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A visible-pump/UV-probe transient absorption is used to characterize the ultrafast dynamics of bacteriorhodopsin with 80-fs time resolution. We identify three spectral components in the 265- to 310-nm region, related to the all-trans retinal, tryptophan (Trp)-86 and the isomerized photoproduct, allowing us to map the dynamics from reactants to products, along with the response of Trp amino acids. The signal of the photoproduct appears with a time delay of [approximately equal to] 250 fs and is characterized by a steep rise ([approximately equal to] 150 fs), followed by additional rise and decay components, with time scales characteristic of the J intermediate. The delayed onset and the steep rise point to an impulsive formation of a transition state on the way to isomerization. We argue that this impulsive formation results from a splitting of a wave packet of torsional modes on the potential surface at the branching between the all-trans and the cis forms. Parallel to these dynamics, the signal caused by Trp response rises in [approximately equal to] 200 fs, because of the translocation of charge along the conjugate chain, and possible mechanisms are presented, which trigger isomerization. ultrafast spectroscopy | retinal proteins | translocation of charge | structural dynamics

Analogous fluorescent states were discovered to be generated in native and locked pigments. This was known through the use of near infrared spectroscopy. Findings have indicated that the primary dynamic process in early light-induced events in bacteriorhodopsin do not involve the C13=C14 torsional coordinate and that other chromophore and protein coordinates must be involved in the very early branching from the excited state to the reactive and nonreactive pathways.

The proton pump mechanism and the final stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570) are known to be strongly pH dependent. In experiments carried out in deionized membrane suspensions, the relative amount of the accumulated O630 intermediate was measured along with the rise and decay constants (Kr and Kd) over a wide pH range. The pH dependencies, the bell curves of Kr and Kd, and the s-shaped curves of the O630 intermediate were then analyzed in relation to the protein residues tagged R1, R2 and R3.

The Asp-85 residue is important to the function of bacteriorhodopsin (bR), acting as a light-driven proton pump. Time-resolved titrations of Asp-85 were undertaken over a broad range of pH and cation concentrations. pH and cation jumps induced the Purple-Blue transition in both directions. It was found that binding/unbinding of metal cations is not rate-determining in Asp-85 titration.

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 [right arrow] Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.

The pKa values of the protonated Schiff base and aspartic 85 are altered due to the geometric constraints in the polyene skeleton by the five-membered rings of different synthetic retinals in the light-induced protonated bacteriorhodopsin. Breaking of the bridged structure where the Schiff base and its aspartic 85 counterion are linked to the water molecules is responsible for the changing pKa values.

The deprotonation or protonation methods of the retinal Schiff base and of the Asp-85 residue in artificial bacteriorhodopsins confirm that the basic relationships of homogenous kinetics are followed by the Schiff base in the pH range between 7 and 9, and is also true for Asp-85 residue located closely to the Schiff base. Of the channels where exchange of protons occur between the Schiff base and the extramembrane medium, Asp-85 is a vital component. The proton transport in the channel is unaffected by the protonation of Asp-85.

Spectroscopic studies of the picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment with a retinal show that C-13=C-14 isomerization is the key step in the BR photocycle. Motion in the C-12=C-13 and C-13=C-14 regions are essential for the formation of K-like intermediate in the native BR-570 photocycle, but are not required to form an intermediate with spectral and kinetic properties similar to that of J-625 in the BR photocycle.

Analysis of the difference spectra and transition temperatures of the conversion of the intermediate M state to bacteriorhodopsin (bR) reveals the presence of at least three equilibrated substates in the M state, which are dependent on pH. Changes in the arrangement of water molecules and conformations lead to these transition states. Generation of another intermediate state, the N state occurs through the transition from (M)a to (M)b substates. This rate-determining step involves the protein state M(V).

Visual pigment bacteriorhodopsin (bR) does not depend on the covalent bond between the protein backbone and retinal chromophore for its basic structure, proton pump activity and photochemical features. The mutant protein k216G with retinal alkylamine Schiff bases is used to prepare bR pigments that lack retinal-Lys-216 covalent bond.

Model compounds which have all-trans- and 13-cis-retinal-protonated Schiff bases with C double bond N anti and syn bond configurations are used in the study of the Schiff base linkage bond configurations of bacteriorhodopsin. A combination of Fourier transform infrared spectroscopy and isotopically labeled chromophores are used in the analysis of the configuration of C double bond N. The analysis reveals that the coupling between C14-C15 stretching frequency and N-H rock is not strong in the Schiff bases in the anti and syn C double bond N configurations.

13C NMR and absorption measurements are performed on retinal protonated Schiff bases in solution and solid state to analyze the effects of Schiff base environment changes, ring-chain s-trans planarity and nonconjugated positive and negative charges. Results show that the Schiff base environment affects the C5 chemical shift of the retinal protonated Schiff base. The nonconjugated charge effect is controlled by the spatial arrangement between the chemical shift and the double bond. Moreover, the absorption maximum of bacteriorhodopsin is controlled by ring-chain s-trans planarity.

The structural alterations during photocycling in the active-site lysine of bacteriorhodopsin (bR) were determined by fourier transform infrared spectroscopy. The active-site lysine was labelled by deuterizing its alpha carbon, and bR was enymatically cleaved then regenerated to observe the vibrational modes during coupling. The results showed that the vibrational modes that exhibit coupling are contributed by the the active-site lysine, amd not retinal. In addition, results indicated the presence of an out-of-plane twist at the C15-N bond.

A comparative study of the ultrafast pump-probe spectroscopy of all-trans protonated Schiff bases (PSB) of free and C(sub 13) double bond C(sub 14) locked retinals is presented. The results revealed that several major features appear to be common and are only marginally effected by locking of the C(sub 13) double bond C(sub 14) to all three PSBs.

A protonation study of retinal model proteins in micellar medium probes into the factors controlling the pKa of the retinal protonated Schiff base in pigments and their photoinduced intermediates. The study shows that a specific angle between the Schiff base and carboxyl group allows water molecules to bridge the groups, both in the ground and excited states. These water structures provide stability to the ion pairs and induce the protonation of the Schiff base by a specific carboxyl group.

Picosecond time-resolved coherent anti-Stokes Raman spectroscopy of intermediate T5.12 is reported.