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.

We demonstrate hole burning on a protein by using an intrinsic aromatic amino acid as a probe. The protein is bovine pancreatic trypsin inhibitor (BPTI), the labeled amino acid is tyrosine. Only one of the four tyrosines could be burned. As an application we present pressure tuning experiments from which the local compressibility around the burned tyrosine probe is determined.

We measured the Stark-e †ect of spectral holes burnt into the long wavelength absorption of phenylalanine dissolved in a glycerolÈwater glass. Within the pH-range investigated we observed two protonationÈdeprotonation transitions, however, they did not change the general pattern of the Stark-e †ect : phenylalanine behaves as a nearly centrosymmetric molecule with no detectable permanent dipole moment up to a Ðeld strength of about 40 kV cm~1.

We investigated a series of structurally and chemically different dye-DNA complexes via the specific behavior of spectral holes under pressure. In all samples chromophore and solvent were the same, only the DNA strands were different. The dominant interaction with the solvent is mainly electrostatic via the strongly polar BF 2 group of the chromophore. The relative change of the dipole moment upon excitation can be estimated from the experimental data. There is a significant color effect in the pressure-induced broadening of the holes, which signals a breakdown of the Gaussian approximation. The origin of this breakdown is associated with ordered structures around the chromophore due to the DNA strands but also because of the formation of ordered solvent cage structures as a consequence of the hydrophobic nature of the chromophore. The various DNA strands show characteristic features in the spectra under pressure that reflect specific structural features.

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