A Stanford University research team has designed the first microscope sensitive enough to track the real-time motion of a single protein down to the level of its individual atoms. Writing in the Nov. 13 online issue of the journal Nature, the Stanford researchers explain how the new instrument allowed them to settle long-standing scientific debates about the way genes are copied from DNA--a biochemical process thats essential to life.
In a second paper published in the Nov. 8 online issue of the journal Physical Review Letters, the scientists offer a detailed description of their novel device, an advanced version of the "optical trap," which uses infrared light to trap and control the forces on a functional protein, allowing researchers to monitor the molecules every move in real time.
"In the Nature experiment, we carried out the highest-resolution measurement ever made of an individual protein," says Steven Block, professor of applied physics and of biological sciences. "We obtained measurements accurate to one angstrom, or one-tenth of a nanometer. Thats a distance equivalent to the diameter of a single hydrogen atom, and about 10 times finer than any previous such measurement."
Determining which model is correct has been a difficult challenge, because until now, no instrument was sensitive enough to track each microscopic step taken by RNAP along DNA during transcription. Thats because conventional optical traps cant measure anything smaller than about 10 angstroms (1 nanometer). However, each base in the DNA ladder--A, T, G or C--is only separated by about 3.4 angstroms. "My lab has been working very hard for the better part of a decade to break the nanometer barrier and attain angstrom-level resolution," Block says.
Light and motion
To achieve that goal, the Block team had to overcome two inherent problems with conventional force clamps: fluctuating signals and bending light waves.
"When you shine a laser through the air, the light beam wiggles around a bit, for the same reason that stars twinkle in the sky," Block explains. "But we want to use that beam to measure the position of something to within the size of an atom, so if the beam moves just 1 angstrom, thats the end of the story. We took all the optics external to the microscope, enclosed them in a sealed box and replaced the air with helium gas, which has a refractive index thats 10 times closer to that of a vacuum than air. So you get, roughly speaking, 10 times less twinkling and an instrument with angstrom-level stability."
In addition to stabilizing the light, the researchers also had to improve the method for detecting force and displacement. Optical force clamps use tiny forces from an infrared laser beam to trap DNA and other molecules. In a conventional force clamp experiment, microscopic beads are attached near the opposite ends of a long DNA molecule--an arrangement that resembles a weight lifters dumbbell. A single RNAP enzyme attached to the surface of one bead then moves along the DNA and churns out a complementary strand of RNA, drawing the ends of the dumbbell closer together as it advances. The two beads that form the dumbbell are usually held near the center two separate optical traps. But graduate student William Greenleaf discovered that if one of the two beads in the dumbbell was placed near the outer edge of its trap, the force on it would remain constant, allowing angstrom-level measurements to be made quickly and efficiently.
"Thats just what you want--a clamp that allows RNAP to move with impunity, but the force itself doesnt change," Block says. "Normally the bead is inside the trap in the center, but right at the edge of the trap we have this magical property where the force is constant."
Unlike conventional instruments, the new force clamp requires no time-consuming computer computations to correct for competing forces. "This new technique is entirely passive, like a thermos that just sits there and keeps something cool," Block says. "All we have to do is shine light on the system and everything takes care of itself. As a result, we were finally able to resolve the minuscule, 3.4-angstrom steps taken by E. coli RNAP as it transcribes a bacterial gene."
Settling the debates
With these innovations in place, the research team appears to have settled some of the fundamental arguments over DNA-RNA transcription. "Quite simply, our experiment rules out both discontinuous-location models," Block says. "Neither the inchworm nor the scrunching model is consistent with our data, and the idea that some have held all along--that RNAP climbs the DNA ladder one base pair at a time--is probably the right answer."
The Stanford group also weighed in on another controversy concerning the actual mechanism that allows RNAP to advance. "RNAP is a molecular motor that starts at one end of the DNA and walks down to the other end," Block explains. "It gets its energy from the chemical reaction that occurs when it copies A, T, G or C. Its as if a machine that lays down asphalt could somehow be powered by the asphalt itself."
Scientists have come up with two different models to explain what drives this molecular motor:
The power stroke model, in which pent up energy thrusts the enzyme forward--like a loaded spring thats periodically released.
The Brownian (or thermal) ratchet model, whereby random thermal energy causes the RNAP enzyme to jiggle back and forth. Each incoming DNA base then locks the enzyme into the forward position so that it cannot jiggle backwards. "It would be as if you were repeatedly bouncing off a wall, and every time you happened to bounce a bit farther away, somebody came in and moved the wall up behind you, so you couldnt bounce so far back. Youd wind up drifting forwards, even though your own motion was mostly random," Block explains.
In the Nature study, Block and his colleagues concluded that the Brownian ratchet model is probably correct for RNAP, even though several other motor proteins are believed to move instead by the power stroke mechanism. "Weve certainly come down hard in favor of the Brownian ratchet camp and against the power stroke camp," Block says. "But does that mean all power stroke models have been ruled out and that all Brownian ratchet models are acceptable? No."
The Block team also applied the new force clamp technology to one the hottest fields in biomedical research--molecular folding. For a protein to function properly, it has to fold into a specific, intricate three-dimensional shape. Diseases such as Alzheimers, Mad Cow and Parkinsons may result when proteins do not fold into their correct 3-D conformation. Medical researchers are trying to solve the mystery of how proteins fold in hopes of some day curing these and other diseases.
In the experiment published in Physical Review Letters, the Block group addressed certain aspects of the general folding problem on a simpler scale by focusing on single DNA hairpins--folded structures that can form when a single strand of DNA pairs with itself instead of with the opposite strand. "Hairpins are wonderful models," Block says. "By keeping the force constant, we were able to measure the folding and unfolding transitions of a single DNA hairpin at the angstrom scale. In the future, this may help us understand and predict what shape a more complex linear protein will assume in three-dimensional space."
The development of an ultra-stable optical trapping system with angstrom resolution is "a major advance," says Charles Yanofsky, the Morris Herzstein Professor of Biological Sciences at Stanford and a pioneer of modern molecular genetics. The new device is like "adding movies to stills in understanding enzyme action," he says.
"This technical achievement will no doubt lead to new information about the molecular machinery that carries out basic cellular processes, particularly those related to replication, transcription and translation," adds Catherine Lewis, a program director in biophysics at the National Institute of General Medical Sciences (NIGMS).
"If I look in my crystal ball and see where this is going, I think this blows open the field of single-molecule biophysics," Block says. "We have achieved a resolution for a single molecule comparable to what a crystallographer typically achieves in a millimeter-sized crystal, which has 1,000 trillion molecules in it. Not only are we doing all this with one molecule at one-angstrom resolution, were doing it in real time while the molecule is moving at room temperature in an aqueous solution."
Block notes that it took "years of careful instrument development, sponsored by the National Institutes of Health, and the construction of a special laboratory built by Stanford University to make this possible, along with the simply outstanding efforts of some incredibly bright and hard-working graduate students and postdocs here at Stanford. I am especially proud of this work."
The Physical Review Letters and Nature papers were supported by NIGMS and by Stanford University.
Mark Shwartz | EurekAlert!
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