January 2010

Anne-Frances Miller: Spinning Toward Success

Miller chose enzymes that use flavins as cofactors as her second interest, because these cofactors, which resemble nucleotides, hearken back to the ancient RNA world and are likely the remnants of the evolutionary ancestors to enzymes. And, as organic molecules, not inorganic metal ions, they have different spectroscopic properties that enable Miller to ask a different set of questions.

Solid-state NMR, which, as implied by the name, examines samples that are solids or frozen solutions, can prevent the molecules under study from moving or reorienting. This allows orientation-dependent properties to be observed in the spectra, and, in Miller’s case, allows the three orientationally distinct components of the chemical shift to be resolved.

Miller has looked at the carbon and nitrogen atoms of the flavin ring system to complement solution NMR studies of the surrounding amino acids of the protein. Most importantly, the solid-state NMR results often can distinguish between effects on different orbitals of the flavin, resulting from different interactions between the flavin and the protein. With that information, she hopes to understand how different protein environments cause the bound flavin to emphasize different reactivities out of its inherently broad repertoire. Meanwhile, solution NMR studies of the surrounding protein address issues such as how some flavoenzymes like nitroreductase have such a broad substrate specificity range.

Beyond these studies, though, Miller is also busy trying to improve on the existing NMR and EPR technologies, so as to give them a broader and more cost-effective appeal.

In discussing her drive to do this, Miller reflects back on when she first came to the U.S. for graduate school. “At the time I left Guelph, there were very few positions available in Canada, as funding for universities was very tight,” she says. “My professors not only repaired laboratory equipment themselves, because they couldn’t afford to get it serviced, they built the equipment themselves as well.”

Considering the perilous nature of today’s economy, such memories resurface. “In a time of tightening budgets, there will be questions about the need to continue to run expensive NMR facilities,” she says, adding that the cost not only reflects the machines but the cryogens and reagents (like heavy isotopes of carbon and nitrogen) required to produce NMR-quality samples. While NMR holds many advantages as a tool for structure determination, it is weak when it comes to sensitivity because the magnetic moments of nuclei are quite small, thus, requiring large amounts of pure protein in each sample.

Some research groups have begun trying to alleviate the sensitivity problem by combining elements of NMR and EPR technology in a new application known as dynamic nuclear polarization. Rather than directly polarizing (or exciting) nuclear magnetic moments, DNP polarizes electrons first, as they have magnetic moments about 660 times that of the 1H magnetic moment. DNP then transfers that polarization to nearby nuclei. “So in theory,” says Miller, “you could have an NMR signal that’s 660 times more powerful than usual, which is mind-boggling.”

Thanks to a sabbatical she took, Miller, in collaboration with Thorsten Maly and Robert G. Griffin at MIT’s magnet lab, has tried to take DNP one step further. “Currently, DNP relies on added free radicals as bearers of the unpaired electrons,” she says, “but I realized that biology provides built-in radicals whose unpaired electrons can be used as sources of polarization. Many flavoproteins can be prepared with the flavin in a radical state, and the flavin molecule is bound in exactly the same way in each molecule. So we know where the polarization starts in every instance, in contrast with the random and uncontrolled locations of exogenous radicals.” Moreover, the flavin radical is often located in the enzyme’s active site.

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Fascinating! - Eric B

 

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