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To bridge the information obtained by crystallography and NMR, Kern's team performed a third experiment—a computer simulation that calculated how fast the molecule could move between the structures seen in the crystal. The simulation only added confusion. It indicated that the enzyme could move between the open and partially closed states seen in the crystal in nanoseconds—that is, thousands of times faster than the milliseconds suggested by NMR.
Kern had an idea of what was going on but needed the help of her brother, a physicist at Germany's Martin Luther University Halle-Wittenberg, to experimentally reveal the inner workings of adenylate kinase. “It was a real collaboration where we were flying back and forth across the ocean,” says Kern, who credits her light-hearted brother for making the partnership lots of fun.
They performed single molecule fluorescence resonance energy transfer (FRET) experiments—tacking fluorescent molecules onto the upper and lower lids of the clam-shaped enzyme and tracking its opening and closing by measuring changes in fluorescence. Huebner had designed and built a unique ultrasensitive laser that allowed precise measurements and time resolution in microseconds.
The siblings found that every few nanoseconds—as the computer simulation had shown—the enzyme twitches partially shut. But every few milliseconds—as suggested by NMR—it closes all the way.
The traditional view had been that an enzyme snaps shut only when it makes contact with its substrate. But Kern and Huebner showed that an enzyme can constantly fidget and take on a new structure without that contact. And it's not just in this one instance—Kern has detected twitching in the handful of other enzymes she's tested so far using NMR. “This is really a paradigm shift,” she says. “We want to encourage scientists to consider that this happens with their own systems. So far, it seems that these short-lived, higher energy states quite often are the biologically active states.”
Returning to her original question on heat-loving adenylate kinase, Kern performed new experiments showing that the hinges between the lids of the enzyme are more rigid in heat-loving bacteria, which slows the enzyme's movements, presumably keeping it from unraveling at high temperatures. The team's findings appeared in two papers online in Nature on November 18, 2007. Next, Kern wants to find out just how the protein manages to switch states without losing its structure.
“The risk of a protein being flexible is that it can fall apart,” she says. “The fascinating part to me is how nature keeps that from happening. These proteins are really living on the edge.”
As for the sibling team, the collaboration continues. “He's simply the best,” says Kern of Huebner. Calling the teamwork between the labs “electrifying,” Kern says, “The combination of these different biophysical techniques provided us with a much deeper understanding of the fundamental principles of protein function. We're already planning new projects together.”