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Crystallization of the nonameric small terminase subunit of bacteriophage P22

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The packaging of viral genomes into preformed empty procapsids is powered by an ATP‐dependent genome‐translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L‐terminase) and small terminase (S‐terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring‐like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase‐dependent DNA translocation. Here, the gene encoding bacteriophage P22 S‐terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S‐terminase assembles into a concentration‐independent nonamer of ∼168 kDa. Nonameric S‐terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P21212, with unit‐cell parameters a = 144.2, b = 144.2, c = 145.3 Å, and diffracted to 3.0 Å resolution. Crystal form II belonged to space group P21, with unit‐cell parameters a = 76.48, b = 100.9, c = 89.95 Å, β = 93.73°, and diffracted to 1.75 Å resolution. Preliminary crystallographic analysis of crystal form II confirms that the S‐terminase crystals contain a nonamer in the asymmetric unit and are suitable for high‐resolution structure determination.
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Keywords: DNA packaging; bacteriophage P22; large terminase; nonamers; small terminase

Document Type: Research Article

Affiliations: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA

Publication date: January 1, 2011

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