Interactions between proteins are one of the main ways in which signals are transferred onward in intracellular signalling pathways. But few methods of studying these interactions can capture the dynamic nature of molecular recognition within signalling complexes.

Peter Lu studies the dynamic interactions of signalling complexes.

A group led by Peter Lu, senior research scientist in the W.R. Wiley Environmental Molecular Sciences Laboratory at Pacific Northwest National Laboratory in Richland, Washington, has now developed a method to do just that.

Using a technique called single-molecule photon stamping spectroscopy, which detects and analyses photons that are emitted as single proteins interact, the group has been able to capture proteins in motion as they flip-flop against each other in a manner somewhat analogous to line flicking in fly-fishing. “This type of behaviour cannot be measured by static structure measurements such as X-ray crystallography or NMR,” says Lu.

His team has used this approach to study the interaction of single molecules of activated Cdc42 with the dye-labelled WASP (Wiskott–Aldrich syndrome protein) biosensor developed in collaboration with Klaus Hahn and his colleagues (see main text above). Their results indicate highly dynamic — rather than static — interactions between this pair of signalling proteins.

Lu's set-up is largely ‘home-built’. Measurements of the interaction of single molecules of Cdc42 with the dye-tagged WASP fragment are made using confocal fluorescence microscopy with laser excitation. Fluorescence photons are directed towards a photon-stamping detector, which records the intensity and duration of the photon peaks; the data are then analysed to determine the dynamics of the protein–protein interaction.

Technical limitations at present are photobleaching of the dye molecule by the excitation laser and movement of the complex away from the laser focal point. Lu is experimenting with methods for confining the complex either using agarose gel or by tethering one of the protein partners to a glass surface.

Lu plans to extend the single-molecule spectroscopy approach to other important biomolecular complexes under physiological conditions, ultimately hoping to study single-molecule protein conformational dynamics in living cells. “Right now, the most interesting part may not be the technique but the new scientific information we learn,” he says.

Diane Gershon