Single-molecule Förster resonance energy transfer (FRET) is a molecular measure that is sensitive to distance changes on a nanometer scale. It has been widely used for investigations of conformational changes of biomacromolecules such as proteins and nucleic acids and their interactions. In most experiments, two-color FRET is used, in which the measured quantity is the transfer efficiency between a single donor and acceptor pair. By adding one extra fluorophore, two additional distances can be measured, from which much more detailed information on molecular structures and dynamics can be obtained. For example, it is possible to monitor conformational changes and molecular interactions simultaneously by attaching two fluorophores to one molecule for monitoring conformational changes and attaching the third to the other molecule for the detection of interactions between the two molecules. In another example, all three dyes can be attached to the same molecule to monitor conformational changes during molecular processes such as folding of nucleic acids or proteins. In three-color FRET studies so far, the timescale of measurement has been slow (tens of milliseconds and longer) primarily due to the complexity of the experiment and relatively low photostability of the fluorophores with absorption and emission at a long wavelength that are used for the third dye. However, there are many molecular processes that occur on a timescale of millisecond and faster. I will present our development of the theory and experimental and analysis methods for fast three-color single-molecule FRET spectroscopy. This method is applied to probing sub-millisecond folding dynamics of an engineered fast-folding protein, a<sub>3</sub>D, and fast binding and folding dynamics of intrinsically disordered proteins.<br/>

Fast three-color single-molecule FRET

2019 Spring National Meeting

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