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Miyasaka Laboratory

Yusuke Satoh

David W. McCamant et al, Science, 2005, 310, 1006-1009

Structural observation of the primary isomerization in vision with femtoseco

nd-stimulated Raman

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VisionThe light reaches the retina through eyes and is changed into signal in retina.Signals are sent to our brains.

Scheme 1. Structure of eye

(Ref. http://www.kiriya-chem.co.jp/q&a/q52.html)

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Retinal

Scheme 2. Structure of Rhodopsin

Opsin is a protein of 7 spiral structures.

A chromophore inside Opsin is Retinal.

11-Cis Retinal changes into all-trans-retinal by light irradiation.

Signal is sent to the optic nerve.

(Ref. http://www.spring8.or.jp/j/user_info/sp8-info/data/5-6-2k/5-6-2k-3-p394.pdf)

NH

NHh

11-cis retinal all-trans retinal

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Past research of retinal

Table 1 Fluorescence lifetime and Transient absorption spectroscopy of retinal

Ref. Chem. Phys. Lett., 2001, 334, 271Science, 1991, 100, 14526

Transient absorption measurement and time-resolved fluorescence detection of 11-cis Retinal ～ 200 fs lifetime of the excited state reported.

Fluorescence lifetime Transient absorption spectroscopy

retinal 200 300 fs～ 200 fs

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Motivation

But fluorescence and electronic absorption spectra do not provide direct information of the molecular structure.

A new time-resolved Raman spectroscopy method is necessary in order to elucidate the dynamics of this isomerization reaction and factors regulating this rapid structural change.

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Contents

・ Introduction

・ Experiment

・ Result and Discussion

・ Summary

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Principle of Spotaneous Raman scattering

Stokes shift Anti-stokes shift

Virtual excited state

Ground state

Raman spectroscopy has been used for the identification of the chemical bond and for the determination of the molecular structure.

Scheme 3. Mechanism of SpotaneousRaman scattering

0± ： Raman scattering

: Raman shift

0 0 － 0 ＋0

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Time-resolved Raman spectroscopy

Pump pulse

Sample

Detector

Intermediate

Scheme. 4 Time-resolved Raman spectroscopy

The simple application of femtosecond laser pulse does not provide detailed information of vibrational spectra.

Probe pulse

Delay 　 time

0

Raman scattering

0 －

Frequency / cm-1

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Resonance Raman and Stimulated Raman

Excited state

Ground state

0 0 －

Resonance Raman Stimulated Raman

Scheme. 5 Resonance Raman and Stimulated Raman

Virtual excited state

0

0 －

(narrow)

+ (0 － (broad)

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Stimulated Raman spectroscopy

Fig. 1. Stimulated Raman spectroscopy 　 (Ref. Rev. Sci. Instrum., 2004, 75, 4971)

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Stimulated Raman system

Fig. 2 Stimulated Raman spectroscopy system(Ref. Rev. Sci. Instrum., 2004, 75, 4971)

Excited pulse: 500 nm, 30 fs fwhmRaman pump: 805 nm, 3 ps fwhmRaman probe: 830 ～ 960 nm, 20 fs fwhm

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Structures of 11-cis Retinal and all-trans Retinal

NH

NHh

11-cis Retinal all-trans Retinal

11-Cis Retinal change into all-trans Retinal by light irradiation.

