A method for measuring picosecond phenomena in photolabile species: the emission lifetime of bacteriorhodopsin

Hirsch, Mitchell D.; Marcus, Michael A.; Lewis, Aaron; Mahr, H.; Frigo, N.J.

doi:10.1016/s0006-3495(76)85783-9

Cited by 53 publications

(21 citation statements)

References 30 publications

(45 reference statements)

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“…These molecules (1 in 10,000 at room temperature, significantly higher as the temperature is lowered) are deflected by any "roughness" in the PDC surface and enter states near zero PDC that are not accessible to molecules in which protein deformation to P,, has taken place. Thus, the emitting state (or possibly even states), with a lifetime of 15 psec at physiological temperatures (30,31) or 40 psec at 77 K (29), is drawn to indicate zero PDC and, as is observed (31), has dynamic properties that are not correlated to the rate of production of the photochemical product at physiological temperatures or at 77 K. This is clearly understood in terms of our three-dimensional illustration in Fig. 2.…”

Section: Experimental Observationsmentioning

confidence: 74%

“…However, the above experiments have not been able to demonstrate that this minimum can be populated from the batho intermediate. Furthermore, room temperature (30) and low temperature (77 K) (29) picosecond emission spectroscopy and picosecond absorption spectroscopy (25,31,32) have shown that the photochemistry occurs directly from the state produced by vertical excitation, whereas emission occurs by a parallel competing pathway from a state or states that are entered by molecules that do not proceed to the batho intermediate.…”

Section: Experimental Observationsmentioning

confidence: 99%

See 1 more Smart Citation

The molecular mechanism of excitation in visual transduction and bacteriorhodopsin

Lewis

1978

Proc. Natl. Acad. Sci. U.S.A.

107

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An electronic theory of excitation is proposed and described in terms of a three-dimensional excited/ ground-state energy surface which elucidates the photochemical and excited-state dynamics of rhodopsins. In this theory the primary action of light is to produce significant electron redistribution in the retinal, thereby generating new interactions that vibrationally excite and perturb the ground-state protein conformation. Thus, light energy causes charge redistribution in the retinal and induces transient charge-density assisted bond rearrangements (such as proton translocation) in the protein structure which is stabilized by subsequent retinal structural alteration. In this theory the isoprenoid chain of the retinal is considered a structurally pliable molecular entity that can generate charge redistributions and can subsequently achieve intermediate conformations or various isomeric states to minimize the energy of the new protein structure generated by light. Thus, the 11-cis to all trans isomerization of the retinylidene chromophore is not considered a primary mechanism of excitation. An alternate biological role for this molecular process (which is eventually completed in all photoreceptors but not in bacterial rhodopsins) is to provide the irreversibility needed for effective quantum detection on the time scale of a neural response. Finally, it will be demonstrated that this mechanism, which readily accounts for the photophysical and photochemical data, can also be restated in terms of the Monod, Wyman, and Changeux terminology suggesting that aggregates of these pigments may function allosterically. This paper proposes a generalized mechanism of excitation in visual transduction and bacteriorhodopsin that accounts for the spectral similarities observed in all rhodopsin-like systems while accounting for their functional diversity. Unlike previous descriptions of the excitation mechanism (1), this theory is based on the effect the excited state of retinal has on the conformational state of the protein. The result of this approach, which views a protein's conformation as a dynamically fluctuating and responding entity, elucidates the photochemical and excitedstate dynamics of rhodopsins in terms of a unique excitedstate/ground-state energy surface. In essence, the theory not only explains a large fraction of the recent data on rhodopsin* and bacteriorhodopsin,t but also demonstrates how the energy used in the subsequent steps of transduction is stored in the photochemical event. Experimental observationsThere are several experimental results that must be accounted for in any mechanism of excitation. In both rhodopsin and bacteriorhodopsin, absorption of a photon produces a high energy species (19,20) that has a red-shifted absorption maximum relative to the parent pigment (21-23) (Fig. 1). This red-shifted species is produced in <6 psec (24, 25) in both these systems. A kinetic argument of Rosenfeld et al. (26), based on the data of Kropf et al. (27), has shown that all batho intermediates lie at ...

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Section: Experimental Observationsmentioning

confidence: 74%

Section: Experimental Observationsmentioning

confidence: 99%

The molecular mechanism of excitation in visual transduction and bacteriorhodopsin

Lewis

1978

Proc. Natl. Acad. Sci. U.S.A.

