Two-photon double resonance spectroscopy of bacteriorhodopsin. Assignment of the electronic and dipolar properties of the low-lying 1A*−g-like and 1B*+u-like π, π* states

111

Quantum mechanics/molecular mechanics calculations based on ab initio multiconfigurational second order perturbation theory are employed to construct a computer model of Bacteriorhodopsin that reproduces the observed static and transient electronic spectra, the dipole moment changes, and the energy stored in the photocycle intermediate K. The computed reaction coordinate indicates that the isomerization of the retinal chromophore occurs via a complex motion accounting for three distinct regimes: (i) production of the excited state intermediate I, (ii) evolution of I toward a conical intersection between the excited state and the ground state, and (iii) formation of K. We show that, during stage ii, a space-saving mechanism dominated by an asynchronous double bicycle-pedal deformation of the C10 ═ C11 ─ C12 ═ C13 ─ C14 ═ N moiety of the chromophore dominates the isomerization. On this same stage a N ─ H∕water hydrogen bond is weakened and initiates a breaking process that is completed during stage iii.photoisomerization | quantum mechanics/molecular mechanics (QM/MM) | retinal proteins T he light-activated proton pump bacteriorhodopsin (bR) is an Archaea receptor contained in the purple membrane of Halobacterium salinarium. As shown in Scheme 1, upon photoexcitation, the all-trans retinal chromophore (PSBT) bounded to Lys216 via a protonated Schiff base linkage is converted to the 13-cis isomer (PSB13). This event triggers a series of conformational changes that ultimately result in a proton translocation from the cytoplasmic to the extracellular domain (1).The bR protein environment affects both the spectroscopic and photochemical properties of PSBT (2-4). First, the absorption maximum (568 nm) is red-shifted with respect to the one observed in solution (440 nm in methanol). Second, timeresolved spectroscopy reveals a dominant excited state lifetime component of 450 fs (5, 6) almost matching the 500-fs time scale for formation of the vibrationally hot primary photoproduct J (7,8). In contrast, PSBT in solution features a biexponential decay dynamics with a dominant (ca. 10-fold longer) 2-ps component (9-11). Finally, although bR photoisomerizes stereoselectively (leading exclusively to PSB13) with high (ca. 67%) quantum yield (9, 12), irradiation of PSBT in solution leads to a mixture of different stereoisomers with a smaller (ca. 25%) total quantum yield (9, 13).Low-temperature spectroscopic studies provided evidence for the existence of a tiny (≤1 kcal mol −1 ) energy barrier on the excited state (S 1 ) potential energy surface of bR (14,15). Such a barrier would explain the 450-fs short-lived quasistationary state observed by Ruhman et al. (5) that is assigned to the fluorescent state I (16-18) and precedes decay to the ground state (S 0 ). Recent experiments by Léonard et al. (19) have also revealed that the photoinduced changes in the permanent dipole moment of PSBT are mirrored by the absorption of Trp86 up to the first stable (i.e., cryogenically trapped) photoproduct K (7). Relative to bR, K bears 11.7-...

Section: Photoisomerization Photoproduct Formation and Retinal Dipolesupporting

confidence: 63%

Aborted double bicycle-pedal isomerization with hydrogen bond breaking is the primary event of bacteriorhodopsin proton pumping

Altoè

Cembran

Olivucci

et al. 2010

111

“…This value is similar to σ 2 reported for bacteriorhodopsin, a microbial proton pump homologous to CR, near its spectral two-photon absorption peak (290 GM) (17). This value is also higher than most σ 2 values reported for fluorophores common to TPLSM [e.g., EGFP, DsRed2; 40-100 GM (18)], implying that TPE stimulation of CR photocurrents should not be excitation-limited at intensities compatible with TPLSM imaging.…”

