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

Abstract

SummaryAn 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 Nterminal 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.Keywords bacteriophage ø29; scaffolding proteins; in vitro capsid assembly; prolate procapsid Correspondence to: Peter E. Prevelige, Jr..

A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

“…regions likely span at least 10 residues of the scaffolding molecule (residues 65-74) and are located upstream of the capsid binding region, which was previously localized to the crystallographically disordered C terminus (14). The correlation between the in vivo and in vitro phenotypes of the S65N scaffolding protein suggested that the connector/scaffolding interactions formed in the complexes in vitro recapitulate those involved in recruiting connector protein in vivo.…”

“…Protein Preparations-The scaffolding and connector proteins were purified as described previously (14,16). The scaffolding deletion mutants ⌬79 -98, ⌬74 -98, ⌬70 -98, and ⌬66 -97 were constructed by introducing a stop codon to replace residue Glu-79, Thr-74, Gln-70, and Lys-66, respectively, using the QuikChange sitedirected mutagenesis kit (Stratagene) with primers 5Ј-gacagacaaacaggaataagatcacaagaaagctgatattagtg-3Ј for ⌬79 -98, 5Ј-gcttttcagacagattggtttgtgagacaaacaggaagaagatc-3Ј for ⌬74 -98, 5Ј-caaatagtaagcttttcagatagattggtttgacagacaaacagg-3Ј for ⌬70 -98, and 5Ј-gatctgatcgtgtcaaatagttagcttttcagacagattgg-3Ј for ⌬66 -97.…”

“…A three-residue loop and a seven-turn helix (␣3) complete the shaft. The C-terminal 15 residues, which interact with capsid protein as determined in the in vitro assembly assay, are disordered in the crystal structure (14).…”

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

“…The microvirus internal scaffolding protein shares many properties and functions with the scaffolding proteins found in other viruses (7,9,10,14,15,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). However, it does not physically construct the procapsid or control morphogenetic fidelity.…”

“…For example, coat proteins from other systems form aberrant assemblies in the absence of scaffolding protein, whereas scaffolding proteins alone are relatively inert. When mixed, the scaffolding proteins control fidelity, suppressing the formation of heterogeneous aberrant structures containing only the coat protein (21,33,(42)(43)(44)(45)(46)(47)(48)(49)(50). In contrast, the X174 external scaffolding protein D self-associates to form large heterogeneous spherical complexes (Fig.…”