Femtosecond and picosecond fluorescence of native bacteriorhodopsin and a nonisomerizing analog

The spectrally and temporally resolved fluorescence properties of native bacteriorhodopsin (bR) and bR reconstituted with a nonisomerizing analog of the retinal Schiff base (bR5.12) are examd. The first attempt to exptl. monitor the excited state relaxation processes in both type of pigments using ultrafast fluorescence spectroscopy is reported. The fluorescence is emitted from retinal mols. in an all-trans configuration. Substantial energy relaxation involves very fast intramol. and intermol. vibrational modes and these are shown to occur on a time scale faster than isomerization. The possible contribution of dielec. interaction between the retinal Schiff base and the protein environment for the excited state energy relaxation is discussed. [on SciFinder (R)

Generation of 10 to 50 fs pulses tunable through all of the visible and the NIR

Noncollinearly phase-matched optical parametric amplifiers (NOPAs) pumped by the blue light of a frequency-doubled Ti:sapphire regenerative amplifier are a convenient source of continuously tunable ultrashort pulses in the visible and near infrared fo

Probing the ultrafast charge translocation of photoexcited retinal in bacteriorhodopsin

The ultrafast evolution of the electric field within bacteriorhodopsin was measured by monitoring the absorption changes of a tryptophan residue after excitation of retinal. The Trp absorption decreases within the first 200 femtoseconds and then recovers on time scales typical for retinal isomerization and vibrational relaxation. A model of excitonic coupling between retinal and tryptophans shows that the signal reflects a gradual rise of the retinal difference dipole moment, which precedes and probably drives isomerization. The results suggest an intimate connection between the progressive dipole moment change and the retinal skeletal changes reported over the same time scale

Probing the ultrafast charge translocation of photoexcited retinal in bacteriorhodopsin

The ultrafast evolution of the electric field within bacteriorhodopsin was measured by monitoring the absorption changes of a tryptophan residue after excitation of retinal The Trp absorption decreases within the first 200 femtoseconds and then recovers on time scales typical for retinal isomerization and vibrational relaxation. A model of excitonic coupling between retinal and tryptophans shows that the signal reflects a gradual rise of the retinal difference dipole moment, which precedes and probably drives isomerization. The results suggest an intimate connection between the progressive dipole moment change and the retinal skeletal changes reported over the same time scale

Absorption spectroscopy of three-dimensional bacteriorhodopsin crystals at cryogenic temperatures: effects of altered hydration

A comparative study of absorption spectroscopy at 100 K has been performed on three-dimensional crystals of bacteriorhodopsin extd. from a lipidic cubic phase and on native purple membrane. A modified microspectrophotometer has been designed which yields absorption data with a high signal-to-noise ratio and remarkable reproducibility. Excellent agreement of the absorption spectra of the three-dimensional crystals and the purple membrane is obsd. provided that a rigorous crystal-handling procedure is followed. This result supports the equivalence of the protein structure in both the cubic phase crystals and the native purple membrane. On the other hand, it is shown that dramatic deviations of the crystal spectrum can be induced by minor changes in the extn. method. Exposure to air at room temp. can lead within a short time to an irreversible dehydration manifested by a distinct species with an absorption max. at 500 nm. Exposure of the crystals to a buffer with lower ionic strength than the crystn. soln. produces a different spectral form with an absorption max. at 477 nm, which was assigned to a distorted protein conformation induced by osmotic stress. The extreme sensitivity of these crystals to exptl. conditions is relevant for x-ray structural studies, in particular as different exptl. treatments are implemented to trap the intermediates of the protein's photocycle. [on SciFinder (R)