title: Reconstitution of the thermostable trimeric phage P22 tailspike protein from denatured chains in vitro.
abstract: Intermediates in the intracellular chain folding and association pathway of the P22 tailspike endorhamnosidase have been identified previously by physiological and genetic methods. Conditions have now been found for the in vitro refolding of this large (Mr = 215,000) oligomeric protein. Purified Salmonella phage P22 tailspikes, while very stable to urea in neutral solution, were dissociated by moderate concentrations of urea at acidic pH. The tailspike protein was denatured to unfolded polypeptide chains in 6 M urea, pH 3, as disclosed by analytical ultracentrifugation, fluorescence, and circular dichroism. Upon dilution into neutral buffer at 10 degrees C, the polypeptides fold spontaneously and associate to form trimeric tailspikes with high yield. Like native phage P22 tailspikes, the reconstitution product is resistant to denaturation by dodecyl sulfate in the cold and displays endorhamnosidase activity. Sedimentation coefficients, electrophoretic mobility, and fluorescence emission maxima of native and reconstituted tailspikes are identical within experimental error. By characterization of intermediates, localization of temperature-sensitive steps, and analysis of the effect of previously identified folding mutations, the reconstitution system described should allow comparison of in vivo and in vitro folding pathways of this large protein oligomer.
authors.affiliation: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
authors.forename: R
authors.initials: R
authors.lastname: Seckler
authors.forename: A
authors.initials: A
authors.lastname: Fuchs
authors.forename: J
authors.initials: J
authors.lastname: King
authors.forename: R
authors.initials: R
authors.lastname: Jaenicke
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012483:Salmonella Phages; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins; D017376:Viral Tail Proteins
chemical_list: D014764:Viral Proteins; D017376:Viral Tail Proteins