107

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“…Considerable effort has been spent in the last years to elucidate the structure and the photochemistry of the purple membrane (for recent reviews, see Oesterhelt, 1976;Henderson, 1977). Most of the work so far has been devoted to the analysis of the photochemical reaction cycle of isolated purple membrane fragments in aqueous phase (Oesterhelt and Stoeekenius, 1973;Oesterhelt and Hess, 1973;Lewis et al, 1974;Dencher and Wilms, 1975;Kung et al, 1975;Lozier et al, 1975;Hirsch et al, 1976;Kaufmann et al, 1976;Lozier et al, 1976;Sherman et al, 1976;Becher and Ebrey, 1977;Goldschmidt et al, 1977;Marcus and Lewis, 1977;Renthal, 1977;Campion et al, 1977). While these studies have revealed the occurrence of a number of intermediate steps in the overall photocycle, the connection between the single photochemical reaction steps and the proton transfer process is not completely clear.…”

Section: Introductionmentioning

confidence: 99%

Photocurrents generated by bacteriorhodopsin on planar bilayer membranes

Bamberg

Apell

Dencher

et al. 1979

Biophys. Struct. Mechanism

142

111

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Abstract. When purple-membrane fragments from Halobacterium halobium are added to one aqueous phase of a positively-charged black lipid membrane, the membrane becomes photoelectrically active. Under normal conditions the steady-state photo-current is extremely low, but increases considerably when the lipid bilayer is doped with proton-permeable gramicidin channels or with a lipophilic acid-base system. These findings indicate that the purple-membrane sheets are bound to the surface of the bilayer, forming a sandwich-like structure. The time-behaviour of the photocurrent may be interpreted on the basis of a simple equivalent circuit which contains the conductance and capacitance of the purple membrane in series with the conductance and capacitance of the lipid bilayer. From the dependence of the photocurrent on the polarization of the exciting light the average angle between the transition moment of the retinal chromophore and the plane of the bilayer was calculated to be about 28 degrees. Furthermore, it was shown that chromophore-free apomembrane binds to the lipid bilayer and that its photoelectrical activity can be restored in situ by adding all-trans-retinal to the aqueous phase.

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“…This method is particularly suited for laser pulses of moderate peak powers but very high repetition rate. Very low light level emissions with picosecond lifetimes were soon measured from bacteriorhodopsin (15 ± 3 ps) [13] and the Sz~So fluorescence ofxanthione (14±2 ps) [14]. Other early investigations using this technique include fluorescence measurements of rhodamine 6G [15], malachite green [16], cresyl violet, and HITC [17] as a function of the solvent viscosity.…”

Section: Historical Background Of the Time-gated Up-conversion Techniquementioning

confidence: 99%

“…Only a few examples will be given which emphasize the advantage of time-resolved fluorescence spectroscopy over time-resolved absorption spectroscopy due to the well-defined ground and excited states. In relation to the biochemical importance of retinyl chromophores in photoreceptor proteins (rhodopsin and bacteriorhodopsin [13]), the fluorescence decay of all-trans retinal in hexane consists of ultrafast (r = 30 fs), fast (r = 370 fs), and slow (r = 33.5 ps) components [69]. The photo active yellow protein from the purple bacterium Ectothiorhodospira halophila contains a thiol ester linked p-coumaric acid.…”

Section: Biological Systemsmentioning

confidence: 99%

Investigation of Femtosecond Chemical Reactivity by Means of Fluorescence Up-Conversion

Mialocq¹,

Gustavsson²

2001

New Trends in Fluorescence Spectroscopy

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Fluorescence spectroscopy is a powerful tool for the study of the reactivity of chemical and biological systems. The fluorescence spectrum, the fluorescence quantum yield, and the fluorescence lifetime of a chromophore depend on its microenvironment and the solute-solvent interactions. Fluorescence lifetimes also depend on the kinetics of possible chemical reactions. Fluorescence measurements in the time domain thus provide detailed information about intraand inter-molecular photophysical and photochemical processes. During the past 30 years the development of laser sources and ultrafast techniques has opened new ways for the study of the fluorescent excited states of molecules in the nano-, pico-, and femtosecond time range. When analyzing the rise and the decay (often multi-exponential) of their fluorescence intensity as a function of time, the pre-exponential factors and the fluorescence time constants have to be determined accurately after a careful deconvolution using a computer-based algorithm. Several techniques are used: phase modulation spectroscopy with a modulated laser pump source on a time scale comparable to the decay times of interest, time-correlated single photon counting under picosecond laser excitation (alternatively nanosecond flashlamp excitation), ultra-fast streak cameras designed for the conversion of time dependent light emission into distance dependent electron impact on a phosphor screen. Because a sufficient resolution is not accessible with those techniques for the measurement of fast decays in the 50 fs-100 ps range, fluorescence up-conversion in nonlinear optical crystals is the technique of choice. Nanosecond and Picosecond Time-Resolved Fluorescence TechniquesThe fluorescence lifetime TF of a molecule is the reciprocal of the rate TF of depopulation of the first excited singlet state following optical excitation from the ground state. In the case of a monoexponential decay TF is given by

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A method for measuring picosecond phenomena in photolabile species: the emission lifetime of bacteriorhodopsin

Cited by 53 publications

References 30 publications

The molecular mechanism of excitation in visual transduction and bacteriorhodopsin

The molecular mechanism of excitation in visual transduction and bacteriorhodopsin

Photocurrents generated by bacteriorhodopsin on planar bilayer membranes

Investigation of Femtosecond Chemical Reactivity by Means of Fluorescence Up-Conversion

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