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SINGLE-MOLECULE


A flying butterfly is one of the most beautiful creatures that the mother nature has presented on the earth. As a child we were all curious how it flied in such an elegant way. We could figure out how a butterfly moved its wings to fly -- flipping them in and out -- just by watching. What if the size of the butterfly is so small that we cannot see them with eyes? What if its size is smaller than the diffraction limit of visible light? How will we be able to track the motion of the wings?



FRET (Forster Resonance Energy Transfer) is a powerful molecular biology tool that has been used for detecting conformational dynamics of macro-molecules such as DNA, RNA and proteins with nanometer resolution. We use two fluorescence dyes whose spectra are similar to each other. After we label the two dyes onto each wing of the "butterfly molecule", the change in color reports in real time on how the butterfly changes its conformation.


When the butterfly sits on grass, it is likely to remain in one conformation, say, with its wings closed. One steady FRET signal will be observed (high FRET, red signal). When it flies, the FRET efficiency will periodically change between high and low states reflecting the way it flies -- flipping its wings in and out over and over again.


Fluorescence detection methods used in conventional molecular biology require to have trillions of proteins emitting signals. When trillions of molecules observed altogether, an ensemble measurement averages out signals erasing off dynamic signature of individual proteins. Instead of detecting fluctuating signals of red and green, all we get is an averaged color between green and red.
        To extract kinetic information, scientists have used several tricks. One is to synchronize the dynamics of the protein population. If we can put all butterflies in one conformation, for example, making them take a nap, initially the FRET signal will be high since all of them will have their wings folded. At time equals to zero, we wake them up to fly and the FRET signal drop since the FRET efficiency will reach an average of folded and unfolded conformations.






While many kinetic studies rely on such synchronization, it is not straightforward to interpret the data, missing many important details. Here comes the single molecule study. The beauty of single molecule studies is to track and observe single butterflies just by watching them. There is no need of synchronization. We can detect hidden details of the dynamics effortless. We can also catch heterogeneity between proteins immediately.




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