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Ultrafast fluorescence upconversion measurements were carried out on the peripheral (LH2) light harvesting antenna complex of Rhodobacter sphaeroides isolated in the detergents N-octyl--D-glucopyranoside and lauryl dimethylamine oxide. The B800 and B850 bands were excited in separate experiments, and the B850 emission was detected in each case. We make use of the recently determined crystal structure of a purple bacterial LH2 complex to simulate our data and to calculate the exciton level structure of the B850 aggregate. The B800 to B850 excitation transfer occurs with a 650 fs time constant. The depolarization of B850 emission follows a wavelength dependent, biexponential decay with time constants 50-90 and 400-500 fs. We can reproduce the non-exponentiality of the depolarization by assuming incoherent hopping between dimeric sites with a 250 cm -1 full width at half-maximum (fwhm) Gaussian distribution of site energies (inhomogeneity). We calculate the homogeneous hopping time between dimers in B850 to be ∼100 fs. The exciton calculations including pigment energetic disorder demonstrate the validity of a hopping picture of excitation transfer within the B850 band at room temperature.

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The dynamics of solvation of an excited chromophore in pure water and in a restricted space with a limited number of water molecules have been studied. The time-dependent Stokes shift of Coumarin 480 (C480) and Coumarin 460 (C460) were measured using femtosecond fluorescence upconversion and time-correlated singlephoton-counting techniques. The system with a limited number of water molecules was an inclusion complex of Coumarin dyes with y-cyclodextrin (yCD). The results of molecular dynamics simulations are compared with the observed solvent response in pure water and in the yCD cavity. The observed relaxation times range from < fs to 1.2 ns. Solvation of C480 in pure water is observed to occur with time constants of <50 and 310 fs. In sharp contrast with the solvation response in pure water, in the case of the C48O/yCD inclusion complex, additional long solvation time constants of 13, 109 and 1200 ps are observed. The stoichiometry, structure and dynamics of the CoumarinlyCD complexes are also discussed.

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Three-pulse echo peak shift measurements were performed on the B875 and B850 bands of detergent-isolated LH1 and LH2 complexes at room temperature. The peak shifts are much larger and decay much faster than typically observed for dye molecules in solution. Simulations of the peak shifts based on the optical transition frequency correlation function, M(t), are presented. M(t) includes contributions from rapid protein fluctuations, vibrational motion, and energy transfer. The model reproduces the room temperature absorption spectra of B850 and B875, shows that the coupling of electronic and nuclear degrees of freedom is much weaker than for dyes in solution, and identifies contributions to the line shapes that may be important to the energy transfer processes. The implications of these results for the extent of electronic delocalization in LH1 and LH2 are also discussed. Although the role of coherence transfer still needs to be understood, the results are shown to be consistent with the use of weak-coupling excitation transfer models of B850 and B875.

The solvent-dependent ground-state conformational equilibrium and excited-state dynamics of 2-(2′-hydroxyphenyl)benzoxazole have been characterized in several solvents on the femtosecond to nanosecond time scales. The only observable ground-state tautomer is the enol, which exists in equilibrium between the syn-and anti-rotational isomers. In the anti-enol isomer, the phenyl hydroxyl group appears to not interact strongly with solvent but rather forms a strong intramolecular hydrogen bond with the benzoxazole oxygen atom. In the syn-enol isomer, the phenyl hydroxyl proton may interact with solvent or form an internal hydrogen bond with the benzoxazole nitrogen atom. Upon excitation, the proton is transferred from the oxygen atom to the nitrogen atom of the internally hydrogen bonded syn-enol isomer in 170 fs, regardless of the solvent. The lifetime of the resulting excited keto tautomer is solvent dependent and on the order of picoseconds. In addition to these dynamics, several additional dynamic processes are detected which may correspond to relaxation of a distorted excited keto tautomer.

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).