Fig. 3 Structure of 11-cis Retinal and all-trans Retinal

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Raman spectra of ground-state Retinal

Fig. 4 Raman spectra of ground-state of 11-cis Retinal(bottom) and all-trans Retinal(top)

・ Raman spectra of 11-cis Retinal(bottom)

1548 cm-1 ・・・ C=C stretch

1100 ～ 1300 cm-1 ・・・ C-C single bond stretch and C-H rocking modes

969 cm-1 ・・・ hydrogen-out-of-plane(HOOP) wagging motion of the C11 and C12 hydrogens

・ Raman spectra of all-trans Retinal(top)

920, 875, and 850 cm-1 ・・・ C11-H, C10-H, and

C12-H wagging

mode

hydrogen-out-of-plane(HOOP):水素の面外変角運動 rocking mode: 横ゆれ変角運動 wagging mode: 縦ゆれ変角運動

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Time-resolved Raman spectra of Retinal

Fig. 5 Time-resolved Raman spectra of Retinal(200 fs ～ 1 ps) and Raman spectra of ground state of 11-cis retinal(bottom) and all-trans retinal(top)

The dispersive HOOP features evolve on the same time scale as the finger-print bands into the expected three positive features of the Bathorhodopsin spectrum.

These data show that there is considerable reactive evolution on the ground-state surface from 200 fs to 1 ps.

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Time Profile of C10-H,C11-H and C12-Hhydrogen wagging frequencies

Fig. 6 Time profile of C10-H, C11-H and C12-H

hydrogen wagging frequency

The HOOP frequency increase by 100 cm-1 with 325 fs time constant.

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Fig. 7 Retinal chromophore structures for reactant

rhodopsin and for photorhodopsin and

bathorhodopsin that reproduce the observed

hydrogen wagging frequencies.

Structures of Retinal, Photorhodopsin and Bathorhodopsin

The Bathorhodopsin structure is twisted by –144° about the C11=C12 and by 31°about the C12–C13 bond.

The Photorhodopsin structure is more highly distorted, in particular about the C9=C

10 (45°), C10–C11 (25°), and C11=C12 (–110°) bonds.

With these larger twists, the overall shape of retinal is much more like that of 11-cis Rhodopsin than all-trans Bathorhodopsin,

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Theoretical and experimental hydrogen wagging frequencies

Fig. 8 Theoretical and experimental hydrogen wagging frequencies for the Photo and Bathorhodopsin structures

Caluculated frequency for Photorhodopsin structure show good agreement with experimental data for the C10-H,C11-H modes.

Vibrational calculations for the Bathorhodopsin structure yielded features in excellent agreement with experimental data, except for an underestimated C11–H wagging frequency.

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The isomerization coordinate for the primary event in vision

Fig. 9 Multidimensional representation of the isomerization coordinate for the primary event in vision

Excited-state of 11-cis Retinal carry the system toward a conical intersection in ～ 50 fs.

From 200 fs to 1 ps , Photorhdopsin changes into Bathorhodopsin on the ground-state surface.

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Summary

・ Excited-state decay (200 fs) through a conical intersection is mediated largely by fast HOOP motion.

・ By 1 ps, vibrational cooling has narrowed, thereby completing the transformation to Bathorhodopsin.

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Stimulated Raman spectroscopy

Fig. 1 Mechanism of stimulated Raman spectroscopy

Amplitude of coherent vibration induced by Raman and probe pulse 　

Heterodyne detection yields a gain feature on top of the probe envelope in the energy domain shifted in energy relative to the Raman pulse according to the frequency of the vibration.

①

①

Stimulated Raman spectroscopy is obtained by this method.

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Retinal

Scheme 2. Structure of Rhodopsin

Opsin is a protein with 7 spiral structures.

A chromophore inside Opsin is Retinal.

11-cis retinal changes into all-trans-retinal by light irradiation.

Signal is sent to the optic nerve.

(Ref. http://www.kiriya-chem.co.jp/q&a/q52.html)

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Feynman diagram

Feynman diagram

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Photoisomerization reaction of Rhodopsin

Photoisomerization reaction of Rhodopsin

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Principle of Raman scattering

Scheme 3. Mechanism of Raman scattering

0± ： Raman scattering

: Raman shiftRaman spectroscopy has been used for the identification of the chemical bond and for the determination of the molecular structure.

(Ref. http://www.natc.co.jp/bunseki/lr.html)

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Wagging mode and Rocking mode