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Tania Baker has used optical trapping techniques to measure the forces involved in protein folding. Next up: the force required for molecular machines to yank protein sections apart.
Force is anything that makes an object change its speed, direction, or shape. In the context of cells, forces are required to move molecules. Quantifying these forces gives scientists a way to compare and contrast different molecular motors. Without measurements of force, they were missing a crucial entry in the equations of how cells use energy.
“The bottom line is that the cell is not just a little bag of concentrated reactants. The cell resembles a factory,” says Carlos Bustamante, an HHMI investigator at the University of California, Berkeley. “There are different centers, each specialized in certain functions. Those centers are primarily made up of protein machines. And those machines work as motors that produce torque and movement and force.”
Today, thanks to techniques that Bustamante helped bring from physics to biology, scientists can quantify the forces and movements generated by molecular motors. They can precisely measure the force it takes to unfold a protein or unwind a strand of DNA. The innovative methods not only provide fascinating insight into the magnitude of force being generated inside living cells, they also offer ways to change this force and study the consequences. Scientists can play tug of war with a strand of DNA or pull a protein backward along a track to see how the molecules behave under stress.
Shining a Light on Force
In the 1980s, Bustamante worked at the University of New Mexico studying how pieces of DNA moved through gels. When coaxed through the gel by an electric current, fluorescently stained DNA strands moved at different speeds depending on their sizes.
“As I was watching this separation, what became evident to me was how elastic these molecules appeared,” he says. “Sometimes a little piece would get caught and the DNA would stretch out, and then it would snap back like a spring.”
He became curious about the elasticity of DNA and how to measure it. How much force would he need to stretch out a strand like the gel was doing?
In his earliest calculations, Bustamante estimated that he needed a tenth of a piconewton to begin to stretch DNA. “A newton is about the weight of an apple on the surface of the earth,” he says. A piconewton is a millionth of a millionth of that, around the weight of a red blood cell.”
To generate this small amount of force, Bustamante attached one end of a DNA strand to a glass coverslip and the other end to a tiny magnetic bead. Then, he used a second magnet, with a known magnetic strength, to tug on the magnetized end of the DNA strand. He could measure how strong the magnet had to be to stretch the DNA by different amounts. It was the first direct measurement of the elasticity of a strand of DNA and was reported in Science in 1992.
Over the next decade, Bustamante and his colleagues refined the method and brought cutting-edge physics to bear. Instead of using magnetism, their techniques relied on optics, or light. These methods allowed them to make more precise measurements and apply even smaller forces.
If a powerful laser shines through a plastic bead, the light beam is slightly deflected at the bead’s surface. This change in direction of the light beam requires a tiny amount of force. And according to Newton’s third law—for every action there is an equal and opposite reaction—this miniscule amount of force pulls the tiny bead toward the center of the beam. Change the intensity of light, and the amount of force exerted on the bead changes.
Physicist Steven Chu, now the U.S. Secretary of Energy, won the 1997 Nobel Prize in Physics for his quantum physics application of this technique, called optical trapping because it traps a particle in the beam of light. Bustamante was among a handful of scientists who pioneered its use in biology for single-molecule studies.
Photo: Jason Grow