[12]
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
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[12] R e s o n a n c e R a m a n a n d I n f r a r e d D i f f e r e n c e S p e c t r o s c o p y of R e t i n a l P r o t e i n s B y FRIEDRICH SIEBERT
Introduction Vibrational spectroscopy such as resonance Raman (RR) and infrared difference spectroscopy (IRD) has mostly been applied to retinal proteins having the retinal covalently bound to the protein via a protonated Schiff base. In all the cases studied, the side chain of a lysine constitutes the amino group. The vertebrate and invertebrate visual pigments and four different proteins located in the plasma membrane of the bacterium Halobacterium halobium belong to this class of proteins. The absorption of light by the chromophore causes its isomerization which, in turn, induces the protein structural changes responsible for the various functions of these systems. The proton pump bacteriorhodopsin, the chlorine pump halorhodopsin, the sensory pigments sensory rhodopsin and P-480 constitute the retinal proteins of Halobacterium halobium. Several reviews on these systems have been published. TM As far as the visual pigments are concerned, the special volume (Vol. 13) ofPhotobiochemistry andPhotobiophysics (1986) and the articles by Balogh-Nair and Nakanishi, 5 Ottolenghi,2 and Sandorfy and VoceUe6 provide a survey. Vibrational spectroscopy can help to determine what are the structures of the chromophores in these pigments and in the intermediates of their photoreactions, what kind of interaction exists between the chromophore and the protein, and what are the structural changes evoked in the protein by the photoreaction. These topics are dealt with in two recent reviews on the application of RR spectroscopy7,8 and in a review on the 1 D. Oesterhelt and J. Tittor, Trends Biochem. Sci. 14, 57 (1989). 2 M. Ottolenghi, in "Advances in Photochemistry" (J. N. Pitts, G. S. Hammond, K. Gollnik, and D. Grosjean, eds.), p. 97. Wiley (Interscience), New York, 1980. 3 W. Stoeckenius and R. A. Bogomolni, Annu. Rev. Biochem. 52, 587 (1982). 4 j. K. Lanyi, Annu. Rev. Biophys. Biophys. Chem. 15, 11 (1986). 5 V. Balogh-Nair and K. Nakanishi, in " N e w Comprehensive Biochemistry, Volume 3: Stereochemistry" (C. Tamm, ed.), p. 283. Elsevier Biomedical, Amsterdam, 1982. 6 C. Sandorfy and D. Vocelle, Can. J. Chem. 64, 2251 (1989). 7 M. Stockburger, T. Alshuth, D. Oesterhelt, and W. G~rtner, in "Spectroscopy of Biological Systems" (J. H. Clark and R. E. Hester, eds.), p. 483. Wiley, New York, 1986. 8 R. A. Mathies, S. O. Smith, and I. Palings, in "Biological Application of Raman Spectrometry, Volume 2: Resonance Raman Spectra of Polyenes and Aromatics" (T. G. Spiro, ed.), p. 59. Wiley, New York, 1987.
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
124
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[12]
application of IRD spectroscopy.9 Such problems may also apply to other retinal-containing proteins. The basic principles of the two methods are described here and some important applications discussed.
Resonance Raman Spectroscopy Normal Raman scattering is a weak effect, and recording a complete spectrum usually requires many hours. However, if the molecule has an absorption band near the wavelength of the probing beam (usually a continuous wave laser), the scattered photon is emitted from the electronic excited state and the scattering cross section for vibrations of this molecule is increased by several orders of magnitude. Resonance Raman spectroscopy has, therefore, the unique advantage of selectivity in that, in the spectrum of such complex systems as retinal proteins, only vibrations of the chromophore will be reflected. In the retinal proteins, however, a photoreaction is evoked by the absorption of light. Resonance Raman scattering is, therefore, always connected with the generation of intermediates of the photoreaction, and special techniques have been developed to obtain spectra of these intermediates as well as of the initial state. The basic methods are described briefly. Depending on the system being investigated, there are two basic techniques for overcoming these difficulties: namely, time-resolved technique and pump-probe technique. The first technique uses either a capillary flow system 1°-12or a rotating cell. 13,14The main purpose of both methods is to continuously bring fresh sample into the laser beam. The laser power has to be low enough to accumulate negligible amounts of photoproducts during the short time the sample resides within the cross-section of the laser beam (usually a few microseconds). The pump-probe technique was first applied to rhodopsin by Oseroff and Callender. 15In this application a photoequilibrium between three species of the photoreaction of rhodopsin can be established at 80 K (rhodopsin, bathorhodopsin, and isorhodopsin). By altering the wavelength of the "pump" laser beam, which usually
9 M. S. Braiman and K. J. Rothschild, Annu. Rev. Biophys. Biophys. Chem. 17, 541 (1988). 10 R. H. Callender, A. Doukas, R. K. Crouch, and K. Nakanishi, Biochemistry 15, 1621 (1976). 11 R. A. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, 1 (1976). ~2R. A. Mathies, T. B. Freedman, and L. Stryer, J. Mol. Biol. 109, 367 (1977). ~3 W. Kiefer and H. J. Bernstein, Appl. Spectrosc. 25, 500 (1971). 14 M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979). 15 A. R. Oseroff and R. H. Callender, Biochemistry 13, 4243 (1974).
[12]
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
125
has the higher intensity, the composition of the photoequilibrium is changed. By tuning the wavelength of the "probe" laser beam to a position where one of the three species contributes most to the absorption spectrum, the RR scattering of this compound will selectively be enhanced. The probe beam should have a lower intensity in order not to alter the composition of the photoequilibrium. For systems which undergo a cyclic photoreaction, such as bacteriorhodopsin and halorhodopsin, the pump-probe technique can be applied at room temperature. The photostationary state will be dominated by the initial state and the intermediate with the slowest decay time. The time-resolved and pump-probe techniques can, of course, be combined. The composition of intermediates can be altered by altering the power and wavelength of the pump laser. Again, by adjusting the probe beam, the RR scattering from the species of interest will be enhanced. In general, however, irrespective of the method employed, the RR spectrum will contain contributions from several species. Employing time-resolved techniques, the RR spectrum of the initial state can usually be obtained by illuminating the sample with a very weak probe beam only. This spectrum can then be used to deduce from the composite spectra the spectra of the intermediates of the photoreaction. By moving the focus of the probe beam from the focus of the pump beam, it is possible to collect spectra at times after the excitation of the sample by the pump beam. The time is given by the distance between pump and probe beam divided by the velocity of the flow. This is an additional method for altering the relative amounts of intermediates in the cross section of the probe beam. Intermediates arising several microseconds up to several milliseconds after excitation can be measured in this way. More technical details about the methods can be found in the review papers mentioned above. One can obtain detailed molecular information by comparing the RR spectra of retinal proteins with those of model compounds. The basic investigation by the Mathies group has contributed greatly to our understanding of the vibrational spectra of isomers of retinal 16'~7and of protonated and unprotonated retinylidene Schiff bases.lS.~9 An essential part of this study was the collaboration with the Lugtenburg group, providing the 16 B. Curry, A. Broek, J. Lugtenburg, and R. A. Mathies, J. Am. Chem. Soc. 104, 5274 (1982). 17 B. Curry, I. Palings, A. Broek, J. A. Pardoen, P. P. J. Mulder, J. Lugtenburg, and R. A. Mathies, J. Phys. Chem. 88, 688 (1984). i8 S. O. Smith, A. B. Myers, R. A. Mathies, J. A. Pardoen, C. Winkel, E. M. M. Van den Berg, and J. Lugtenburg, Biophys. J. 47, 653 (1985). 19 B° CLlrry, I. Palings, J. A. Pardoen, J. Lugtenburg, and R. A. Mathies, Adv. Infrared Raman Spectrosc. 12, ll5 (1985).
