title: Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike.
abstract: 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 beta-helix during monomer collapse, while G244R is primarily an assembly defect.
authors.affiliation: Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Lefebvre
authors.forename: Noelle K
authors.initials: NK
authors.lastname: Comolli
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Gage
authors.forename: Anne Skaja
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013696:Temperature; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage