Fluorescent proteins (FPs) have many applications in biomedical research, but are not widely used directly as biosensors because the chromophore resides inside the �-barrel structure of the protein and is thus remarkably stable and relatively insensitive to the external environment. In previously published work, we showed that quenching of Forster Resonance Energy Transfer (FRET) from UV-excited endogenous tryptophan (W) residues to the chromophore was sensitive to the environment and could be used to detect nitroaromatic explosives like TNT and RDX, even though these agents have virtually no effect on the normal visual excitation and emission (Fig. 1).  Still, examination of crystal structures of the seven different FPs used indicated the vast majority of the indole chromophore surfaces of the W side chains, like the FP chromophores themselves, resided predominantly within the �-barrel structure. This means that quenching agents have only moderate accessibility to enable collisional quenching. In contrast, due to the hydrophilic phenolic functional group, most of the surfaces of the many tyrosine (Y) residues are on exterior of the FP. We obtained a DNA construct encoding for a W-less GFP and have shown that Y-to-chromophore FRET exists in this protein. Furthermore, the Y-to-chromophore FRET can be quenched by nitroaromatic explosive simulants. We are currently investigating the relative sensitivity and selectivity of FRET from Y to that of W, as well as examining the palette of quenching agents that can be detected by this methodology. Our next goal is to see if quenching of two-photon excited Y-to-chromophore FRET can be used for in vivo biosensing.

Investigating tyrosine FRET in fluorescent protein for biosensing applications

247th National Meeting (2014)

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