Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR

被引:16
作者
Abramov, Gili [1 ]
Goldbourt, Amir [1 ]
机构
[1] Tel Aviv Univ, Sch Chem, Raymond & Beverly Sackler Fac Exact Sci, IL-69978 Tel Aviv, Israel
基金
以色列科学基金会;
关键词
Magic angle spinning; Solid state NMR; T7; bacteriophage; dsDNA; Chemical shift assignment; Virus; ANGLE-SPINNING NMR; DOUBLE-STRANDED DNA; RESONANCE ASSIGNMENT; SECONDARY STRUCTURE; HYDROGEN-BONDS; C-13; NMR; EXPERIMENTAL CONSTRAINTS; RNA NUCLEOSIDES; AMYLOID FIBRILS; HIGH-RESOLUTION;
D O I
10.1007/s10858-014-9840-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply the magic-angle spinning (MAS) solid-state NMR (SSNMR) technique to study a uniformly C-13 and N-15 labeled wild-type T7 phage. We describe the details of the large-scale preparation and purification of an isotopically enriched phage sample under fully hydrated conditions, and show a complete C-13 and a near-complete N-15 nucleotide-type specific assignment of the sugar and base moieties in the 40 kbp dsDNA of T7 using two-dimensional C-13-C-13 and N-15-C-13 correlation experiments. The chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS SSNMR was found to be extremely useful in determining the structures of proteins in native-like environments, its application to nucleic acids has lagged behind, leaving a missing C-13 and N-15 chemical shift database. This work therefore expands the C-13 and N-15 database of real B-form DNA systems, and opens routes to characterize more complex nucleic acid systems by SSNMR.
引用
收藏
页码:219 / 230
页数:12
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