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Green Fluorescent Protein

Not surprisingly, the theoretical understanding of biophysical processes is a very active field of research. However, and in spite of the large amount of experimental work in photo-active molecules, the theoretical description of the interaction of these molecules with external time-dependent fields is very much in its infancy. Many biological processes rely on a subtle interplay between optical absorption in the photo-active center and its coupling to internal vibrational modes and to the environment (hosting protein and solvent). The most famous of these processes is vision, that is triggered by a photo-isomerization mechanism. Another paradigmatic case is the green fluorescent protein (GFP). This molecule has become a unique tool in various kinds of biomedical research due to its fluorescence and inertness when attached to other proteins.

Several members of the ETFS are currently involved in the study of the processes of light absorption and luminescence of the GFP and its mutants. It is hoped that a better understanding of the basic processes will allow for the design of novel mutants of the GFP or of other completely new chromophores with enhanced properties.

The study of the optical properties of biochromophores has developed into an important and active field of research. The rationale is clear, as the absorption and emission of light by biomolecules are at the center of crucial biophysical processes -- such as vision or photosynthesis, and has led to various important technological applications. Among photo-active proteins, and due to its unique photophysical properties, the family composed by the green fluorescent protein (GFP) and its mutants has attracted a considerable amount of attention during the last decade. These molecules have been used as important and versatile fluorescent markers, with widespread applications in the field of biotechnology. One of the originalities of the GFP resides in the fact that the chromophore responsible for the photophysics of the protein, 4-(p)-hydroxybenzylidene-imidazolidin-5-one, is completely generated by an autocatalytic, post-translational cyclization and oxidation of the --Ser66--Tyr66--Gly67-- triad, without the need of any external cofactor. Thus, all the information needed to synthesize the biochromophore is encoded in the corresponding gene. Furthermore, the GFP can be easily attached to other proteins without changing its own absorption properties. This unique characteristic, due to the protective cage-like secondary structure of the protein, makes the GFP an ideal candidate for a biological marker.

With the widespread use of the GFP, there has been an increasing demand for the ability to visualize different proteins in vivo that require multicolor mode imaging. This has triggered intensive research aimed at the development of GFP-mutant forms with different optical responses. A mutant of the chromophore of particular interest is the Y66H variant, in which Tyr66 is mutated to His. The resultant protein exhibits fluorescence shifted to the blue range, and is for that reasonoften referred to as the blue emission variant of the GFP, or the blue fluorescent protein.

Absorption spectrum of the GFP. Theory vs exp.
Calculations on the chromophores of the green fluorescent protein, the blue fluorescent mutant (Y66H), and the photoactive yellow protein have appeared recently. Some of these carried out by members of the proponent team. To illustrate the usefulness of these calculations, we take the case of the GFP. The main excitation peaks, calculated using time-dependent density functional theory (TDDFT), for the neutral and anionic forms are at 3.01 and 2.67 eV, respectively, in really good agreement with the measured excitation energies, located at 3.05 and 2.63 eV. Furthermore, the comparison to the experimental spectrum allows for the clear assignment of the measured peaks to either the neutral or anionic forms of the GFP. Finally, from the calculations it is possible to extract a 4:1 ratio for the concentration of the neutral/anionic forms in vivo, which is very close to the estimated experimental ratio of 80% neutral and 20% anionic.

Note that the process of light absorption and emission is quantum mechanical, and it is not therefore, accessible using the classical mechanics techniques usually applied in biochemistry. However, the size of the problem (that involves thousands of atoms), and its complexity forbid the use of pure quantum mechanical approaches. Therefore, the solution of such problems requires a mixture of approaches.

  • T. Wilson and J. W. Hastings, Annu. Rev. Cell Dev. Biol. 14 197-230 (1998).
  • V. Pieribone and D. F. Gruber, Alone in the Dark: the Revolutionary Science of Biofluorescence (The Belknap Press of Harvard University Press, Cambridge, Massachussetts).
  • M.A.L. Marques, et al, Phys. Rev. Lett. 90, 258101 (2003); X. Lopez, et al, J. Am. Chem. Soc. 127, 12329-12337 (2005).