Probing the Decay Coordinate of the Green Fluorescent Protein: Arrest of Cis-Trans Isomerization by the Protein Significantly Narrows the Fluorescence Spectra - Analytical Methods & Tools for Lyophilization

Probing the Decay Coordinate of the Green Fluorescent Protein: Arrest of Cis-Trans Isomerization by the Protein Significantly Narrows the Fluorescence Spectra
Received August 14, 2005
Web Release Date: January 13, 2006
Solomon S. Stavrov, Kyril M. Solntsev, Laren M. Tolbert, and Dan Huppert*
J. Am. Chem. Soc.
ACS Publications
Copyright? 2006 American Chemical Society
Contribution from the Sackler Faculty of Medicine, Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Tel Aviv University, Tel Aviv 69978, Israel, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, and Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel
huppert@tulip.tau.ac.il
Abstract:
The fluorescence spectra of the wild-type green fluorescence protein (wt-GFP) and the anionic form of p-hydroxybenzylidenedimethylimidazolone (p-HBDI), which models the protein chromophore, were obtained in the 80-300 K temperature range in glycerol/water solvent. The protein spectra have pronounced and well-resolved vibronic structure, at least at lower temperatures. In contrast, the chromophore spectra are very broad and structureless even at the lowest temperatures. Analysis of the spectra shows that the experimentally observed red-shift of the protein spectrum upon heating is apparently caused by quadratic vibronic coupling of the torsional deformation (TD) of the phenyl single bond of the chromophore to the electronic transition. The broad spectra of the chromophore manifest the contribution of different conformations in the glycerol/water solvent. In particular, the lowest-temperature spectrum reflects the distribution over the same TD coordinate in the excited electronic state, which essentially contributes to the asymmetry of the spectrum. Upon heating, motion along this coordinate leads to a configuration from which the radiationless transition takes place. This narrows the distribution along the TD coordinate, causing a more symmetric fluorescence spectrum. We were able to reconstruct the broad, structureless fluorescence spectra of p-HBDI in glycerol/water solutions at various temperatures by convoluting the original wt-GFP spectra with the function describing the distribution of the transition energies of the p-HBDI chromophore. Thus, both the fluorescence broadening and increase in radiationless transition upon removal of the protein chromophore to bulk solvent are consistent with decay by a barrierless TD of the phenyl single bond.
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