The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation. Study holds ProTherm entries: 19610, 19611, 19612 Extra Details: P22 tailspike; protein folding; stability; mutation; ionic interaction
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||3|
|Proteins||Tail spike protein ; Tail spike protein|
|Assays/Quantities/Protocols||Experimental Assay: Cm|
|Libraries||Mutations for sequence YSIEADKKFKYSVKLSDYPTLQDAASAAVDGLLIDRDYNFYGGETVDFGGKVLTIECKAKFIGDGNLIFTKLGKGSRIAGVFMESTTTPWVIKPWTDDNQWLTDAAAVVATLKQSKTDGYQPTVSDYVKFPGIETLLPPNAKGQNITSTLEIRECIGVEVHRASGLMAGFLFRGCHFCKMVDANNPSGGKDGIITFENLSGDWGKGNYVIGGRTSYGSASSAQFLRNNGGFERDGGVIGFTSYRAGESGVKTWQGTVGSTTSRNYNLQFRDSVVIYPVWDGFDLGADTDMNPELDRPGDYPITQYPLHQLPLNHLIDNLLVRGALGVGFGMDGKGMYVSNITVEDCAGSGAYLLTHESVFTNIAIIDTNTKDFQANQIYISGACRVNGLRLIGIRSTDGQGLTIDAPNSTVSGITGMVDPSRINVANLAEEGLGNIRANSFGYDSAAIKLRIHKLSKTLDSGALYSHINGGAGSGSAYTQLTAISGSTPDAVSLKVNHKDCRGAEIPFVPDIASDDFIKDSSCFLPYWENNSTSLKALVKKPNGELVRLTLATL|