Section: Resultsmentioning

confidence: 48%

Two-photon excitation of channelrhodopsin-2 at saturation

Rickgauer

Tank

2009

268

300

We demonstrate that channelrhodopsin-2 (CR), a light-gated ion channel that is conventionally activated by using visible-light excitation, can also be activated by using IR two-photon excitation (TPE). An empirical estimate of CR's two-photon absorption crosssection at λ = 920 nm is presented, with a value (260 ± 20 GM) indicating that TPE stimulation of CR photocurrents is not typically limited by intrinsic molecular excitability [1 GM = 10 −50 (cm 4 s)/ photon]. By using direct physiological measurements of CR photocurrents and a model of ground-state depletion, we evaluate how saturation of CR's current-conducting state influences the spatial resolution of focused TPE photostimulation, and how photocurrents stimulated by using low-power scanning TPE temporally summate. We show that TPE, like visible-light excitation, can be used to stimulate action potentials in cultured CR-expressing neurons.optogenetics | photostimulation | laser-scanning microscopy C hannelrhodopsin-2 (CR) is a microbially derived cation channel that transiently opens in response to blue-light illumination (1). Several modified forms of the channel, each encoded by a single gene, have been expressed heterologously in excitable mammalian neurons to recapitulate this light-gated conductance. It has been shown that in CR-expressing neurons, action potentials can be triggered by single-photon excitation, using blue-light illumination, with millisecond temporal resolution (2-7). Where multiple cells are photosensitized, the optical resolution with which each cell can be independently excited determines the precision of optical neuronal interrogation.Spatially localized excitation of individual cells is, however, difficult to achieve in thick biological tissue by using single-photon excitation. Brain tissue, for example, is an optically scattering medium that defocuses light, reducing the spatial definition and intensity of a focused beam or projected spatial pattern with increasing tissue depth (8). Additionally, excitation cannot be confined to a single z-focal plane of interest perpendicular to the axis of illumination (but see ref. 9), a geometry typically favored for in vivo studies of neural circuits.Two-photon laser-scanning microscopy (TPLSM) using a focused IR laser beam is the method of choice by which to achieve spatially localized fluorescence excitation deep in scattering tissue, providing an intrinsic optical section around the plane of focus (10, 11). We hypothesized that an analogous method to activate CR photocurrents by using two-photon excitation (TPE) would confer a much higher degree of spatial precision for targeted neuronal photostimulation than is currently possible by using single-photon (blue-light) illumination.Here we use whole-cell recordings of photocurrents in cultured cells to provide an initial biophysical characterization of TPE of CR. At typical light intensities used for TPLSM, we find that brief (<25 ms) CR photocurrents are described by a singlephotocycle model incorporating depletion of the CR ground state....

“…However, experiments with modified bR containing nonisomerizable retinal analogs challenged this model by showing that the initial photo-induced events are not associated with retinal isomerization (8)(9)(10). In fact, in an early alternative hypothesis (11), the essential process was proposed to be dielectric relaxation of the protein as a response to sudden polarization upon retinal excitation (11)(12)(13). Recent molecular dynamics calculations (14) and experiments (15,16) support this view.…”

mentioning

confidence: 74%

Resonant optical rectification in bacteriorhodopsin

Groma

Colonna

Lambry

et al. 2004

The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans3cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary lightinduced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility (2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics.R etinal proteins play an essential role in a broad range of light-driven biological processes, including vision (1), energy transduction (2), and circadian control (3). All these functions involve both the conversion of light energy into charge separation and retinal isomerization, but the interplay of these processes is the subject of intense debate. The retinal protein of which the initial photochemistry is most extensively studied is the photosynthetic protein bacteriorhodopsin (bR). This protein colors the purple membrane of halobacteria and acts as a light-driven proton pump by means of a multistep process termed the photocycle (2). In the traditional model for the initial transduction step in this cycle is directly light-driven trans3cis isomerization of retinal (4-6); this model has been recently extended by including excited-state skeletal stretching (5, 7). However, experiments with modified bR containing nonisomerizable retinal analogs challenged this model by showing that the initial photo-induced events are not associated with retinal isomerization (8-10). In fact, in an early alternative hypothesis (11), the essential process was proposed to be dielectric relaxation of the protein as a response to sudden polarization upon retinal excitation (11-13). Recent molecular dynamic...