Stalled Folding Mutants in the Triple β-Helix Domain of the Phage P22 Tailspike Adhesin

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple β-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple β-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540–548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540–544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple β-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.