Photophysics and Dihedral Freedom of the Chromophore in Yellow, Blue, and Green Fluorescent Protein

Abstract

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).

Smaller 145th residue makes fluorescent protein non-fluorescent: Fluorescence lifetimes of enhanced yellow fluorescent protein (eYFP) Y145 mutants and H148 mutants

“…Furthermore, a previous study has demonstrated the existence of a photoswitchable FP that has a rigid structure in the fluorescent state and a flexible structure in the dark state [11]. Also, correlation between the dihedral freedom in the chromophore and the fluorescence quantum yield for some FPs has been suggested based on calculations [12]. The fluorescence mechanisms of these specific FPs and CPs are rather clear, but direct comparison between these proteins appears to be difficult.…”

Beta-Barrel Scaffold of Fluorescent Proteins

“…Upon excitation of the chromophore, the π-conjugation of the bridge is reduced and the phenolic ring of the chromophore can rotate freely. In this case, the protein matrix does not allow the chromophore to gain the perpendicularly twisted conformation that is postulated to be the main pathway of nonradiative energy dissipation (Megley et al, 2009). Thus, the microenvironment of the chromophore should be rigid enough in FPs with high quantum yield.…”

A diabatic three-state representation of photoisomerization in the green fluorescent protein chromophore

“…Presumably, the protein must inhibit displacement along all bridge torsion coordinates in order to preserve the emitting state. This "multiple pathway problem" has been highlighted by Zimmer and co-workers, [26][27][28][29] However, these models cannot address the twisted intramolecular charge-transfer ͑TICT͒ character that accompanies excited-state torsion. 23,24 Straightforward extension of theories of enzymatic catalysis 30 would suggest that electrostatic interactions between the TICT states and the protein may contribute to suppression or control of the photoisomerization.…”