126
STRUCTURE AND ANALYSIS
[12]
13C- and 2H-labeled retinals. 2°,21 By developing an empirical molecular force field, almost all bands in the RR spectra could be assigned to specific vibrations of the molecules and characteristic bands for the various retinal isomers could be deduced. The effects of forming the Schiff base and its protonation have also been studied. Since retinals, as well as their protonated and unprotonated Schiff bases, are photolabile, the capillary flow technique proved to be essential. In Fig. l, the RR spectra of bacteriorhodopsin under various illumination conditions using the rotating cell technique are shown. 14Spectrum (a) in Fig. 1 was collected with a weak probe beam of 514 nm only, reflecting the light-adapted species of bacteriorhodopsin only, BR568, which has an absorption maximum at 568 nm. By applying a broad pump beam of 514 nm and a coaxial probe beam of 450 nm, an almost pure spectrum of the long-lived intermediate M412 could be obtained [spectrum (b) in Fig. 1], having an absorption maximum at 412 nm. Only the shoulder at 1530 cm -1 indicates a small percentage of the initial state BR568. If the intensity of the probe beam is increased, spectrum (c) in Fig. 1 is obtained. This spectrum contains, in addition to BR568, the M412 intermediate, as can be seen from the band at 1567 cm -1. I f a pump beam of 450 is now applied, driving the M412 intermediate back to BR568, spectrum (d) (Fig. 1) is obtained. These experiments demonstrate the principles of the pump-probe technique in combination with time-resolved techniques. The spectra (Fig. 1) are all dominated by a strong band between 1500 and 1600 cm -1, caused by the ethylenic vibration of the retinal. Its position is correlated with the absorption maximum of the retinylidene Schiff base, owing to the dependency of the ethylenic force constants on ~r-electron delocalization. 22 The band at 1642 cm -l (Fig. la) was shown to shift to 1624 cm -1 in 2H20, demonstrating that it is caused by the C = N stretching vibration of the Schiff base and that "the Schiff base is protonated. The vibrations of this group have been especially investigated, both theoretically and experimentally. 23-28It appears unclear whether protonation increases or de2o j. Lugtenburg, Pure Appl. Chem. 57, 753 (1985). 21 j. Pardoen, C. Winkel, P. Mulder, and J. Lugtenburg, Recl. Tray. Chim. Pays-Bas 103, 135 (1984). 22 M. E. Heyde, D. Gill, R. G. Kilponen, and L. Rimai, J. Am. Chem. Soc. 93, 6776 (1971). 23 H. Deng and R. H. Callender, Biochemistry 26, 7418 (1987). 24 H. S. R. Gilson, B. H. Honig, A. Croteau, G. Zarrilli, and K. Nakanishi, Biophys. J. 53, 261 (1988). 25 T. Baasov, N. Friedman, and M. Sheves, Biochemistry 26, 3210 (1987). 26 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7131 (1986).
[12] RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS I
1
I
I
127
r-"-
~8
C
-
a
g,~g
,,o,~
!
p_.
~ A
o~
,
=
-
f
I
1
I
I
I
800
1000
1200
1400
1600
Av (cm-') FIG. 1. Pump-probe resonance Raman spectra of bactefiorhodopsin at room temperature using a rotating cell system. (a) Spectrum of the initial state BR568 taken with a weak probe beam of 514 nm only; (b) spectrum of the long-lived intermediate M412, taken with a 514-nm pump beam a 457-nm probe beam; (c) spectrum obtained with strong probe beam of 514 nm, showing the presence of BR568 and M412; (d) as (c), but with an additional pump beam of 457 nm, driving the M412 intermediate back to BR568. [Reproduced from M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979).]
