Complete Chemical Shift Assignment of the ssDNA in the Filamentous Bacteriophage fd Reports on Its Conformation and on Its Interface with the Capsid Shell
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- 作者：Omry Morag ; Gili Abramov ; Amir Goldbourt
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：February 12, 2014
- 年：2014
- 卷：136
- 期：6
- 页码：2292-2301
- 全文大小：821K
- ISSN：1520-5126
文摘
The fd bacteriophage is a filamentous virus consisting of a circular single-stranded DNA (ssDNA) wrapped by thousands of copies of a major coat protein subunit (the capsid). The coat protein subunits are mostly 伪-helical and curved, and are arranged in the capsid in consecutive pentamers related by a translation along the main viral axis and a rotation of 36掳 (C5S2 symmetry). The DNA is right-handed and helical, but information on its structure and on its interface with the capsid is incomplete. We present here an approach for assigning the DNA nucleotides and studying its interactions with the capsid by magic-angle spinning solid-state NMR. Capsid contacts with the ssDNA are obtained using a two-dimensional 13C鈥?sup>13C correlation experiment and a proton-mediated 31P鈥?sup>13C polarization transfer experiment, both acquired on an aromatic-unlabeled phage sample. Our results allow us to map the residues that face the interior of the capsid and to show that the ssDNA鈥揷apsid interactions are sustained mainly by electrostatic interactions between the positively charged lysine side chains and the phosphate backbone. The use of natural abundance aromatic amino acids in the growth media facilitated the complete assignment of the four nucleotides and the observation of internucleotide contacts. Using chemical shift analysis, our study shows that structural features of the deoxyribose carbons reporting on the sugar pucker are strikingly similar to those observed recently for the Pf1 phage. However, the ssDNA鈥損rotein interface is different, and chemical shift markers of base pairing are different. This experimental approach can be utilized in other filamentous and icosahedral bacteriophages, and also in other biomolecular complexes involving structurally and functionally important DNA鈥損rotein interactions.