Structure of Bacteriophage P22 Portal Protein in Relation to Assembly:  Investigation by Raman Spectroscopy

Abstract

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.

Raman spectroscopy of proteins: from peptides to large assemblies

“…90 The portal assembly is a molecular motor associated with one five-fold vertex of the icosahedral procapsid and its major component is a dodecahedral portal protein (Gp1). 91 The assembly of the portal dodecamer from its monomeric subunits was studied by Raman spectroscopy. 91 Only minute changes in the protein secondary structure were detected.…”

“…91 The assembly of the portal dodecamer from its monomeric subunits was studied by Raman spectroscopy. 91 Only minute changes in the protein secondary structure were detected. On the other hand, aromatic side-chains and cysteine sulfhydryls substantially changed their environments and hydrogen bonding patterns.…”

“…90,93 Hence the dodecamer assembly is most likely mediated by similar domain movements. 91 The role of individual cysteines was subsequently examined by a combination of site-directed mutagenesis and Raman spectroscopy. 94 Cysteine 173 was found essential for the correct folding and assembly of the dodecamer while Cys283 !…”

Hybrid organic-inorganic nanoparticles: controlled incorporation of gold nanoparticles into virus-like particles and application in surface-enhanced Raman spectroscopy

“…Time-resolved Raman spectroscopy therefore seems to be the ideal tool to elucidate the assembly pathway as well as structural intermediates in icosahedral viruses. While the assembly of some bacteriophages has been probed by Raman spectroscopy (20)(21)(22)(23)(24)(25), this technology has not yet been employed in the characterization of viral parasites of mammals. This is probably due to the fact that these viruses are difficult to obtain in the quantities required for Raman spectroscopy, while bacteriophages can easily be obtained in quantities of several hundred milligrams.…”

“…Raman spectroscopy is a very powerful technique to probe structure and conformational changes in molecules, although it has only seldomly been applied to such large macromolecules as viruses (20)(21)(22)(23)(24)(25). Another big advantage of Raman spectroscopy is its insensitivity to water, making it possible to probe molecules in solution.…”