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Need More Resolution?

February 2008--Only by visually detailing the nooks and crannies within each protein can an investigator truly understand that molecule's bioactivity. And in this particular arena, X-ray crystallography has long been the 3-D standard. However, prospective structure seekers do have a second tool at their disposal: nuclear magnetic resonance or NMR spectroscopy, which provides its own unique “spin.” 

 As the name implies, NMR exploits the inherent magnetic properties of certain atoms—isotopes with an odd number of protons or neutrons like hydrogen, 13C, 15N and 31P. “These nuclei behave like tops spinning about an axis, much like the earth,” explains Ananya Majumdar, director of Hopkins’ NMR facility. “When we place them in a magnetic field and shine radio waves on them, the nuclei absorb electromagnetic energy and emit signals that are recorded as an NMR spectrum.” Majumdar adds that the signals sent by each atom depend on both the atom itself and all the atoms in the immediate vicinity. “Every molecule, therefore, has a unique NMR spectrum, which we use to build its structure.”

The key advantage NMR holds over crystallography, says Pharmacology’s Jim Stivers, “is that you can determine the structure of a biomolecule in solution as opposed to trapping it in a solid crystalline state. This enables you to not only define the physical shape of a molecule, but also how it moves over time.”

 That ability to detect motion explains why NMR resonates with researchers like Stivers. His lab studies the mechanisms behind DNA repair, a dynamic process in which enzymes scan our genetic code for errors and then remove them. One such repair enzyme known as uracil DNA glycosylase specifically plucks out uracils, bases that are supposed to be in RNA not DNA. Stivers has been intrigued as to how this enzyme operates, since the UDG crystal structure suggested that it recognizes uracil only when it’s pulled out from the DNA duplex.

The question that arose was whether the enzyme actively pops out the uracil, or if it finds the mistake passively. “DNA breathes, using thermal energy to open and close,” says Stivers, “so the enzyme may just happen to land on a uracil while it is in an open state, or alternatively, it could somehow find the uracil and actively pull it out of the DNA duplex.” Stivers wondered if NMR could resolve this problem.

He reasoned that, like proteins, DNA bases contain magnetic nuclei. While concealed in a DNA duplex, these protons are used, via Watson-Crick base pairing, to hold the DNA together. However, once exposed, they can interact with water. “We used a little trick where we magnetically label the water protons so that they align in the opposite direction from the DNA base protons,” says Stivers. “And when water protons come in contact with an exposed base, the base protons flip their polarity as well, which we can detect with NMR.”

He tried this trick with and without UDG and found that the enzyme did not speed up the breathing process, which provided strong evidence for the passive DNA damage recognition model. He also determined the enzyme’s structural and dynamic changes during base inspection and found that if it grabbed onto a normal DNA base, then it held on only briefly before releasing it, while uracil had just the right shape to get pushed farther into the enzyme pocket and excised from the DNA strand—a multistep process that only could be inferred from the UDG crystal structure. Stivers currently is preparing similar tricks to examine the back-and-forth between DNA and the enzymes that unwind it, topoisomerases. The dynamic properties of topoisomerase-DNA complexes play an important role in the binding of some anticancer drugs.

Stivers hopes that NMR’s popularity will keep growing to keep pace with X-ray crystallography, since the two methods are complementary; the highly detailed snapshots produced by crystal structures provide a great starting point to ask biological questions that NMR might be used to answer. While crystallography of biomolecules is a fairly established methodology, NMR remains a maturing field that requires at least two to three years’ training to properly execute and comprehend the experiments. Fortunately, Hopkins is doing a lot to make that happen. “Our facility and training program, led by Ananya, is top-notch. Students who use NMR in their labs get practical, hands-on courses and are given the opportunity to really grasp the methods,” he says. “I wish I’d had such focused training when I was learning the ropes.”

--Nick Zagorski

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