Dr. Jasper J. van Thor Profile

Dr. Jasper J. van Thor

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Proceedings Article | 23 February 2006 Paper

Ultrafast and low-barrier motions in the photoreactions of the green fluorescent protein

Jasper van Thor, Michael Towrie, Kate Ronayne, Georgi Georgiev, J. Timothy Sage

Proceedings Volume 6098, 609806 (2006) https://doi.org/10.1117/12.651278

KEYWORDS: Green fluorescent protein, Chromophores, Picosecond phenomena, Luminescence, Absorption, Spectroscopy, X-rays, Crystals, Infrared spectroscopy, Crystallography

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Green fluorescent protein (GFP) is highly fluorescent with blue excitation despite the creation of a buried charge resulting from photoionization of the chromophore and neutralization of Glu 222. These major electrostatic rearrangements do not lead to rapid internal conversion processes. A competing phototransformation reaction, which ionizes the chromophore and decarboxylates Glu 222, causes electrostatic and structural changes which are very similar to those in the fluorescence photocycle. The X-ray crystallography and IR spectroscopy of phototransformed GFP provides evidence for relaxations involving protein, chromophore, solvent, and CO₂. Time-resolved and static infrared spectroscopy and X-ray crystallography to 1.85 Å resolution identify structural mechanisms common to phototransformation and to the fluorescence photocycle. A detailed study of the ps time-resolved IR absorption during the fluorescence photocycle has been reported. Through global fitting the species associated difference spectra were determined showing the vibrational response to excited state proton transfer in addition to the transient population of a late ground state photocycle intermediate 'I₂'. We additionally used pump-dump-probe spectroscopy to dump the deprotonated radiative state 'I*' and directly provide the I₂-I* difference spectrum, which strongly resembled its photocycle counterpart. We discuss spectral markers that specifically report on structural relaxation during the photocycle which may be compared with structural relaxations in the competing phototransformation reaction.

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