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, forming a single turn of a triple 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 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 beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.
Weigele
Peter R
PR
Department of Biology Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Haase-Pettingell
Cameron
C
Campbell
Phillip G
PG
Gossard
David C
DC
King
Jonathan
J
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics; D002499:Centrifugation, Density Gradient; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D015723:Gene Library; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013696:Temperature; D013698:Templates, Genetic; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018829:Adhesins, Bacterial; D004220:Disulfides; D017376:Viral Tail Proteins