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Pressure dissociation studies provide insight into oligomerization competence of temperature‐sensitive folding mutants of P22 tailspike
Author(s) -
Lefebvre Brian G.,
Comolli Noelle K.,
Gage Matthew J.,
Robinson Anne Skaja
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03579304
Subject(s) - trimer , chemistry , dimer , mutant , monomer , protein folding , biophysics , folding (dsp implementation) , dissociation (chemistry) , crystallography , biochemistry , organic chemistry , biology , polymer , electrical engineering , gene , engineering
Several temperature‐sensitive folding ( tsf ) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild‐type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer. The effects of pressure on two different tsf mutants, G244R and E196K, were explored. Pressure treatment of both G244R and E196K aggregates produced a folded trimer. E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein. In contrast, trimer formation from pressure‐treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment. The center‐of‐mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates. Taken together, these results suggest that E196K has a primary defect in formation of the β‐helix during monomer collapse, while G244R is primarily an assembly defect.