Ultrafast spectroscopy of the visual pigment rhodopsin.

186

168

Femtosecond transient absorption measurements of the ci-trans isomerization of the visual pigment rhodopsin clarify the interpretation of the dynamics of the first step in vision. We present femtosecond time-resolved spectra as well as kinetic measurements at specific wavelengths between 490 and 670 nm using 10-fs probe pulses centered at 500 and 620 nm following a 35-fs pump pulse at 500 nm. The expanded spectral window beyond that available (500-570 nm) in our previous study [ Vision is initiated when a photon is absorbed by the seven a-helical, membrane-bound protein called rhodopsin (1). After optical excitation, the 11-cis-retinal prosthetic group of rhodopsin is converted to an all-trans primary photoproduct in an efficient and apparently barrierless isomerization reaction ( Fig. 1). This product, which was first identified as bathorhodopsin in low-temperature absorption experiments, has an absorption maximum of =z560 nm (2). (13) with 35-fs pump and 10-fs probe pulses centered at 500 nm. Subsequently, Callender and co-workers (14) presented 300-fs time resolution data at 620 nm, which they interpreted in terms of a 200-fs twisting process on the excited-state surface, followed by a 3-ps excited-state to ground-state photoproduct transition. To resolve this disagreement, we present here a more complete series of femtosecond experiments on rhodopsin in which the probe wavelength range is extended to the red by using 10-fs pulses centered at 620 nm. Narrow-bandwidth kinetic traces at a selection of probe wavelengths as well as differential spectral measurements extending from 490 to 670 nm have been obtained. These results establish, in agreement with our earlier report, that the formation time for the rhodopsin photoproduct is 200 fs, and they provide key evidence that clarifies the interpretation of the primary photochemistry of vision. EXPERIMENTAL PROCEDURESThe method for generating 35-fs pump pulses at 500 nm as well as 10-fs probe pulses centered at 500 and 620 nm have been described elsewhere (12,13,15). The experimental configuration used to obtain differential transmittance spectra and time-resolved measurements is identical to that used in ref. 13. Briefly, the average pump power is ==3 ,W at a repetition rate of400 Hz. The pump (==0.5 mJ/cm2) and probe (O0.04 mJ/cm2) beams are crossed in a 0.3-mmjet offlowing bovine rhodopsin (15 OD/cm in ammonyx-LO). The flow rate is sufficient to replace the photolyzed sample between successive pump pulses. Two methods are used to measure transient changes in absorption (AT/T) as a function ofprobe delay time after photolysis of the rhodopsin. Differential spectra at particular pump-probe delay times are obtained by dispersing the probe pulse in a spectrometer after it passes through the sample and imaging it onto a dual diode array. Kinetic information at specific wavelengths is obtained by filtering the probe pulse after the sample (-"7-nm bandpass) and combining differential detection with lock-in amplification as the pump-probe delay time is continu...

Section: Methodscontrasting

confidence: 52%

Section: Methodsmentioning

confidence: 53%

See 1 more Smart Citation

The first step in vision occurs in femtoseconds: complete blue and red spectral studies.

Peteanu

Schoenlein

Wang

et al. 1993

186

168

“…This species cannot be isolated even at low temperature, and it is identified through a red-shifted, 570-nm max a . Furthermore, the excited-state or ground-state nature of photo Rh is still a matter of debate (40)(41)(42). Given the limited knowledge provided by our computations on the structure of the S 1 reaction path and S 0 relaxation path we cannot presently assign such an entity.…”

Section: Resultsmentioning

confidence: 98%

Structure, initial excited-state relaxation, and energy storage of rhodopsin resolved at the multiconfigurational perturbation theory level

