Chromophore Formation in the Green Fluorescent Protein [dataset]

Dan Gurnon
2009 SciVee   unpublished
The fluorescence emission from green fluorescent protein (GFP) is known to be heavily influenced by hydrogen bonding between the core fluorophore and the surrounding side chains or water molecules. Yet how to utilize this feature for modulating the fluorescence of GFP chromophore or GFP-like fluorophore still remains elusive. Here we present theoretical calculations to predict how hydrogen bonding could influence the excited states of the GFP-like fluorophores. These studies provide both a new
more » ... provide both a new perspective for understanding the photophysical properties of GFP as well as a solid basis for the rational design of GFP-based fluorophores. The green fluorescent protein (GFP) has been an indispensable tool for bioimaging and bioengineering since it was discovered in the jellyfish Aequorea victoria 1-4 . Tagged with GFP, living cells and tissues can be monitored via fluorescence microscopy non-invasively 5 . GFP is a 238-aa protein with a molecular weight of about 26.9 kDa. The core of GFP, 4-(4-hydroxybenzylidene)-1,2-dimethyl-imidazolinone (p-HOBDI), is enclosed at the centre of an 11-stranded β-barrel, and spontaneously forms a complex by binding to amino acid residues Ser65-Tyr66-Gly67 after a multistep reaction 6 . The remaining residues fold into a host scaffold or matrix, providing an intricate microenvironment which is essential for keeping the fluorophore HOBDI highly emissive 7 . Given that the synthetic replication of the three-residue-led moiety with exactly the same chemical structure as p-HOBDI only produces weak to no fluorescence in both solutions and polymer films, it is apparent that the key to the highly emissive state relies on the specificity of the surrounding protein matrix 8 . A generally accepted notion is that the protein skeleton increases the quantum yield of the fluorophore by restraining its rotational freedom 9,10 . In addition, conformations in the ground/excited state 11 , intermolecular interactions such as electrostatic interactions, hydrogen bond formation, π-π stacking, Van der Waals forces, medium viscosity, as well as many other parameters have been shown to influence the rate of non-radiative decay 12-16 . The degenerate contributions make it difficult to cast deep insights to tune the luminescence properties. As a result, it is truly essential to modulate the fluorescence of the GFP and GFP-like chromophore at the molecular and atomic level through theoretical understanding. Among better-studied aspects of GFP, the equilibrium between the neutral and ionized states of the fluorophore is regulated by a hydrogen bonding network 17 . It is well documented that the hydrogen bonding interactions between the chromophore and neighbouring side chains or water molecules could significantly affect the photochemical process. The excited-state proton transfer, which is the dynamics of the hydrogen bonding would contribute to generate an intense main fluorescence emission band 18 . Grigorenko et al. 19 simulated the proton transfer from the neutral or ionized structure to the surrounding amino acid side chains, demonstrating the significant role of hydrogen bonding network on the stability and emission of the chromophore. Oltrogge and Boxer et al. 20 have verified the significance of low-barrier hydrogen bonding in color tuning of chromophore fluorescence by modifying the acidity of halide-substituted chromophores.
doi:10.4016/11251.01 fatcat:3ceddtqbizgahck4lzkp3zrzsm