Molecular Dissection of Ø29 Scaffolding Protein Function in an in Vitro Assembly System

Chi yu Fu, Marc C. Morais, Anthony J. Battisti, Michael G. Rossmann, Peter E. Prevelige

Research output: Contribution to journalArticle

29 Scopus citations

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, Δ 1-9, Δ 1-15 and Δ 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, Δ 79-97 and Δ 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.

Original languageEnglish (US)
Pages (from-to)1161-1173
Number of pages13
JournalJournal of Molecular Biology
Volume366
Issue number4
DOIs
StatePublished - Mar 2 2007

Keywords

  • bacteriophage ø29
  • in vitro capsid assembly
  • prolate procapsid
  • scaffolding proteins

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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