© 2004 AAAS

includes residues 201 to 321 of WASP, which contains the CRIB domain essential for Cdc42 binding. (Reprinted with permission from P. Nalbant, Science, 305:1615–9, 2004.)

Scientists at the Pacific Northwest National Laboratory in Richland, Wash., have used an established technique to observe real-time interactions between single protein molecules for the first time. Ultimately, the PNNL method – single-molecule photon stamping spectroscopy – will be used to study signaling events in living cells. At present it offers more information about protein-protein interaction dynamics than can be obtained with conventional structural biology techniques such as nuclear magnetic resonance (NMR) and X-ray crystallography.

"Protein conformation fluctuation dynamics is a stochastic process," says lead investigator H. Peter Lu, a staff scientist at PNNL. "Experimentally you have to study this process under physiological conditions, and to be able to follow [it] one molecule at a time, otherwise those stochastic...


The researchers found that the two proteins fluctuated between bound and loosely associated states, in support of the so-called fly-casting mechanism of protein-protein interaction. Proposed by Peter Wolynes at the University of California, San Diego,2 this theory states that weak interactions between relatively unstructured proteins initiate protein-protein binding. In the case of WASP and Cdc42, the interaction between the two proteins is dynamic; rather than being rigid, highly structured domains, both targets fluctuate, Lu explains. "Like fly fishing, you keep casting to that position, never tightly binding to that position, but also never completely dissociated from that position," he says.

Klaus Hahn, professor of pharmacology, University of North Carolina-Chapel Hill School of Medicine, developed the environmentally sensitive I-SO dye used in the experiment. Whereas most fluorescent labels require genetic modification of target proteins, I-SO attaches covalently to the protein of interest and changes its fluorescence during a binding event. "You put [a] domain with the dye hanging on it into a live cell, and it will respond to the endogenous protein, so you don't have to overexpress endogenous material, [or] modify the endogenous material to see what's going on," Hahn explains.

Hahn hopes to build on what he's learned so far to extend the method to single-molecule detection in live cells. "What I want to do with [Lu] in the future is use that information to build much better biosensors, and also to do the things he did in vitro [and] in vivo, to individual cells and look at single molecules undergoing signaling reactions," says Hahn.

Although not quite there, Hahn's team has recently used the I-SO-coupled WASP biosensor to visualize the average activation of endogenous Cdc42 in living cells.3Cdc42 is known to control cell extensions in fibroblasts; using the biosensor, Hahn's team found that the molecule was activated within growing fibroblast extensions, a location that was surprisingly microtubule-dependent. "I think [such uses] will open the door to a lot of signaling pathways that people couldn't study before, largely due to the brightness of the dye," says Hahn.

The biosensor really adds to the field, says Mark Philips, professor at New York University School of Medicine, because it enables the observation of endogenous signaling proteins. But he cautions also that the technique's reliance on microinjection may be cumbersome for some labs. "This kind of technology is absolutely critical in terms of taking the next step in understanding huge swaths of cell biology," says biochemist Michael Rosen of the University of Texas Southwestern Medical Center, Dallas.

In the end, it's the generalization of the method that gives it such potential. Says Philips, "This is not an idea limited to GTPases and their effectors."

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