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FEATURES: Force Factor
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“In bulk studies, researchers didn’t get an unwinding signal at all in the presence of ATP,” says Wang. “So we didn’t know this was happening.” She thinks the slippage is an adaptation by the cell to slow unwinding of DNA when there aren’t nucleotides around to quickly bind and pair with each single strand. Since dTTP is a nucleotide that can be incorporated into the strands, its binding to the helicase signals that there are plenty of nucleotides around and the single strands won’t be left hanging.
DNA Unwinding by T7 Helicase Helicase T7 encircles a single strand of DNA as it unwinds double-stranded DNA. Each of the six helicase subunits is shown in a different color. The affinity of a subunit to DNA is represented by a small hook: a closed hook for higher affinity and an open hook for lower affinity. When both ATP and dTTP are present, a subunit may bind to either an ATP (lower affinity to DNA, open hook) or a dTTP (higher affinity to DNA, closed hook). The helicase will not slip as long as at least one subunit does not release the DNA.
Molecular Tug of War
The first applications of optical trapping in biology revolved around DNA and RNA. Since Bustamante had already calculated the elasticity of the molecules, these measurements provided a starting point for other experiments on the forces exerted on or by the nucleic acids. But scientists soon wanted to manipulate proteins on their own—to study how they can alter their complex conformations, unfold, and interact with each other. Optical trapping offered a way to do this physical wrangling.
At the Massachusetts Institute of Technology (MIT), HHMI investigator Tania Baker, as part of a long-term collaboration with HHMI scientific review board member Bob Sauer, runs experiments that are similar to those Michelle Wang set up to study T7 helicase. But Baker studies how one molecular machine, ClpXP, unfolds entire proteins rather than DNA strands.
“Proteins are designed to be really stable in cells, but there are critical times when the cell needs to unfold them,” says Baker. Unfolding a protein inactivates it if the protein is no longer needed, has to cross a membrane, or needs to be remodeled, she explains.
ClpXP, like helicase T7, is ring shaped, and pulls proteins through its center. But it doesn’t always move at a steady rate—some proteins, or parts of proteins, are harder to unfold and cause ClpXP to stall, while other proteins or sections unravel easily, especially once a neighboring bit is unfolded. Baker wanted a way to study the range of speeds at which ClpXP moves as it unwinds different parts of a protein.
So she collaborated with biophysicist Matt Lang, then at MIT and now at Vanderbilt University, to devise an optical trap setup. Two traps held either end of a protein strand in place. As ClpXP unfolded the protein, the strand lengthened—measurable by determining the distance between the traps. So far, she’s shown that the technique works, quantifying the stop–start motion of the unfolding process. Next, she’ll use it to tackle the tougher question of what determines how hard it is for ClpXP to yank protein sections apart.
“We do a lot of biochemistry and a lot of structural studies, and now this is another tool to study this family of enzymes,” says Baker. “One of the things this protein does is create force, so it’s important to study that aspect of it.”
Not all motors in the cell are pulling molecules apart. Some are vehicles, carrying cellular supplies from one location to another. A neuron, for example, has a long process—the axon—that can extend up to one meter. Proteins, membranes, and chemicals must move rapidly from one end of the axon to the other, requiring a molecular motor.
In 1985, HHMI investigator Ron Vale of the University of California, San Francisco, discovered kinesin, the molecular motor that transports materials through neurons on filaments called microtubules. In his early experiments, Vale could watch kinesin moving a plastic bead along microtubules under a microscope and later could follow the movement of the motor by single-molecule fluorescence microscopy. But in their natural state, molecular motors of the neuron need to produce a reasonable amount of force to drag their cargos through the dense environment of the cytoplasm. Vale found optical traps to be a useful tool for studying this force.
Dynein Optical Trapping
Arne Gennerich, a postdoc in the Vale Lab, explains how he uses an optical trapping microscope to analyze dynein.
Walking with Kinesin
Watch kinesin as it uses energy from ATP to move stepwise along a microtubule.
“It’s like learning how an engine works by studying how it performs under different loads,” says Vale.
In his latest experiments, optical traps have allowed him to push and pull a single kinesin molecule along microtubules and observe how it responds. Unexpectedly, he found that simply pulling on the kinesin causes it to take regular steps along the microtubule, even in the absence of the chemical energy that it usually needs to produce movement. He also found that he could pull the molecule backward along microtubules, but it takes more force. The difference in the required force provides clues about how kinesin works and how it moves in the correct direction.
The Force of Innovation
While optical traps have answered some questions posed by biologists and given them a way to quantify force in their systems, the method has also led to more questions.
Video: Chris Pelkie and Daniel Ripoll / Cornell Theory Center