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The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

ABSTRACT: Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (?) scaffolding protein gpNu3. The purified protein possesses significant ?-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T(m)=40.6±0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K(d,app)~50 ?M) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the ? major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.

SUBMITTER: Medina EM

PROVIDER: S-EPMC3247018 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

Medina Eva Margarita EM Andrews Benjamin T BT Nakatani Eri E Catalano Carlos Enrique CE

Journal of molecular biology 20110729 4

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to ...[more]

PMID: 21821043

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Project description:During ?X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ?X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE:Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ?X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

Dataset Information

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

Publications

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

Similar Datasets

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