Out of Focus: Language Barrier
While you won’t catch more than a hint of a Canadian accent in speaking with Miller these days, she admits to having had occasional communication “challenges” when she first moved from Guelph to New Haven, Conn. This led to one of her more bizarre graduate school experiences. One day, while returning from school, she was approached and accosted by a pair of youths who demanded her bicycle. “Their accent was so strong and foreign to me that I could barely understand them,” she says. Add in the fact that she came from a small, quiet college town, and she was not prepared for such a situation. “So rather than run away immediately (and lose my bike), I responded with a polite, if scared, refusal. Then they had trouble comprehending me. After several back-and-forth exchanges in which I can remember thinking I was completely crazy to be insisting on retaining my bicycle and repeating ‘I beg your pardon’ (because I still could not understand their English), instead of fleeing back up the street, one of them cracked a smile.” She says, “This whole conversation was probably the last thing they expected and in retrospect, it really was humorous. Once it had become a joke, they waved me on and I rode off. I would, nonetheless, not recommend this as a general strategy.”
Other recent spectroscopic analysis has revealed insights into how superoxide dismutase controls the movement of the electrons between the active site metal ion and substrate. “Proteins do not have good means of controlling electrons directly,” Miller says. “But we found that the big bridge by which superoxide dismutases regulate the sources and destinations of the transferred electrons is the protein’s exceptional control over protons, because the protons have a very big influence over where the electrons go.”
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.