Rotational-echo double resonance NMR characterization of DNA packaging in bacteriophage T4 and of Saccharomyces cerevisiae lumazine synthase-inhibitor complexes. Tsyr-Yan Yu

ISBN: 9780549839101

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150 pages


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Rotational-echo double resonance NMR characterization of DNA packaging in bacteriophage T4 and of Saccharomyces cerevisiae lumazine synthase-inhibitor complexes.  by  Tsyr-Yan Yu

Rotational-echo double resonance NMR characterization of DNA packaging in bacteriophage T4 and of Saccharomyces cerevisiae lumazine synthase-inhibitor complexes. by Tsyr-Yan Yu
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Bacteriophage T4 is a large-tailed E. coli virus whose capsid is 120 nm x 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 15N and [&egr--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{lcub}31P{rcub} and 31 P{lcub}15N{rcub} 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.-The 15N{lcub}31P{rcub} REDOR NMR spectra of three metabolically stable phosphonate reaction intermediate analogues complexed to Saccharomyces cerevisiae lumazine synthase have been obtained at 30 MHz and 50 MHz. Distances from the phosphorus atoms of the ligand groups to the side-chain nitrogens of Lys92, His97, Arg136, and His148 have been determined.

These distances were used as restraints in molecular dynamics simulations which began with the X-ray crystal coordinates of one of the intermediate analogues complexed with the enzyme. The resulting models indicate that mobility of the Lys92 side chain could facilitate phosphate exchange in the enzyme-catalyzed reaction.-We present a McKay-OConnor-Potter design of a four-frequency transmission-line probe, which has smaller size and better efficiency than the typical design of a McKay transmission-line probe.

For this probe, we present a simple design to control a commercial NMR transmitter to achieve active control of the power level of the applied radio-frequency field.



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