title: Molecular dissection of ø29 scaffolding protein function in an in vitro assembly system.
abstract: An in vitro assembly system was developed to study prolate capsid assembly of phage ø29 biochemically, and to identify regions of scaffolding protein required for its functions. The crowding agent polyethylene glycol can induce bacteriophage ø29 monomeric capsid protein and dimeric scaffolding protein to co-assemble to form particles which have the same geometry as either prolate T=3 Q=5 procapsids formed in vivo or previously observed isometric particles. The formation of particles is a scaffolding-dependent reaction. The balance between the fidelity and efficiency of assembly is controlled by the concentration of crowding agent and temperature. The assembly process is salt sensitive, suggesting that the interactions between the scaffolding and coat proteins are electrostatic. Three N-terminal ø29 scaffolding protein deletion mutants, Delta 1-9, Delta 1-15 and Delta 1-22, abolish the assembly activity. Circular dichroism spectra indicate that these N-terminal deletions are accompanied by a loss of helicity. The inability of these proteins to dimerize suggests that the N-terminal region of the scaffolding protein contributes to the dimer interface and maintains the structural integrity of the dimeric protein. Two C-terminal scaffolding protein deletion mutants, Delta 79-97 and Delta 62-97, also fail to promote assembly. However, the secondary structure and the dimerization ability of these mutants are unchanged relative to wild-type, which suggests that the C terminus is the likely site of interaction with the capsid protein.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, AL35294, USA.
authors.forename: Chi-yu
authors.initials: CY
authors.lastname: Fu
authors.forename: Marc C
authors.initials: MC
authors.lastname: Morais
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Battisti
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rossmann
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
mesh_terms: D017100:Bacteriophage P22; D001435:Bacteriophages / Q000737:chemistry*; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D012996:Solutions; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D012996:Solutions; D015678:Viral Structural Proteins; D011092:Polyethylene Glycols; D012965:Sodium Chloride