128
STRUCTURE AND ANALYSIS
[ 12]
creases the C~---N force constant. Also, the contribution of the NH bending vibration to the C ~ N frequency is not completely understood. The understanding of this coupling is especially important, since it would provide a means to investigate the interaction of the protonated Schiff base group with its environment. Figure 2 demonstrates the pump-probe technique at -160 °, where the primary photoreaction of rhodopsin has been investigated) 9 Spectrum (A) in Fig. 2 was obtained with a probe beam of 585 nm. The sample was also illuminated with an all-lines argon laser pump beam. If the spectrum was recorded without the pump beam [spectrum (B), Fig. 2] only isorhodopsin was present. A pure rhodopsin spectrum was obtained by the capillary flow technique [spectrum (C), Fig. 2]. If appropriate amounts of spectra (B) and (C) are subtracted from spectrum (A), a pure spectrum of bathorhodopsin, the first intermediate which can be stabilized at low temperature, could be obtained. The downshift of the ethylenic mode (1545 versus 1536 cm -~) is in accordance with the red-shifted absorption maximum of bathorhodopsin. The strong bands below I000 cm -1 are of special interest. They could be assigned to hydrogen-out-of-plane (HOOP) vibrations of the retinal (i.e., the hydrogens move perpendicular to the polyene plane), and the high intensities were attributed to twists around single bonds of the polyene.3°,31 This means that the chromophore, already isomerized from 11-cis to all-trans, could not yet adopt a relaxed planar configuration owing to steric interaction with the protein, which is rigid at low temperature. A similar effect was observed for the 80 K photoproduct of bacteriorhodopsin, the K intermediate, 32and interpreted in a similar way. A peculiarity in the bathorhodopsin spectrum is the anomalous decoupling of the 1I- and 12-HOOP vibrations, causing the band at 920 cm-~; it was attributed to a specific interaction with a negatively charged residue or dipole of the protein. In recent illustrative applications of RR spectroscopy, demonstrating the selectivity of the method, the visual pigments in the retina of toad, anglefish, gecko, and bullfrog have been investigated) 3 In this case, the 27 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7241 (1986). 28 j. j. L6pez-Garriga, S. Hanton, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7251 (1986). G. Eyring and R. A. Mathies, Proc. Natl, Acad. Sci. U.S.A. 76, 33 (1979). 3o G. Eyring, B. Curry, A. Broek, J. Lugtenburg, and R. Mathies, Biochemistry 21, 384 (1982). 3~ I. Palings, E. M. M. Van den Berg, J. Lugtenburg, and R. A. Mathies, Biochemistry 28, 1498 (1989). 32 M. Braiman and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 79, 403 (1982). 33 B. A. Barry and R. A. Mathies, Biochemistry 26, 59 (1987).
[12]
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
I
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
123
[12] R e s o n a n c e R a m a n a n d I n f r a r e d D i f f e r e n c e S p e c t r o s c o p y of R e t i n a l P r o t e i n s B y FRIEDRICH SIEBERT
Introduction Vibrational spectroscopy such as resonance Raman (RR) and infrared difference spectroscopy (IRD) has mostly been applied to retinal proteins having the retinal covalently bound to the protein via a protonated Schiff base. In all the cases studied, the side chain of a lysine constitutes the amino group. The vertebrate and invertebrate visual pigments and four different proteins located in the plasma membrane of the bacterium Halobacterium halobium belong to this class of proteins. The absorption of light by the chromophore causes its isomerization which, in turn, induces the protein structural changes responsible for the various functions of these systems. The proton pump bacteriorhodopsin, the chlorine pump halorhodopsin, the sensory pigments sensory rhodopsin and P-480 constitute the retinal proteins of Halobacterium halobium. Several reviews on these systems have been published. TM As far as the visual pigments are concerned, the special volume (Vol. 13) ofPhotobiochemistry andPhotobiophysics (1986) and the articles by Balogh-Nair and Nakanishi, 5 Ottolenghi,2 and Sandorfy and VoceUe6 provide a survey. Vibrational spectroscopy can help to determine what are the structures of the chromophores in these pigments and in the intermediates of their photoreactions, what kind of interaction exists between the chromophore and the protein, and what are the structural changes evoked in the protein by the photoreaction. These topics are dealt with in two recent reviews on the application of RR spectroscopy7,8 and in a review on the 1 D. Oesterhelt and J. Tittor, Trends Biochem. Sci. 14, 57 (1989). 2 M. Ottolenghi, in "Advances in Photochemistry" (J. N. Pitts, G. S. Hammond, K. Gollnik, and D. Grosjean, eds.), p. 97. Wiley (Interscience), New York, 1980. 3 W. Stoeckenius and R. A. Bogomolni, Annu. Rev. Biochem. 52, 587 (1982). 4 j. K. Lanyi, Annu. Rev. Biophys. Biophys. Chem. 15, 11 (1986). 5 V. Balogh-Nair and K. Nakanishi, in " N e w Comprehensive Biochemistry, Volume 3: Stereochemistry" (C. Tamm, ed.), p. 283. Elsevier Biomedical, Amsterdam, 1982. 6 C. Sandorfy and D. Vocelle, Can. J. Chem. 64, 2251 (1989). 7 M. Stockburger, T. Alshuth, D. Oesterhelt, and W. G~rtner, in "Spectroscopy of Biological Systems" (J. H. Clark and R. E. Hester, eds.), p. 483. Wiley, New York, 1986. 8 R. A. Mathies, S. O. Smith, and I. Palings, in "Biological Application of Raman Spectrometry, Volume 2: Resonance Raman Spectra of Polyenes and Aromatics" (T. G. Spiro, ed.), p. 59. Wiley, New York, 1987.
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
124
STRUCTURE AND ANALYSIS
[12]
application of IRD spectroscopy.9 Such problems may also apply to other retinal-containing proteins. The basic principles of the two methods are described here and some important applications discussed.
Resonance Raman Spectroscopy Normal Raman scattering is a weak effect, and recording a complete spectrum usually requires many hours. However, if the molecule has an absorption band near the wavelength of the probing beam (usually a continuous wave laser), the scattered photon is emitted from the electronic excited state and the scattering cross section for vibrations of this molecule is increased by several orders of magnitude. Resonance Raman spectroscopy has, therefore, the unique advantage of selectivity in that, in the spectrum of such complex systems as retinal proteins, only vibrations of the chromophore will be reflected. In the retinal proteins, however, a photoreaction is evoked by the absorption of light. Resonance Raman scattering is, therefore, always connected with the generation of intermediates of the photoreaction, and special techniques have been developed to obtain spectra of these intermediates as well as of the initial state. The basic methods are described briefly. Depending on the system being investigated, there are two basic techniques for overcoming these difficulties: namely, time-resolved technique and pump-probe technique. The first technique uses either a capillary flow system 1°-12or a rotating cell. 13,14The main purpose of both methods is to continuously bring fresh sample into the laser beam. The laser power has to be low enough to accumulate negligible amounts of photoproducts during the short time the sample resides within the cross-section of the laser beam (usually a few microseconds). The pump-probe technique was first applied to rhodopsin by Oseroff and Callender. 15In this application a photoequilibrium between three species of the photoreaction of rhodopsin can be established at 80 K (rhodopsin, bathorhodopsin, and isorhodopsin). By altering the wavelength of the "pump" laser beam, which usually
9 M. S. Braiman and K. J. Rothschild, Annu. Rev. Biophys. Biophys. Chem. 17, 541 (1988). 10 R. H. Callender, A. Doukas, R. K. Crouch, and K. Nakanishi, Biochemistry 15, 1621 (1976). 11 R. A. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, 1 (1976). ~2R. A. Mathies, T. B. Freedman, and L. Stryer, J. Mol. Biol. 109, 367 (1977). ~3 W. Kiefer and H. J. Bernstein, Appl. Spectrosc. 25, 500 (1971). 14 M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979). 15 A. R. Oseroff and R. H. Callender, Biochemistry 13, 4243 (1974).
[12]
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
125
has the higher intensity, the composition of the photoequilibrium is changed. By tuning the wavelength of the "probe" laser beam to a position where one of the three species contributes most to the absorption spectrum, the RR scattering of this compound will selectively be enhanced. The probe beam should have a lower intensity in order not to alter the composition of the photoequilibrium. For systems which undergo a cyclic photoreaction, such as bacteriorhodopsin and halorhodopsin, the pump-probe technique can be applied at room temperature. The photostationary state will be dominated by the initial state and the intermediate with the slowest decay time. The time-resolved and pump-probe techniques can, of course, be combined. The composition of intermediates can be altered by altering the power and wavelength of the pump laser. Again, by adjusting the probe beam, the RR scattering from the species of interest will be enhanced. In general, however, irrespective of the method employed, the RR spectrum will contain contributions from several species. Employing time-resolved techniques, the RR spectrum of the initial state can usually be obtained by illuminating the sample with a very weak probe beam only. This spectrum can then be used to deduce from the composite spectra the spectra of the intermediates of the photoreaction. By moving the focus of the probe beam from the focus of the pump beam, it is possible to collect spectra at times after the excitation of the sample by the pump beam. The time is given by the distance between pump and probe beam divided by the velocity of the flow. This is an additional method for altering the relative amounts of intermediates in the cross section of the probe beam. Intermediates arising several microseconds up to several milliseconds after excitation can be measured in this way. More technical details about the methods can be found in the review papers mentioned above. One can obtain detailed molecular information by comparing the RR spectra of retinal proteins with those of model compounds. The basic investigation by the Mathies group has contributed greatly to our understanding of the vibrational spectra of isomers of retinal 16'~7and of protonated and unprotonated retinylidene Schiff bases.lS.~9 An essential part of this study was the collaboration with the Lugtenburg group, providing the 16 B. Curry, A. Broek, J. Lugtenburg, and R. A. Mathies, J. Am. Chem. Soc. 104, 5274 (1982). 17 B. Curry, I. Palings, A. Broek, J. A. Pardoen, P. P. J. Mulder, J. Lugtenburg, and R. A. Mathies, J. Phys. Chem. 88, 688 (1984). i8 S. O. Smith, A. B. Myers, R. A. Mathies, J. A. Pardoen, C. Winkel, E. M. M. Van den Berg, and J. Lugtenburg, Biophys. J. 47, 653 (1985). 19 B° CLlrry, I. Palings, J. A. Pardoen, J. Lugtenburg, and R. A. Mathies, Adv. Infrared Raman Spectrosc. 12, ll5 (1985).
