechanism of this protein, and obtain a processive folding route on which the protein nucleates in the N-terminal region of the helix.