Low-Temperature Photochemistry and Photodynamics of the Chromophore of Green Fluorescent Protein (GFP)

Abstract

Hole-burning and temperature-dependent spectroscopy in green fluorescent protein (GFP) have been shown in the literature to be valuable tools in unraveling the photodynamics of this protein. It would be straightforward to perform similar experiments on the isolated GFP chromophore to differentiate between properties that are affiliated with the chromophore itself and those that are rather determined by the protein cage. To this end, we performed temperature-dependent and hole-burning spectroscopy on the organically synthesized chromophore of GFP in alcohol solutions. It can be shown that many of the spectral features described for the GFP protein are also observed in the chromophore dissolved in alcohol and alcohol glasses. In addition to the neutral state A and the anionic state B of the chromophore, additional states (I states) are observed. Analogous to those of the GFP protein, these states are assigned to unrelaxed anionic environments and are distinguished from the relaxed environment by different arrangements of the hydrogen bonds to the matrix. The comparison of the spectroscopic and kinetic properties of the GFP chromophore in alcohol solution with the protein has implications for the understanding of the photodynamics of GFP. We demonstrate that the protein cage alters the properties of the chromophore significantly. In particular, it can be concluded that proton transfer in the protein proceeds along better-defined reaction trajectories compared to those of the chromophore embedded in an amorphous matrix. Furthermore, the electron−phonon coupling of the chromophore of GFP in the amorphous lattice is higher compared to that in the protein, which is indicative of differences in the ground- and excited-state potential surfaces. The protein cage exerts restrictions upon the chromophore that may also be responsible for the high fluorescence quantum yield in the protein even at room temperature.

Solution pKa Values of the Green Fluorescent Protein Chromophore from Hybrid Quantum-Classical Calculations

“…8 These observations were attributed to the inhibition of rapid internal conversion involving motion with considerable amplitude. It was established 55 that the free chromophore in low-temperature alcoholic solution shows a spectral and photochemical reaction pattern similiar to that of the chromophore embedded in the protein matrix. In a poly(vinyl alcohol) (PVA) matrix, even ESPT is recoverd (P. Schellenberg, private communication).…”

Potential Energy Landscape of the Photoinduced Multiple Proton-Transfer Process in the Green Fluorescent Protein: Classical Molecular Dynamics and Multiconfigurational Electronic Structure Calculations

“…Green fluorescent protein (GFP) is an important fluorescent marker in cell biology, exhibiting a complex photophysics and photochemistry that has been the subject of many investigations during the last 10 years . Experimental − as well as theoretical − works have been mainly devoted to shedding light into the complex photophysics of the protein, arising from the internally caged chromophore and the nearby residues. A great part of the interest on GFP comes from the multiple proton relay system that operates in it after photoexcitation.…”

“…Experimental studies on the photophysics and photochemistry of GFP are usually interested in the overall cycle occurring upon photon absorption. The number of experiments concerning the three-step proton wire operating inside the protein is enormous, − whereas there is comparatively less theoretical work on the simulation and modeling of the three proton-transfer processes that constitute the photoactivated proton wire. Some theoretical studies have focused on the nonradiative deactivation pathways of the photoexcited chromophore, ,− involving mainly bond torsions that lead to a loss of planarity.…”

Internal Conversion in the Chromophore of the Green Fluorescent Protein: Temperature Dependence and Isoviscosity Analysis

“…We will return to the viscosity dependence below. Other groups have investigated the spectroscopy of compounds similar to I as models for GFP spectroscopy. − In particular Schellenberg and co-workers have demonstrated that there are significant differences in the Raman spectra of I and GFP, while I has a low-temperature photochemical hole-burning behavior which is rather similar to that observed in GFP. , …”