REDOR NMR Characterization of DNA Packaging in Bacteriophage T4

Abstract

Bacteriophage T4 is a large-tailed E. coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 minutes during viral infection. We have isolated 50-mg quantities of uniform 15 N and [ε-15 N]lysine-labeled bacteriophage T4. We have also introduced 15 NH 4 + into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo-and cryoprotected lyophilized T4 using 15 N{ 31 P} and 31 P{ 15 N} rotational-echo double resonance. The results of these experiments have shown that: (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH 2 groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid, are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.

Structure, Assembly, and DNA Packaging of the Bacteriophage T4 Head

“…Solid-state nuclear magnetic resonance analysis of phage T4 particle shows that DNA is largely B form and allows its electrostatic interactions to be tabulated (Yu and Schaefer, 2008). This study reveals high-resolution interactions bearing on the internal structure of the phage T4 head.…”

High Resolution 31P NMR Spectroscopy Generates a Quantitative Evolution Profile of Phosphorous Translocation in Germinating Sesame Seed

“…More advanced 31 P NMR methods such as heteronuclear NMR experiments involving 31 P for studying the large-scale structure 26 or complex biochemical reactions 27 in biological systems were also demonstrated to be feasible and powerful. Gambhir et al .…”

Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

“…Detailed structural information on viral genomes in native states is not easily accessible and the secondary structures of encapsulated DNAs were primarily studied by Raman spectroscopy, circular dichroism, and more recently by ssNMR [42,43,45]. Prior studies on tailed dsDNA phages show that the mechanisms of viral genome packaging and release are similar presumably since their genomes are highly condensed to a comparable degree [75].…”

“…In a ssNMR study of the icosahedral phage T4, Yu and Schaefer [43] used 15 N{ 31 P} and 31 P{ 15 N} REDOR [88] experiments on lyophilized 15 N-lysine-labeled phage, and on unlabeled phage supplemented with 15 NH 4 + ; they observed correlations between positively charged amines and the negatively charged genomic phosphates. These experiments provided evidence for the partial charge balancing of the DNA in T4 and suggested electrostatics as a driving force for DNA packaging.…”

“…We describe the experimental approaches to NMR studies of capsid structure and dynamics, DNA packing and protein-DNA interactions, and discuss their outcomes. The results presented here are based on a series of publications from recent years [37][38][39][40][41][42][43][44][45] and some new insights into silver-doped fd phage particles. While this article discusses bacteriophage viruses, in recent years a variety of ssNMR techniques have been successfully applied to study structure and dynamics of HIV viral capsids [46,47] and most recently proton detected experiments combined with fast MAS were utilized to study measle-virus capsids [48], both of which have human hosts.…”