title: In vitro unfolding/refolding of wild type phage P22 scaffolding protein reveals capsid-binding domain.
abstract: The scaffolding proteins of double-stranded DNA viruses are required for the polymerization of capsid subunits into properly sized closed shells but are absent from the mature virions. Phage P22 scaffolding subunits are elongated 33-kDa molecules that copolymerize with coat subunits into icosahedral precursor shells and subsequently exit from the precursor shell through channels in the procapsid lattice to participate in further rounds of polymerization and dissociation. Purified scaffolding subunits could be refolded in vitro after denaturation by high temperature or guanidine hydrochloride solutions. The lack of coincidence of fluorescence and circular dichroism signals indicated the presence of at least one partially folded intermediate, suggesting that the protein consisted of multiple domains. Proteolytic fragments containing the C terminus were competent for copolymerization with capsid subunits into procapsid shells in vitro, whereas the N terminus was not needed for this function. Proteolysis of partially denatured scaffolding subunits indicated that it was the capsid-binding C-terminal domain that unfolded at low temperatures and guanidinium concentrations. The minimal stability of the coat-binding domain may reflect its role in the conformational switching needed for icosahedral shell assembly.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: B
authors.initials: B
authors.lastname: Greene
authors.forename: J
authors.initials: J
authors.lastname: King
mesh_terms: D001665:Binding Sites; D002213:Capsid / Q000378:metabolism*; D002478:Cells, Cultured; D002942:Circular Dichroism; D006358:Hot Temperature; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011489:Protein Denaturation; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000378:metabolism; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D015678:Viral Structural Proteins; C111088:scaffolding protein, bacteriophage P22; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase