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Protein fishing in America: The movie

27.08.2004


Experiment proves that ’fly-fishing mechanism’ theory of protein-to-protein communication holds water



Proteins pass messages to other proteins much like fly-fishermen flicker their lines against water, or so a current leading theory holds. The repeated weak slapping of protein surfaces against one-another is the critical first step in a chain of events that rule all subsequent cellular behavior.

But this vital exchange between single molecules has defied direct observation because that line-flicking and message-passing happen randomly at such a small scale.


A Pacific Northwest National Laboratory team led by H. Peter Lu, using a technique called single-molecule photon stamping spectroscopy, has now observed real-time interactions of single proteins. Their experimental evidence, reported Thursday at the 228th national meeting of the American Chemical Society, supports the fly-fishing theory of protein communication.

"In the past five years, the field of protein-protein interaction dynamics has exploded," said Lu, a staff scientist at the Department of Energy laboratory in Richland, Wash. "Measurements to date have been snapshots of proteins. But to do dynamic measurements, to capture proteins in motion, this is unique."

Techniques such as nuclear magnetic resonance and x-ray crystallography reveal structural details about proteins and positions of their atoms at a particular time in space. They provide structural reference points, but to contrive interactions, many images have to be gathered at different times, events averaged out and a narrative flow imposed. The effect is akin to cutting a cartoon into a thousand frames tossing the pieces from the ceiling like confetti, then gathering them off the floor and reassembling them. Try making sense of that.

"It’s not an observation in real time," Lu said. "You’re measuring many proteins at a time, and you get information about two states and two states only: binding or not binding. How the binding and not binding are linked is hidden information." Lu’s single-molecule spectroscopy technique gathers and analyzes photons emitted as single proteins interact. This raw data may not produce what we would think of as a motion picture, but for Lu it is just as good. It enables him to construct--in true sequence and in real time--the position and continuous motion of the single molecules for the millisecond they flip-flop against each other.

So far, Lu’s team has looked at two different sets of proteins selected their importance in intracellular signaling--one called Cdc42 that activates with a protein known as WASP and calmodulin, a regulatory protein important in cells that depend on calcium for cell signaling, that can bind with various protein species.

Lu’s group has assembled an elaborate instrument at PNNL’s W.R. Wiley Environmental Molecular Sciences Laboratory. In the instrument, fluorescent-dye-tagged proteins are embedded in a gel and probed by a continuous-beam, or ultrafast, laser. The protein fluorescence fluctuates when molecules interact with one-another. The light emissions and fluctuations from the laser-excited molecules are captured and measured by an inverted fluorescence microscope across a field of about 250 nanometers; individual photons are directed toward a device called a photon-stamping detector that yields key information on each detected photon. "The detection is highly sensitive and precise," Lu said. "One molecule doesn’t have many photons give out. We need to capture as many photo-physical properties as we possibly can."

On this project, Lu’s group collaborated with molecular biologists Klaus Hahn from the Scripps Research Institute and Thomas Squier from Pacific Northwest National Laboratory.

Bill Cannon | EurekAlert!
Further information:
http://www.pnl.gov

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