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Scientists reveal new folding pathways for a single molecule

Using individual dye-labeled molecules, Stanford University researchers have revealed never-before-seen folding patterns in RNA enzymes. The study, by a team of physicists and biochemists, proves that single-molecule experiments are powerful tools for examining the complex behavior of biological molecules.

"This is a significant experiment for several reasons," said Stanford physics Professor Steven Chu, a 1997 Nobel Prize winner for his work using lasers to cool and trap atoms. "We have shown that an individual enzyme can be loosely tethered to a surface so that its biological activity is not altered. Furthermore, we showed that the molecule can be unfolded and then refolded back to its biologically active state. By studying how individual molecules fold and unfold, we have observed folding pathways that have never been seen before."

The research was a collaboration in which physicists led by Chu and biochemists led by Associate Professor Daniel Herschlag. The results were published in the June 16 issue of Science in a paper by Chu, Herschlag and researchers Xiaowei Zhuang (physics), Laura Bartley (biochemistry), Hazen Babcock (physics), Rick Russell (biochemistry) and Taekjip Ha (physics). The collaboration, where physicists and biochemists conducted the biochemistry and physics measurements together, is characteristic of Stanford's new interdisciplinary research initiative known as Bio-X.

The team used a single-molecule technique to monitor the folding of an RNA enzyme, or ribozyme, found in the protozoa Tetrahymena. Although the majority of enzymes are proteins that are made when DNA is transcribed to RNA, which is then translated to proteins, some RNA molecules themselves can catalyze enzymatic reactions. Discovered by Nobel Prize winner Thomas Cech of the University of Colorado, Boulder, the Tetrahymena ribozyme was the first catalytic RNA molecule ever identified.

Three-dimensional folding is essential for enzymes to function. Enzymes whether made of RNA or proteins start out as linear chains that coil and kink into three-dimensional, biologically active structures. The final form minimizes the overall free energy of the structure, but the pathways to the folded state are still largely unknown. A multitude of folding pathways and transient states can take shape as the enzyme folds. Once folded, the molecules could flop between a number of alternate states, some of which may not be biologically active, researchers have conjectured. By following the biological activity and folding of individual molecules, the Stanford team hopes to observe behavior that could easily be hidden in experiments using samples containing many molecules.

The scientists also hope the single-molecule work will help them understand a paradox first posed by the late Cyrus Levinthal of MIT: If the folding molecule were to try all possible configurations in its attempt to find its minimum energy state, the proteins or RNA would take ages to fold. Clearly, that doesn't happen. There has to be some control of the process. Chu and his colleagues hope their studies will shed light on the theory that the folding process is governed by "energy landscapes" and "folding funnels" that direct the protein and RNA into their minimum energy state.

Biochemists traditionally look at millions of molecules in a single experiment. But in previous single-molecule studies of polymer dynamics (published in Science in 1998), Chu showed that the same molecule exposed to the same conditions can take very different paths to a new equilibrium state. Previous studies missed the fact that identical molecules could take different paths.

In the present study, the Stanford researchers wanted to see whether single-molecule techniques could help them visualize RNA folding. The Tetrahymena ribozyme has been used as a model system to understand RNA folding. When the enzyme folds into its final state, an arm-like appendage swings down towards the body as when a figure skater draws in his or her arms to begin a spin. With the arm docked to the body, the ribozyme becomes active and splices part of the arm away from the main body of the enzyme.

To visualize this local folding process, the Stanford researchers affixed the enzyme to a surface and verified that the rate at which the single molecules cut RNA was equal to the rate seen in bulk experiments with free-floating molecules. They then attached two fluorescent dyes, placing a green dye on the arm and a red dye on the body. This technique, called fluorescence resonance energy transfer, or FRET, uses specially designed dyes that can transfer energy to one another. When the arm (bearing the green dye) swings close to the body (bearing the red dye), the green dye transfers its energy to the red dye, causing the red dye to glow more brightly. With the aid of an optical microscope and an extremely sensitive photodetector, these researchers measured the rate at which the dyes wink from green to red and back again as the molecule folds and unfolds. "You can visualize the folding process one molecule at a time. This is a great way to reveal folding dynamics," said Zhuang.

In addition to looking at this local folding step, the researchers examined overall folding of the Tetrahymena ribozyme. Using the same set of dyes, the team traced the folding of the enzyme from a pre-folded condition referred to as the "secondary structure" to the final folded state. They discovered that the ribozyme folded at two distinct rates a fast rate that produced one folded ribozyme per second and a much slower rate of one folded ribozyme in approximately one minute. This slower rate was observed earlier in bulk studies, indicating that the modifications of the molecule (dye labeling and surface immobilization) did not affect the folding rate. However, the fast rate had not been observed before it was a previously unknown pathway. Researchers also were able to confirm the previously published results that the ribozyme could become trapped in a misfolded state that took hours to disentangle before going on to fold properly. The researchers discovered that the slow and misfolding routes share common first steps that only later branch away from each other. The fast folding route branches much earlier, but also can get hung up in misfolding.

Single-molecule techniques have the potential to be used to study everything from gene expression to how nerve cells talk to each other, said Chu, who is also collaborating with researchers across the country and in Europe. Meanwhile, the team's next step is to apply single-molecule FRET to finding out more about what the intermediate steps in RNA folding look like. Said Herschlag: "We are still pushing the limits of what can be done with this technique."




By Catherine Zandonella

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