Vale, for instance, now wants to know how kinesin’s structure changes while it’s stepping along microtubules. The atomic details of protein structure can be obtained by x-ray crystallography but are not visible under a light microscope; thus optical traps alone do not provide data on structural changes.

“I’m fascinated by the idea of putting these two worlds of x-ray crystallography and light microscopy together,” says Vale. “What are the real structural changes that are occurring during force generation?”

HHMI investigator Taekjip Ha, at the University of Illinois at Urbana–Champaign, has developed a technique that offers a way to pair structural data with the force control of optical trapping.

In 1996, Ha developed a method to determine the proximity of two fluorescent molecules based on the light they give off. The technique, called fluorescence resonance energy transfer (FRET), had been around for decades, but he showed that it could be used on two single molecules, rather than as an average. The fluorescent tags can be attached to two molecules or two parts of a molecule. As the two tags come closer together or move apart, the fluorescence changes. He uses FRET as a measure of distance, and therefore movement, between any molecules or parts of molecules.

In a test of the method, Ha collaborated with Pyle to uncover details of how one particular helicase—from the hepatitis C virus—unwinds DNA. Its DNA-unwinding function is vital for the virus to make new DNA and infect cells. The scientists attached fluorescent tags to two strands of DNA and attached the strands to optical traps. As the helicase moved along the double strand, separating it, the researchers could observe the unwinding of the DNA, base pair by base pair, as the fluorescent tags got farther apart.

The pair discovered that the helicase unwinds three base pairs at a time, then releases tension in the strand, letting it relax, before unwinding three more. The discovery could help them understand how to block the helicase from helping the virus replicate.

Next, they want to know how much force this unwinding takes. So Ha is combining FRET with experiments measuring force. By measuring how the distance between two parts of a protein changes as a result of force, scientists can get a fuller picture of how unfolding or conformational changes happen.

Combining FRET with Optical Trapping
This video shows how the Ha lab is using a combination of high resolution optical trapping with single molecule fluorescence detection. DNA (blue) is tethered between two trapped beads (beads are gray, traps are red). A helicase motor molecule (center) on the DNA is labeled with a single fluorophore (glowing orange) that is excited and detected by a confocal microscope (green). In order for this new instrument to work, the trap and fluorescence excitation lasers have to be rapidly cycled on and off in sequence (called interlacing). This movie shows one complete cycle.

And Bustamante is now using similar techniques to probe the basics of numerous biological processes, including protein folding. He’s using optical traps and fluorescent tags to see what happens when a protein strand is stretched and then released, allowing it to fold into its preferred conformation.

He’s also applying the method to nucleosomes—clumps of proteins that control the structure of DNA within a chromosome and influence when genes are expressed. He’s already looked at the interaction between polymerases—enzymes that move along DNA strands—and nucleotides. His team discovered that when polymerases encounter a nucleosome, they pause, not having enough force to unravel the DNA from the nucleosome. Instead, the protein waits for the clump to spontaneously unravel. If he can use optical traps to pull apart a fluorescent chromosome, Bustamante says, he can observe its higher-order structure and the forces that proteins within the nucleosomes exert on the nucleotides.

“It’s natural that as optical trapping starts to mature, we now want to combine these techniques with others,” says Bustamante. “I think in the future we will see even more hybrid experiments that combine optical trapping with other methods.”

He’s happy to see his technique mature and change, he says, if it means applying it to more biological questions.

“There are so many unknowns inside the cell,” he says. Optical trapping lets researchers get a physical handle on those unknowns. While scientists can’t reach inside cells and feel for themselves the forces at work, optical trapping has become their hands that work to sense and manipulate these forces.

Video: Matthew Comstock

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