Du-Jeon Jang

Geminate recombination of molecular iodine. The role of A and A′ states

Picosecond absorption spectroscopy has been used to study the geminate recombination of I2 in room temperature solution. In all solvents studied (CCl4, CH2Cl2, CHCl3, hydrocarbons, and ethylene glycol) a transient absorption band in the 575–800 nm region was observed, and assigned to transitions originating from A(3Π1) and/or A′(3Π2) states. No spectral shift of this band was observed throughout its lifetime, making an assignment to a vibrationally unrelaxed ground state unlikely. Branching ratios for ground state vs A and A′ state recombinations were found to depend upon solvent viscosity. These results are explained in terms of the internuclear separation obtained upon dissociation. The geminate recombination rates into A or A′ states were found to be >(30 ps)−1 and ∼(90–140 ps)−1 into the ground state. At present we can not determine whether surface hopping to the ground state or subsequent diffusion is the rate limiting step in direct ground state recombination. Intersystem crossing rates from the A, ...

The thermal stability of native, delipidated, deionized and regenerated bacteriorhodopsin

Differential scanning calorimetry curves and circular dichroism spectra were determined for native bacteriorhodopsin (bR), deionized bR, acid blue and acid purple bR, 75% delipidated bR, deionized-delipidated bR and Mg2+ regenerated deionized bR. The effects of the different perturbations on the thermal stability (melting temperature) and the apparent helical content of the protein were examined. These perturbations have more influence on the deprotonation efficiency and the color change of retinal than on the thermal stability and the apparent helical content of the protein. Although the addition of Mg2+ to deionized bR restores the photochemical cycle, it does not restore the thermal stability to that of the native material.

ON THE MULTIPLE CYCLES OF BACTERIORHODOPSIN AT HIGH pH

Bacteriorhodopsin (bR) shows at least two parallel photocycles at pH 10.5, suggesting that more than one form of bR exists in alkaline bR sample. Upon the absorption of visible light, the different forms of bR at high pH yield different parallel intermediates: M412 with two rise and two decay components; and R, an extremely fast rising and extremely slow decaying intermediate with an absorption peak at 350 nm. The kinetics and spectra do not agree with the proposal of Kouyama el al. (1988, Biochemistry 27,5855–5863) that the 350 nm‐absorbing species is the N intermediate which follows M412 and that the slow decaying M412 is an M‐like photoproduct of N. Our results basically agree with the proposal of Dacshazy et al. (1988, Proc. Natl. Acad. Sci. USA 85,6358–6361) that the fast and slow decaying M412 intermediates and R are in independent photocycles arising from different forms of bR. The different forms of bR are probably in dynamic equilibrium with their ratios controlled by pH and ionic strength.

EFFECTS OF METAL CATIONS, RETINAL, AND THE PHOTOCYCLE ON THE TRYPTOPHAN EMISSION IN BACTERIORHODOPSIN

Abstract— The picosecond fluorescence kinetics of tryptophan residues in bacteriorhodopsin and some perturbed analogs are measured to study the different tryptophan environments and their changes upon metal cation removal, retinal removal, and M412 trapping. In bacteriorhodopsin, the emission shows four decay components designated Or, C2r, C3r, and C4r in order of increasing lifetimes. The emission wavelength of C3r and C4r is near that found in aqueous solution, while that of C1r is the shortest. The removal of retinal triples the total emission intensity and reduces the number of components to two, suggesting that the observed variation of the lifetimes in bacteriorhodopsin results from the variation of the energy transfer efficiency between different tryptophans and retinal. We conclude that the Or and C2r emission is from the closest tryptophans to the retinal. The quenching of the C3r emission by all metal cations, including those that cannot act as energy acceptors, e.g. Ca2+, is attributed to protein conformation changes caused by metal cation binding which leads to a stronger energy transfer coupling between tryptophans and retinal. The additional quenching of the C2r emission in Eu3+bound bacterioopsin is proposed to result from direct energy transfer between tryptophans and Eu3+.

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr−) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results or NMR, UV Raman as well as electron cryo‐microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp‐182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbation of the lowest energy absorption of Trp‐182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

High‐resolution picosecond transient absorption spectrometer utilizing dye emission and a streak camera

A high‐resolution picosecond transient absorption spectrometer utilizing dye emission and a streak camera is presented and compared with other methods of picosecond transient absorption measurements. Typical transient absorption and ground‐state bleach recovery kinetics measured with this spectrometer are shown. Single wavelength transient absorption or ground‐state bleach recovery kinetics are determined on the basis of a single laser shot with this spectrometer, so that the samples are relatively free from decomposition by irradiation. Excellent kinetics may be obtained from the near UV to the near IR and are not subject to interference from luminescence of samples. The sensitivity of this spectrometer is very high and it is reasonably easy and convenient to set up and use.

Absence of tryptophan fluorescence quenching by metal cations in delipidated bacteriorhodopsin

The addition of metal cations to deionized bacteriorhodopsin (bR) quenches the steady‐state tryptophan (Trp) fluorescence intensity and reduces the decay times of some of its picosecond components. Similar quenching processes of the Trp emission are not observed in deionized 75% delipidated bR (dLbR). The results are discussed in terms of conformational changes taking place in the protein upon delipidation.

Decay of the tryptophan fluorescence anisotropy in bacteriorhodopsin and its modified forms

In this work we study the decay of the polarization of the Trp fluorescence in native bacteriorhodopsin (bR), deionized bR (dlbR), and the retinal-free form of bR, bacterioopsin (bO), using picosecond laser/streak camera system. Two types of depolarization processes are observed, one around 250 ps, which is temperature independent around room temperature, and the other in the 1-3-ns range, which is sensitive to temperature and certain bR modifications. This suggests the presence of at least two different environments for the eight Trp molecules in bR. Native bR and deionized bR gave the same depolarization decay times, suggesting that the removal of metal cations does not change the microenvironment of the emitting Trp molecules. The slow component is faster in bO than in bR, suggesting a change in the environment of the Trp molecules upon the removal of the retinal chromophore. All these results are discussed in terms of the different mechanisms of Trp fluorescence depolarization. A comparison between the depolarization decay in rhodopsin and bR is made.