How Green Fluorescent Proteins Fluoresce

Berkeley researchers have identified the mechanism that makes green fluorescent protein (GFP) light up the way it does. They used femtosecond lasers to image the molecules as they undergo structural changes that cause them to fluoresce.

The transfer of a positively-charged hydrogen atom – a bare proton – along a reaction chain in GFP generates a green flash of light. The laser snapshots show that when the light absorber, or chromophore, nestled in the middle of the protein barrel absorbs an incoming photon of blue light, it starts vibrating, and the electrons start sloshing around the chromophore until it is aligned just right for the proton to hop via a water molecule to a nearby amino acid in the protein. From there, it continues down the reaction chain, creating a state with a negatively charged chromophore that emits green light.
Previous studies had shown that after the chromophore absorbs blue light, it undergoes proton transfer, and green light is emitted. In the current study, Mathies, Fang and their colleagues could actually resolve the early stage of this proton transfer reaction, taking snapshots of the vibrational wagging of the chromophore skeleton in sync with the electron cloud in the chromophore sloshing back and forth. However, the wagging oscillation might have stopped after a few picoseconds, when the chromophore and its vicinity are aligned just right for the proton to hop off down the reaction chain, and the whole protein shines bright green – which it does in its own good time, in about 3 nanoseconds.
Femtosecond stimulated Raman spectroscopy on GFP involves hitting the protein molecule with an approximately 80 femtosecond pulse of ultraviolet light, which excites many vibrational modes in the molecule, and then a one-two punch of picosecond red and femtosecond white light to stimulate Raman emission. The spectrum of emitted signals tells researchers the vibrational modes of various parts of the molecule. If the molecule is in the middle of a reaction, the emitted light at different time delays tells the researcher the various steps the molecule goes through during the reaction.

Berkeley Lab press release: Vibrations key to efficiency of green fluorescent protein …
Abstract in Nature: Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy