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Getting Microscopic: Investigating molecular issues at the source

BY A. J. HOGG

Published April 10, 2006

Imagine grabbing a strand of protein with tweezers made from a beam of light and extracting it from the surface of a cell the same way you would pull a loose thread from a sweater. That's just one technique used by University researchers to learn about the structure and function of proteins in single-molecule research.

Chemistry Prof. Nils Walter is attempting to set up a center at the University to encourage this kind of single-molecule research. His goal is to bring people who have experience in using single-molecule technology together with people whose research would benefit from that technology. They include researchers from engineering, the sciences and the medical school.

No other university has a single-molecule center with this kind of range under one roof. Walter said that the University is good at fostering collaboration, but the center would stimulate further joint research. In order to drum up interest, he is organizing a Single Molecule Symposium, to be held at the Alumni Center May 18 to 20.

Walter's research focuses on ribonucleic acid, or RNA, which regulates the proteins made in a cell.

Scientists have a good understanding of how DNA makes RNA, which in turn makes a protein, but by looking at individual RNA molecules, it turns out RNA itself can be biologically active.

Walter uses nanotechnology in order to study RNA in his research.

Looking at a single molecule

To watch what a single RNA molecule does, Walter uses single-molecule fluorescence spectroscopy. He attaches two fluorophores, or organic dyes, to specific locations on the molecule. When exposed to one particular frequency of light, fluorophores emit a different frequency, making them detectable. In Walter's research, he uses one fluorophore that gives off green light and a second that emits red light. When the two fluorophores are far apart, the green emits light more intensely, but when they are close together, the green dims as energy is transferred from the green fluorophore to the red one; and more red light is emitted. By measuring the color of the light coming from the molecule, he determines how close the two parts of the molecule are, which tells him how the molecule is folding.

Proper folding is vital for proteins to function. A wrong fold changes the shape of the protein, which can alter how it interacts with other molecules. If the folding goes wrong, the protein can stop working, or worse, act in harmful way. Alzheimer's disease is caused by a misfolded protein.

Scientists use fluorophores with proteins as well as with RNA to determine how protein folding changes over time.

Many times, the protein is able to fold from one configuration to another, and may spend only a small amount of time in a particular configuration.

"It can flip-flop," Walter said.

When looking at many molecules at once, you can only see the average configuration, but the advantage of looking at individual molecules is that you can see the unusual fold.

"One hundred times one thing happens, once or twice another thing happens," he explained. Walter said it is these rare occurrences that scientists are just now learning about through current research.

In addition to observing the protein, researchers can introduce mutations and observe changes in the folding properties of the protein.

Once researchers introduce a known change in a protein, they can observe the effects of a new "version" of the protein.

Walter said the real question is, "How likely is it for this particular version to become biologically active?"

Optical tweezers

Another single-molecule research technique is optical tweezers - a way to manipulate individual molecules without touching them. By focusing a laser onto a small, one-micron diameter clear plastic sphere, researchers can trap it within the beam. If the sphere moves out of the beam, the diffractive index - how the beam bends as it passes through the sphere - changes and light, behaving like a particle, moves the sphere back into place.

"You get a recoil," Walter said.

By stringing a protein between two spheres - one end trapped by a laser in the optical tweezers, and the other held still - you can measure the required force to displace the protein. Imagine a spring, with one end bound to a wall and the other held in a person's hand. If the spring contracts, it pulls toward the wall, and the person can feel the force exerted by the spring.

Scientists can do this at the molecular level. For example, rather than a spring, scientists can use a strand of DNA. Because single-stranded DNA is more folded and more coiled than double-stranded, scientists can measure how fast an enzyme chews up one strand of the double-stranded DNA.

This technique works with any enzymes that can move on a given target that can be tethered on the spheres.

"Then you learn something about the forces these enzymes can exert on their substrates," Walter said.

Atomic force microscopy

A third way of looking at single molecules is a technique called atomic force microscopy. Using this method, researchers set a cantilever with a very fine point (similar to a very small version of the needle on a record turntable) to measure the surface of a cluster of molecules. The top of the cantilever is reflective. When a laser reflects off the cantilever, a photodiode measures how much and in which direction the cantilever moves. The measurement is accurate to one nanometer.

The cantilever moves over the surface in meanders, scanning it to give an image of the surface topography.

Individual molecules can also be pulled from the surface with the tip. Sometimes a molecule sticks to the cantilever and gets pulled out of the surface. By measuring the force it takes to pull the molecule off, researchers can learn about how the molecule is folded or threaded in the surface.