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!
Studying fundamental particles in materials
17.01.2017 | Max-Planck-Institut für Struktur und Dynamik der Materie
Seeing the quantum future... literally
16.01.2017 | University of Sydney
Researchers from the University of Hamburg in Germany, in collaboration with colleagues from the University of Aarhus in Denmark, have synthesized a new superconducting material by growing a few layers of an antiferromagnetic transition-metal chalcogenide on a bismuth-based topological insulator, both being non-superconducting materials.
While superconductivity and magnetism are generally believed to be mutually exclusive, surprisingly, in this new material, superconducting correlations...
Laser-driving of semimetals allows creating novel quasiparticle states within condensed matter systems and switching between different states on ultrafast time scales
Studying properties of fundamental particles in condensed matter systems is a promising approach to quantum field theory. Quasiparticles offer the opportunity...
Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.
As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...
At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).
Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...
Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.
The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...
10.01.2017 | Event News
09.01.2017 | Event News
05.01.2017 | Event News
17.01.2017 | Earth Sciences
17.01.2017 | Materials Sciences
17.01.2017 | Architecture and Construction