Studies of Single Green Fluorescent Proteins (GFP) in Gels

Figure 1: 100 frames of the emission from an individual Green Fluorescent Protein molecule (100 ms exposures) in a polyacrylamide gel, showing the blinking process, overlaid with the crystal structure of GFP.

Movie: Click here to see a QuickTime animation (1.1MB) of 100 frames of the emission from an individual molecule of Green Fluorescent Protein (100 ms exposures) in a polyacrylamide gel.

R. M. Dickson, A. Cubitt (Aurora Biosciences), R. Y. Tsien (Howard Hughes Investigator), and W. E. Moerner

Single molecule studies of individual green fluorescent protein molecules have yielded the first example of a room temperature single molecule optical switch.1 A protein composed of 238 amino acids, GFP folds such that the chromophore is isolated inside a compact barrel structure. 2,3 Stabilized and influenced by the three dimensional structure in the immediate environment of the chromophore, small changes to individual amino acids can yield large changes in photophysical behavior. By studying two different red-shifted GFP mutants (T203Y and T203F) which differ only by the presence of a hydroxyl group near the chromophore, slight differences in the photophysical properties were observed. In addition to the differ ences between the mutants, dynamics only discernable on the single molecule level were observed, both mutants exhibiting abrupt changes in fluorescence intensity as a function of time.1 This blinking behavior likely results from ground state dynamics between at least two states of the chromophore, only one of which is capable of being excited at 488 nm and producing fluorescence. Additionally, a much longer lived state is accessible through excited state processes. Thermally stable in the dark for many minutes, this long-lived dark state can be excited at 405 nm to regenerate the original fluorescent state. 1

Interpretation of the states involved in the blinking and switching processes are facilitated by the known crystal structures of wild type3 and red shifted mutant (S65T) of GFP. 2 The different absorption peaks observed in these two proteins of known structure are clearly explained by the observed different chemical states of the chromophore. Present in the wild type structure, the neutral form of the chromophore exhibits high energy absorption and emission peaks. The presence of the anionic chromophore of S65T, on the other hand, is suggested by long wavelength absorption and emission peaks. The absorption and emission spectra of the mutants T203Y and T203F each have two sets of transitions which are indicative of the two distinct chromophore states being simultaneously accessible. Interconversion should be possible through a proton transfer between the chromophore and the surrounding amino acid residues. 4 Since the optical switching we observed employs wavelengths absorbed by the neutral and anionic forms of the chromophore, the protein likely toggles between these two states. Mutation of individual amino acid residues near the chromophore should allow fine-tuning of the potential barrier separating the anionic and neutral states, thus altering their relative ground and excited state stabilities.

The observation of an optical switch at room temperature in which each molecule is individually addressable and can be read quasi-nondestructively is an exciting development. Further single molecule and bulk studies must be performed in order to better understand the natures of the blinking and switching states.5 Optimization and control of these processes could lead to new developments enabling observation of faster protein dynamics within cells. A well defined starting time would be produced by switching GFP-labeled proteins within a small focal volume into their fluorescent state; the subsequent protein dynamics could then be followed. Since each of these molecules has been shown to be an optical storage element, long term developments may include storage devices based on single molecule technology.

Figure 2: 2.4 micron x 2.4 micron image of individual Green Fluorescent Protein molecules in a polyacrylamide gel.


  1. R. M. Dickson, A. B Cubitt, R. Y. Tsien, and W. E. Moerner, "On/Off Blinking and Switching Behavior of Single Green Fluorescent Proteins," Nature, 388, 355 (1997).
  2. Ormo, M., et al., Science 273, 1392-5 (1996).
  3. Yang, F., Moss, L.G. & Phillips, J., G. N., Nature Biotech. 14, 1246-51 (1996).
  4. Brejc, K., et al., Proc. Nat. Acad. Sci., USA 94, 2306-11 (1997).
  5. W. E. Moerner, "Those Blinking Single Molecules," Science 277, 1059 (1997).