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Carlos Bustamante devised optical trapping to physically manipulate molecules and study their behavior. Michelle Wang used the technique to watch DNA unwinding in the presence of two different energy sources—and saw distinctions that no one had picked up before.
The small plastic bead used in optical trapping can be attached to a strand of DNA or a protein and pulled using the force generated by the laser beam. To stretch a piece of DNA, Bustamante could attach a plastic bead in place of the magnet, put it under the laser, and slowly move the laser in one direction.
“We knew we were doing experiments that hadn’t been done before,” says Bustamante. “But we came to realize that besides just learning what the elasticity of DNA was, these techniques offered the chance to learn about other interesting things in the cell.”
Suddenly, Bustamante had a way to physically manipulate any molecule that he wanted. He imagined using optical traps to pull proteins apart or drag motors along pieces of DNA. Today, these experiments are reality, and optical trapping is the go-to way for biologists to push and pull on individual molecules to study their behavior.
Measuring Moving Parts
At Yale University, HHMI investigator Anna Pyle studies the shuffling movements of RNA helicases along strands of RNA, the genetic material that translates DNA codes into proteins. As they move, some RNA helicases push other molecules off the RNA. Other helicases are required to unwind double strands of RNA or to recognize foreign RNA brought into a cell by a virus.
“At their core, all these proteins work by opening and closing, shuffling along an RNA strand,” says Pyle. “But that behavior is coupled to all sorts of different functions in the cell.”
Having studied the biochemistry and structure of helicases, Pyle wanted to quantify the force it took for the proteins to move along RNA. In collaboration with Bustamante, she used an optical trap to tug on a helicase as it moved along an RNA strand. As the helicase moved, the optical trap exerted an increasing amount of force on the bead attached to the helicase. The scientists could measure these forces through the laser beam holding the bead in place.
“Getting these numbers on force serves as a real window into basic thermodynamics of these motors,” says Pyle. Biochemists like to think of chemical reactions in terms of equations, she says, and force has been a missing number in those equations. She can now use her initial results to compare the force used by different helicases or to see how a mutation changes the force a helicase can generate, and thus, its function.
Optical trapping experiments by Michelle Wang, an HHMI investigator at Cornell University, upended ideas on how one protein works. In the 1990s, Wang was part of a Princeton University team that used the technique to measure forces of a DNA-based motor protein for the first time. Today, Wang has turned her attention to T7 helicase, a molecular motor that separates double-stranded DNA into two single strands by pulling the DNA through the center of its donut-shaped structure. Other proteins then add nucleotides—the building blocks of DNA—to turn the single strands into two double strands.
T7 helicase can bind two forms of cellular energy. One, called ATP, is the most common currency of energy in cells. Breaking ATP’s chemical bonds releases energy used in many molecular motors. The other form, called dTTP, is both an energy-storing molecule and a DNA nucleotide. Previous experiments with T7 helicase showed that when only ATP was present, the helicase didn’t unwind DNA. But the studies looked at many strands of DNA and helicases at once, averaging how fast the motors moved. Wang wasn’t convinced the collective results told the whole story, since she knew ATP could bind to the helicase.
“When you do an experiment like that, you don’t know the behavior of each molecule and it’s hard to interpret,” says Wang. “It’s like trying to analyze a whole bunch of runners going at different speeds. But instead of measuring the speed of each runner, you measure how long it takes until the last runner crosses the finish line.”
Wang’s team devised optical traps to hold two DNA strands in place, so they could track the unwinding progress of the T7 helicase one molecule at a time. In the presence of dTTP, their associated helicase unwound the DNA at a consistent rate. With only ATP present, the helicase unwound the strands at a faster rate but constantly slipped backward on the DNA, never getting to the end. The group published their work October 6, 2011, in Nature.
Photos: Wang: Kevin Rivoli/AP, ©HHMI; Bustamante: Andrew Nagata