REDOR NMR Characterization of DNA Packaging in Bacteriophage T4


Journal of Molecular Biology Volume 382, Issue 4, 17 October 2008, Pages 1031-1042

ABSTRACT
Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120 x 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform 15N- and [e-15N]lysine-labeled bacteriophage T4. We have also introduced 15NH4+ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using 15N{31P} and 31P{15N} 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 --NH2 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.

Structures, atom-numbering schemes, and 15N isotropic chemical shifts for adenosine, guanosine, 2'-deoxythymidine, and cytidine.

Conformations of B-DNA (PDB code 1d56) and A-DNA (PDB code 1vj4). Phosphorus atoms (blue) and nitrogen atoms (red) are highlighted.

Experimental (circles) and calculated (continuous lines) 15N{31P} REDOR dephasing (DS/S0) for the 127-ppm N1 peak of thymine and guanosine of phage T4 DNA, assuming an A-DNA model (red) or a B-DNA model (black). Estimated errors are within the symbols.

15N{31P} REDOR dephasing (DS/S0) for the 9-ppm amine peak as a function of dipolar evolution time for uniform 15N and [e-15N]lysine-labeled bacteriophage T4. The observed dephasing (filled circles and squares) of [e-15N]lysine-labeled phage T4 (100 mg lysine/L media, red; and 200 mg lysine/L, black) is consistent with that calculated (red line) assuming a single 15N--31P distance of 3.5 A. The observed dephasing (open circles) of uniform 15N-labeled phage T4 is consistent with that calculated assuming an 15N--31P distance of 3.5 (+-0.2) A for 25 (+-3)% of the amines, and 5.5 A for 16%.

31P{15N} dbFSR spectra of uniform 15N-labeled phage T4 after 128 Tr of dipolar evolution. The full-echo spectrum (S0) is at the bottom of the figure and the REDOR difference spectrum (DS) is at the top. The DANTE carrier frequency was centered at the 9-ppm amine peak. The spectra on the left show sidebands resulting from magic-angle spinning at 6250 Hz. The NMR signal was digitized at 64 times the spinning speed so that the Fourier transform of every 64th time-domain data point resulted in the rotor-synchronized spectra on the right. The spectra were the result of the accumulation of 316,968 scans.

 

dbFSR pulse sequence developed by Kaustov et al. (reference 24). N/2 is an integral multiple of eight and the phase cycling for each eight-pulse unit is xy8. Each DANTE Pi/2 pulse is composed of n/2 rotor-synchronized Pi/n RF pulses. The small CSA of the --NH2 moiety allows multiple pulses per rotor period.