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.
“So, instead of having to analyze an entire protein, you can take a shortcut and focus your measurements just on the active site,” she continues. This “smart” DNP, as Miller refers to it, should make the technique more applicable than ever, as a researcher won’t need large quantities of protein or even a pure sample. Only protein molecules containing the flavin would be evident in a DNP-NMR spectrum.
Miller, A.-F. (2008) Redox Tuning Over Almost 1 V in a Structurally-conserved Active Site: Lessons from Superoxide Dismutase. Acc. Chem. Res. 41, 501-510.
Koder, R. L.,Walsh, J. D., Pometun, M. S., Dutton, P. L., Wittebort, R. J., and Miller, A.-F. (2006) 15N Solid-state NMR Provides a Sensitive Probe of Oxidized Flavin Reactive Sites. J. Am. Chem. Soc. 128, 15200–15208.
Vance, C. K. and Miller, A.-F. (1998) A Simple Proposal that can Explain the Inactivity of Metal-substituted Superoxide Dismutases. J. Am. Chem. Soc. 120, 461-467.
Miller, A.-F., Halkides, C. J., and Redfield, A. G. (1993) An NMR Comparison of the Changes Produced by Different Guanosine 5’-Triphosphate Analogs in Wild-type and Oncogenic Mutant p21ras. Biochemistry 32, 7367–7376.
Miller, A.-F. and Brudvig, G. W. (1989) Manganese and Calcium Requirements for Reconstitution of Oxygen-Evolution Activity in Manganese-Depleted Photosystem II Membranes. Biochemistry 28, 8181–8190.
Nick Zagorski (firstname.lastname@example.org) is a science writer at ASBMB.