The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photoexcitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit at positions 64-69 (1, 2). We have investigated the electronic absorption and ultra-fast emission from GFP and have discovered surprisingly rich photophysics and photochemistry, interesting in their own right and relevant to applications and modifications of this system.The recent interest in this protein builds on many years of effort. The precise chromophore structure and conformation are still subject to investigation, and a three-dimensional structure of the protein is not yet available (3). The chromophore absorption in the visible region consists of two broad bands shown in Fig. 1A. Although these two bands could correspond to transitions from the ground to the first and second singlet excited states of the same chromophore, their ratio depends on conditions such as pH, temperature, and ionic strength (4) suggesting that two different, interconvertible forms of the chromophore may be present. By examining the excited state dynamics of the chromophore, both in its native state and following deuterium exchange, we demonstrate that two different forms are present and that their interconversion in the excited state depends on proton motion. Because GFP fluorescence is insensitive to oxygen quenching, it has been suggested that the chromophore is in a solvent inaccessible region (5). The fluorescence is stable in...
Gas-phase intramolecular electron transfer was studied in molecules of the type D-SgA where D was a positive charge donor, A was a positive charge acceptor, and Sp was a rigid, inert spacer. In our experiments, the donor was always naphthalene, the acceptors were benzene, indole, and biphenyl, and the spacers were cyclohexane and androstane. The naphthalene chromophore was selectively ionized by means of resonantly enhanced two-photon ionization via the naphthalene SI state.The location of the charge was monitored by observing the resonantly enhanced multiphoton dissociation spectrum of the ion. When the charge was localized on the naphthalene, the naphthalene ion D2 -Do spectrum was observed as an action spectrum (ion yield at a given mass versus wavenumber). When the charge had transferred to the acceptor, the naphthalene ion spectrum was not observed. Using this technique we attempted to measure the charge-transfer lifetime by measuring the intensity of the naphthalene ion action s p t r u m as a function of the delay between the ionizing and dissociating lasers. We found that in all cases where charge transfer was thermodynamically possible, the charge-transfer lifetime was less than 2 ns, the time resolution of the experiment. IntroductionIn the last decade charge transfer in bichromophores consisting of a donor and an acceptor separated by a rigid spacer has been actively studied both theoretically3 and e~perimentally.~~~ Charge-transfer rates are not diffusion limited in these systems as they are in intermolecular systems. Intramolecular charge transfer also serves as a good model for biological charge transfer. So far all intramolecular chargetransfer studies have been camed
Intramolecular electronic energy transfer has been observed in one isomer of the bichromophoric molecule D-SpA where the donor (D) is naphthalene, the spacer (Sp) is cyclohexane, and the acceptor (A) is dimethylaniline (DMA). Because the emission spectra of both the donor and acceptor overlap, the emission wavelength could not be used as a diagnostic for energy transfer. However, the donor and acceptor have very different fluorescence lifetimes, and large changes in the fluorescence lifetimes of the vibronic states of naphthalene due to the presence of DMA could be used as a measure of the mixing and energy transfer between the two chromophores. The methyl groups of DMA cause the density of states to rise very rapidly at energies close to the zero-point level, and in the cis isomer this leads to energy transfer from naphthalene vibronic levels that are only slightly above the zero-point level of DMA. The energy-transfer rate was measured as a function of the excited vibronic-state energy. The onset of energy transfer in cis isomers occurs at a lower energy than in the trans isomer, corresponding to a lower density of states.
Intramolecular electronic energy transfer was observed in bichromophoric molecules with the general structure donor-spacer-acceptor where indole is the donor, naphthalene is the acceptor, and cyclohexane is the inert spacer. Measurements were performed in a supersonic jet where both the absorption and emission spectra of the two chromophores were well-resolved and indole could be selectively excited with essentially no excitation on naphthalene. The emission produced on excitation of indole showed strong naphthalene emission and relatively weak indole emission. Moreover, the fluorescence lifetime was similar to the fluorescence lifetime of naphthalene, indicating that the energy transfer occurred on a time scale faster than the fluorescence lifetime of the donor.Energy transfer was observed in several conformers of the bichromophore, but the rates were similar for each. The relative energy-transfer rates of various vibronic levels was determined by measuring the ratio of the naphthalene and indole emission intensities. The energy-transfer rate was found to be fairly constant as a function of vibrational state until the vibrational levels of indole became isoenergetic with the naphthalene S2 state. From this point, the rate increased and then decreased with greater excitation energy. A modification of the theory of mediated intersystem crossing was used to account for this effect.
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