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Flexible or Tight? Protein Interactions Seen in Real Time Using Photon Stamp Technique

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As a technique for studying molecular structures and interactions, single-molecule photon-stamping spectroscopy is far less popular than x-ray crystallography or nuclear magnetic resonance. But physical chemist Peter Lu believes that the method will one day be sought after as a way of gaining a deeper understanding of signaling and networking processes.

Lu, a senior research scientist at the Department of Energy’s Pacific Northwest National Laboratory in Richland, Wash., is one of the first researchers to apply single-molecule spectroscopy to analyzing the dynamic interactions of subcellular components such as proteins and DNA. The method allows data to be gathered in real time, instead of in a frozen, snapshot state, as is the case with x-ray crystallography and NMR.

“With the photon technique, there are a number of advantages,” said Lu, who spoke about the photon-stamping method at a meeting of the American Chemical Society last week in Philadelphia. “One is that there is time-sequence and time-domain information. Secondly, the data can be measured under physiologically relevant conditions, instead of in a high-concentration crystal state as with x-ray crystallography.”

In Lu’s photon-stamping technique, a continuous-beam, ultrafast laser bombards fluorescently labeled molecules of interest, such as enzymes, protein complexes, or DNA. The molecules’ fluorescence fluctuates when they interact with one another.

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 records key information on each detected photon.

“It’s like you’re sending and receiving mail,” Lu said of the photons. “You know when it’s sent out and when it’s received. The detection is highly sensitive and precise.”

By monitoring molecules in real time using photon stamping, researchers can see if molecular interactions are tight or loose and flexible, and in what order molecules form and de-form complexes. That kind of information gives scientists a better picture of how biological networking works, Lu said.

“Right now the pharmaceutical [researchers] are still focusing on simple yes-or-no answers to molecular interactions,” he said. “This goes beyond that. It tells how they interact, and it can even be used to study competitive interactions. It tells who’s interacting with who first, and whose interaction is more stable. I think this type of dynamic interaction will eventually find its application in the pharmaceutical research field.”

Some scientists have likened dynamic molecular interactions to fly-fishing: As a protein loosely folds and unfolds, it acts like a fly-fisher casting bait out into the cytosol over and over again.

“It’s a very dynamic process,” said Lu. “The molecules are very flexible, probing each other and sensing — that’s what they do.”

So far, Lu’s research team has used photon stamping to analyze the interactions of XPA, a protein involved in recognizing DNA lesions; Cdc42, a cell-signaling protein that very effectively recognizes different types of proteins; and T4 lysozyme, an enzyme involved in cutting bacterial cell walls.

Lu’s studies have thus far been carried out in solutions similar to the cytosol, but in the future, Lu’s group may try to adapt the technique so that it can be used in living cells.

“In the living cell, the environment is actually very crowded. It’s not just a liquid solution, all isolated,” Lu said. “Most proteins will attach to some other component, whether it’s a mitochondria or some other part of the cell. By analyzing reactions in the cell, the molecules could be relevant to many other biological processes.”

Since photon stamping involves the detailed study of a small number of molecules at a time, the technique is unlikely to ever become a high-throughput method, like the methods used in proteomics, Lu said, but the technique fits in well as a complementary way of studying molecular interactions.

“Some day you probably will need to go one step further to understand one particular process, and this will be very complementary,” he said.

— TSL

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