Protein fishing in America:...( ule spectroscopy technique gathers and ...)

ule 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.

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Contact: Bill Cannon
cannon@pnl.gov
509-375-3732
DOE/Pacific Northwest National Laboratory
26-Aug-2004

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