ABSTRACT
Weigele, P., Haase-Pettingell, C., Campbell, P.G., Gossard, D.C. and King, J. (2005) J. Mol.Biol.
Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin
The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine constituting a single turn of a tiple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-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 paritally folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540 to 548, where the sister chains begin to wrap around each other. From this set, we isolated and characterized mutants 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 to 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 protrimer to native trimer transition were also destabilized in their native state. These data suggested that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.
