title: An aggregation-prone intermediate species is present in the unfolding pathway of the monomeric portal protein of bacteriophage P22: implications for portal assembly.
abstract: The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.
authors.affiliation: Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.
authors.forename: Carolina A C A
authors.initials: CA
authors.lastname: Braga
authors.forename: Danielle
authors.initials: D
authors.lastname: Carvalho
authors.forename: Flavio Alves
authors.initials: FA
authors.lastname: Lara
authors.forename: Juliana Reis
authors.initials: JR
authors.lastname: Cortines
authors.forename: Sean D
authors.initials: SD
authors.lastname: Moore
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
authors.forename: Debora
authors.initials: D
authors.lastname: Foguel
mesh_terms: D017100:Bacteriophage P22 / Q000502:physiology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006874:Hydrostatic Pressure; D008027:Light; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013696:Temperature; D014508:Urea / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D014764:Viral Proteins; D014508:Urea