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4.2: Proton transfer in green fluorescent protein (GFP)

Some marine organisms, such as plankton, fish and jellyfish sometimes emit light produced in their organisms via chemical reactions. The process is called bioluminescence (also commonly observed in fireflies) and the most famous organism in which it is observed is a jellyfish called Aequoria Victoria. It is not completely clear, why it luminesces; in fact, it rarely does, when left alone. However, it is easy to light them up by externally prodding them. It is known, that the light originates in a chemically active protein called aequorin, which emits bluish light (spectral maximum at around 469 nm [14]). However, when the jellyfish is disturbed, it emits green light (508 nm). This shift in wavelength is caused by another protein called green fluorescent protein (GFP), to which the energy of aequorin is transferred and which then emits green photons. This protein is unique in the sense that it is optically active in the visible range without any prosthetic groups (pigments), such as bacteriochlorophylls or carotenoids, participating in photosynthesis. In GFP, the fluorophore is formed autocatalytically, from the primary sequence of aminoacid residues [15]. This property of GFP makes it a valuable tool of molecular biology (so valuable, in fact, that Nobel Prize in Chemistry was awarded for it in 2008). Biochemists learned to clone this protein and express it in bacteria [16]. Genetic manipulations allow fusing the gene of GFP to the gene of virtually any protein of interest and that protein is thereby marked with a green fluorescent tag, allowing optical observation of its movements within a cell or organism. For example, making a mouse mutant with epithelium protein fused with GFP has resulted in mice with green fluorescent skin. Thus, GFP is an all-natural biological marker, which is not poisonous, and the properties of which can be controlled accurately [14]. It is therefore important to understand the biophysical basis of its fluorescence, because they open the way of making its analogues with properties required for molecular biology research.

GFP is a water-soluble protein Its three-dimensional structure was determined in 1996 by Ormo and coworkers [17]. The protein globule is a barrel of beta-sheets (Fig. 22A, B) that encapsulates and isolates the autocatalytically formed chromophore (Fig. 22C) from the environment. The chromophore itself consists of a phenole ring, with the OH group protonated inside the protein, and imidazole ring with two nitrogen atoms. The p-conjugated bond system, responsible for the absorption in the visible range, spans both rings. GFP absorbs violet light (around 400 nm), however, the emission is in the green (508 nm). It took some time to understand the reason for such a huge Stokes shift, but the fluorescence upconversion experiments performed by M.Chattoraj et al. finally solved the mystery of green GFP fluorescence [18].


Fig. 1) Three-dimensional structure of GFP viewed from ‘the side’ (A) and from the ‘top’ (B). Beta-sheets are shown in green ribbons, and the chromophore is indicated as ball and rod structure. C: Chemical formula of synthetic analogue of GFP chromophore.



The main result of their investigation is shown in Fig. 23. The researchers recorded upconverted time-resolved fluorescence at 460 nm and 508 nm. Two samples were investigated: one ‘normal’ GFP, another – dissolved in a buffer, where D2O was used instead of water. In such conditions, the proton on the phenol ring of GFP is exchanged to deuterium (i.e. becomes two times heavier). From the data, we see that fluorescence at 460 nm appears immediately after the excitation (Fig. 23A,B), which implies that it comes from the same state that absorbs 400 nm photon. In contrast, none of the samples (neither hydrogenated, nor deuterated) shows any 508 nm fluorescence at t=0 (Fig. 23C,D). After about 20 ps in normal GFP and 100 ps in deuterated GFP, initial 460 nm fluorescence decays and the intensity at 508 nm grows in (incidentally, this is the wavelength corresponding to the steady-state fluorescence maximum of GFP). Two important experimental facts here are a) the newly created fluorescence is significantly more intense than the initial signal at 460 nm, and b) the rate of growth of 508 nm fluorescence depends on whether the phenol ring of GFP chromohore has hydrogen or deuterium attached to it.  These led M.Chattoraj and coworkers to a double conclusion: a) green fluorescence of GFP occurs from a state different from the one that absorbs the photon and b) this state is formed from the initially excited state of GFP via the proton transfer (otherwise its appearance would not be influenced by the effective mass of this proton, i.e. there would be no difference in signals observed in H2O and D2O buffers).


Fig. 2) Fluorescence upconversion experiments in GFP: A, C: fluorescence intensity as a function of time in ‘normal’ GFP. Panel A shows the kinetics measured at460 nm, whereas panel C shows the fluorescence recorded at 508 nm. B,D – the same experiments as A and C, performed in GFP dissolved in D2O, where the OH proton of phenol ring is replaced by twice heavier deuterium. Reproduced from [18].


This important insight, provided by time-resolved fluorescence experiments, has led to a better understanding of the mechanism of green fluorescence of GFP. In addition, the experimentalists were handed a new interesting toy: a protein molecule, wherein light produces proton transfer. Proton transfer rate observed in this experiment was 8 ps (this is the decay time of 460 nm fluorescence, and the corresponding growth rate of 508 nm fluorescence, see Fig. 23 A, C). These molecules are widely known in chemistry (they are called photoacids), however, this study showed that they are important in biology too.