Andruniów

Ferré²,

Olivucci³

2004

240

343

We demonstrate that a ''brute force'' quantum chemical calculation based on an ab initio multiconfigurational second order perturbation theory approach implemented in a quantum mechanics͞ molecular mechanics strategy can be applied to the investigation of the excited state of the visual pigment rhodopsin (Rh) with a computational error <5 kcal⅐mol ؊1 . As a consequence, the simulation of the absorption and fluorescence of Rh and its retinal chromophore in solution allows for a nearly quantitative analysis of the factors determining the properties of the protein environment. More specifically, we demonstrate that the Rh environment is more similar to the ''gas phase'' than to the solution environment and that the so-called ''opsin shift'' originates from the inability of the solvent to effectively ''shield'' the chromophore from its counterion. The same strategy is used to investigate three transient structures involved in the photoisomerization of Rh under the assumption that the protein cavity does not change shape during the reaction. Accordingly, the analysis of the initially relaxed excited-state structure, the conical intersection driving the excitedstate decay, and the primary isolable bathorhodopsin intermediate supports a mechanism where the photoisomerization coordinate involves a ''motion'' reminiscent of the so-called bicycle-pedal reaction coordinate. Most importantly, it is shown that the mechanism of the Ϸ30 kcal⅐mol ؊1 photon energy storage observed for Rh is not consistent with a model based exclusively on the change of the electrostatic interaction of the chromophore with the protein͞counterion environment.photoisomerization ͉ quantum mechanics ͉ molecular mechanics ͉ retinal ͉ vision T he visual pigment rhodopsin (Rh) (1, 2) is a G proteincoupled receptor containing an 11-cis retinal chromophore (PSB11) bounded to a lysine residue (Lys-296) via a protonated Schiff base linkage (see Scheme 1). While the biological activity of Rh is triggered by the light-induced 11-cis 3 all-trans isomerization of PSB11, this reaction owes its efficiency (e.g., short time scale and quantum yields) to the protein cavity (1). Accordingly, investigation of the environment-dependent properties of PSB11 is a prerequisite for understanding the Rh ''catalytic'' effect. The equilibrium geometry, absorption maxima ( max a ), and fluorescence maxima ( max f ) are indicators of the environment effect. In fact, whereas the geometry of PSB11 is nearly planar in a crystal (3), in bovine Rh it has a helical conformation (4). Similarly, the 445-nm max a observed for PSB11 in methanol (5) is red-shifted to 498 nm in Rh (1, 2): an effect known as the opsin shift.The Rh fluorescence band ranges from 530 to 780 nm (6). The max f has been reported (6) to be excitation wavelength-dependent, shifting from 595 to 704 nm when the excitation wavelength is shifted from 472 to 568 nm. This observation is consistent with the idea that the emission arises from a nonstationary unrelaxed excited-state population. In methanol solution the PSB11...

“…After photon absorption, a series of at least seven intermediates making up the RhRT photosequence appears. These room-temperature intermediates have been identified primarily via transient absorption spectroscopy (3)(4)(5)(6)(7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

Bathorhodopsin structure in the room-temperature rhodopsin photosequence: picosecond time-resolved coherent anti-Stokes Raman scattering.

Popp

Ujj²,

Atkinson³

1996

Structural changes in the retinal chromophore during the formation of the bathorhodopsin intermediate (bathoRT) in the room-temperature rhodopsin (RhRT) photosequence (i.e., vision) are examined using picosecond time-resolved coherent anti-Stokes Raman scattering. Specifically, the retinal structure assignable to bathoRT following 8-ps excitation of RhRT is measured via vibrational Raman spectroscopy at a 200-ps time delay where the only intermediate present is bathoRT. Significant differences are observed between the C==C stretching frequencies of the retinal chromophore at low temperature where bathorhodopsin is stabilized and at room temperature where bathorhodopsin is a transient species in the RhRT photosequence. These vibrational data are discussed in terms of the formation of bathoRT, an important step in the energy storage/transduction mechanism of RhRT.It is well-recognized that the energy storage/transduction mechanism describing vision under physiological conditions occurs at room temperature in the transmembrane protein rhodopsin (RhRT) (1-3). After photon absorption, a series of at least seven intermediates making up the RhRT photosequence appears. These room-temperature intermediates have been identified primarily via transient absorption spectroscopy (3-9).The initial processes in the RhRT photosequence have been investigated by monitoring absorbance changes with time resolution of e 10-13 s and with an emphasis on excited electronic state lifetimes and the isomerization kinetics associated with the retinal chromophore (4, 5). Many characteristics of these processes, as well as their mechanistic interpretations, remain under discussion (4-7).The RhRT photosequence is well characterized kinetically over the 10-9-to 101-s regime (8,10,11 (14,15,(17)(18)(19)(20)(21)(22)(23). Lowering the protein temperature is thought to reduce the probability with which specific activation barriers along the reaction coordinate can be crossed. Since such an interruption of the Rh photosequence at low temperatures may also alter the protein environment, which is itself intimately involved in the energy storage/ transduction mechanism, it is unclear whether lowtemperature data, and the mechanistic models based on them (1,14,(18)(19)(20)(21)(22), are relevant to the RhRT photosequence and, thereby, to vision.The major experimental limitation to recording vibrational spectra from the RhRT photosequence has been its photoirreversibility. The efficient photodissociation of RhRT into free retinal and opsin after seconds ensures that the sample rapidly disappears after illumination, and, therefore, any spectroscopic data must be recorded efficiently and with an excellent signal-to-noise ratio (S/N). This is especially challenging in the case of vibrational data.CARS spectroscopy using picosecond pulsed excitation is used in this study to successfully address these experimental issues. The high S/N available from CARS means that highquality vibrational data can be obtained efficiently from small quantities of RhRT. T...