Measuring Dynamic Protein Interactions
Interactions between proteins have long been recognized as one of the main methods of communication within and between cells. Accurate measurements of the signaling process have been difficult in the past because the conventional approaches, such as x-ray crystallography and nuclear magnetic resonance, measure stable molecules, not molecules undergoing changes. A group led by H. Peter Lu, senior research scientist at Pacific Northwest National Laboratory, has developed a method to do measure interactions between molecules.
Single-molecule photon stamping spectroscopy detects and analyses photons that are emitted as single proteins interact. With this technique, Lu's group has captured proteins in motion as they flip-flop against each other, transferring energy and, therefore, information. The team studied the interaction of single molecules of activated Cdc42 with the dye-labeled WASP (Wiskott-Aldrich syndrome protein) biosensor. Their results indicate highly dynamic interactions between this pair of signaling proteins.
Measurements of the interaction of single molecules of Cdc42 with the dye-tagged WASP fragment were 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 analyzed 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.