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On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications.

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the pi-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT-type motion.

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.

Green fluorescent protein (GFP) and GFP-like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore. According to quantum mechanical calculations, the excited state of chromophore model systems has significant dihedral freedom, which may lead to fluorescence quenching intersystem crossing. Molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green, yellow, and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state. Most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield. According to our calculations, the dihedral freedom of the systems studied (BFP > A5 > YFP > GFP) increases in the inverse order to the quantum yield. In all simulations, the chromophore undergoes a negatively correlated hula twist (also known as a bottom hula twist mechanism).