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Polarized Absorption Spectra of Green Fluorescent Protein Single Crystals:  Transition Dipole Moment Directions

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journal contribution
posted on 13.12.2002, 00:00 by Federico I. Rosell, Steven G. Boxer
Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments (m⃗) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting Förster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (λmax = 398 nm) and anionic (λmax = 478 nm) forms of the chromophore were found to subtend angles of ∼26° and 13° (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.