Resonantly Enhanced Multiphoton Ionization Spectrum of the Neutral Green Fluorescent Protein Chromophore

Abstract

The photophysics of the green fluorescent protein is governed by the electronic structure of the chromophore at the heart of its β-barrel protein structure. We present the first two-color, resonance-enhanced, multiphoton ionization spectrum of the isolated neutral chromophore in vacuo with supporting electronic structure calculations. We find the absorption maximum to be 3.65 ± 0.05 eV (340 ± 5 nm), which is blue-shifted by 0.5 eV (55 nm) from the absorption maximum of the protein in its neutral form. Our results show that interactions between the chromophore and the protein have a significant influence on the electronic structure of the neutral chromophore during photoabsorption and provide a benchmark for the rational design of novel chromophores as fluorescent markers or photomanipulators.

Combined quantum‐mechanical molecular mechanics calculations with NWChem and AMBER: Excited state properties of green fluorescent protein chromophore analogue in aqueous solution

“…Our calculated absorption values are 3.69 and 3.61 eV for the cis and trans conformers, respectively, and seem to confirm to be within these measurements. Our numbers are also in line with previously calculated values of 3.71 eV (CAM‐B3LYP) and 3.58 eV (CASPT2) for the cis conformer of HBDI.…”

“…The experiment using the charge group by Rajput et al proposed the absorption at 3.35 eV . Recently, Greenwood et al applied an alternative approach and reported the absorption value to be 3.65 eV. Our calculated absorption values are 3.69 and 3.61 eV for the cis and trans conformers, respectively, and seem to confirm to be within these measurements.…”

“…However, the charged group technique by Rajput et al places it at 3.35 eV for the neutral form . Recently, Greenwood et al have applied an alternative method and reported a new maximum absorption value of the neutral form to be 3.65 eV . In aqueous phase the absorption value was determined to be 3.33–3.35 eV .…”

ortho and para chromophores of green fluorescent protein: controlling electron emission and internal conversion

“…Knowing that the environment of the chromophore plays a pivotal role in determining the spectral properties of GFP, which are important for imaging applications, led to numerous investigations to determine how structural modifications to the protein, such as changing single amino acid residues, can be used to tune the absorption or fluorescence properties. 4 , 16 Recently, there has also been a great deal of interest in determining how the structure and environment of the isolated chromophore influences the absorption properties 17 , 18 and dynamics. 19 For example, variants of the isolated chromophore in which the hydroxyl group was moved from the para position to the ortho position were found to be weakly fluorescent in cyclohexane ( Φ ≈ 0.003).…”

GFP Chromophores from L‐Phenylalanine: Synthesis, Photophysical and Thermal Properties

“…The GFP and its family have a profound impact on the versatility of techniques available to cell biologists studying different diseases like cancer and other genetic defects –. Thus, there is an immense interest in determining how the structure and environment of the isolated chromophore influences the absorption properties– and dynamics . Complete understanding of the reaction mechanism of GFP chromophore biosynthesis and reaction pathway and identification of intermediates would be highly beneficial to chemists and biologists.…”