126
STRUCTURE AND ANALYSIS
[12]
13C- and 2H-labeled retinals. 2°,21 By developing an empirical molecular force field, almost all bands in the RR spectra could be assigned to specific vibrations of the molecules and characteristic bands for the various retinal isomers could be deduced. The effects of forming the Schiff base and its protonation have also been studied. Since retinals, as well as their protonated and unprotonated Schiff bases, are photolabile, the capillary flow technique proved to be essential. In Fig. l, the RR spectra of bacteriorhodopsin under various illumination conditions using the rotating cell technique are shown. 14Spectrum (a) in Fig. 1 was collected with a weak probe beam of 514 nm only, reflecting the light-adapted species of bacteriorhodopsin only, BR568, which has an absorption maximum at 568 nm. By applying a broad pump beam of 514 nm and a coaxial probe beam of 450 nm, an almost pure spectrum of the long-lived intermediate M412 could be obtained [spectrum (b) in Fig. 1], having an absorption maximum at 412 nm. Only the shoulder at 1530 cm -1 indicates a small percentage of the initial state BR568. If the intensity of the probe beam is increased, spectrum (c) in Fig. 1 is obtained. This spectrum contains, in addition to BR568, the M412 intermediate, as can be seen from the band at 1567 cm -1. I f a pump beam of 450 is now applied, driving the M412 intermediate back to BR568, spectrum (d) (Fig. 1) is obtained. These experiments demonstrate the principles of the pump-probe technique in combination with time-resolved techniques. The spectra (Fig. 1) are all dominated by a strong band between 1500 and 1600 cm -1, caused by the ethylenic vibration of the retinal. Its position is correlated with the absorption maximum of the retinylidene Schiff base, owing to the dependency of the ethylenic force constants on ~r-electron delocalization. 22 The band at 1642 cm -l (Fig. la) was shown to shift to 1624 cm -1 in 2H20, demonstrating that it is caused by the C = N stretching vibration of the Schiff base and that "the Schiff base is protonated. The vibrations of this group have been especially investigated, both theoretically and experimentally. 23-28It appears unclear whether protonation increases or de2o j. Lugtenburg, Pure Appl. Chem. 57, 753 (1985). 21 j. Pardoen, C. Winkel, P. Mulder, and J. Lugtenburg, Recl. Tray. Chim. Pays-Bas 103, 135 (1984). 22 M. E. Heyde, D. Gill, R. G. Kilponen, and L. Rimai, J. Am. Chem. Soc. 93, 6776 (1971). 23 H. Deng and R. H. Callender, Biochemistry 26, 7418 (1987). 24 H. S. R. Gilson, B. H. Honig, A. Croteau, G. Zarrilli, and K. Nakanishi, Biophys. J. 53, 261 (1988). 25 T. Baasov, N. Friedman, and M. Sheves, Biochemistry 26, 3210 (1987). 26 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7131 (1986).
[12] RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS I
1
I
I
127
r-"-
~8
C
-
a
g,~g
,,o,~
!
p_.
~ A
o~
,
=
-
f
I
1
I
I
I
800
1000
1200
1400
1600
Av (cm-') FIG. 1. Pump-probe resonance Raman spectra of bactefiorhodopsin at room temperature using a rotating cell system. (a) Spectrum of the initial state BR568 taken with a weak probe beam of 514 nm only; (b) spectrum of the long-lived intermediate M412, taken with a 514-nm pump beam a 457-nm probe beam; (c) spectrum obtained with strong probe beam of 514 nm, showing the presence of BR568 and M412; (d) as (c), but with an additional pump beam of 457 nm, driving the M412 intermediate back to BR568. [Reproduced from M. Stockburger, W. Klusmann, H. Gattermann, G. Massig, and R. Peters, Biochemistry 18, 4886 (1979).]
128
STRUCTURE AND ANALYSIS
[ 12]
creases the C~---N force constant. Also, the contribution of the NH bending vibration to the C ~ N frequency is not completely understood. The understanding of this coupling is especially important, since it would provide a means to investigate the interaction of the protonated Schiff base group with its environment. Figure 2 demonstrates the pump-probe technique at -160 °, where the primary photoreaction of rhodopsin has been investigated) 9 Spectrum (A) in Fig. 2 was obtained with a probe beam of 585 nm. The sample was also illuminated with an all-lines argon laser pump beam. If the spectrum was recorded without the pump beam [spectrum (B), Fig. 2] only isorhodopsin was present. A pure rhodopsin spectrum was obtained by the capillary flow technique [spectrum (C), Fig. 2]. If appropriate amounts of spectra (B) and (C) are subtracted from spectrum (A), a pure spectrum of bathorhodopsin, the first intermediate which can be stabilized at low temperature, could be obtained. The downshift of the ethylenic mode (1545 versus 1536 cm -~) is in accordance with the red-shifted absorption maximum of bathorhodopsin. The strong bands below I000 cm -1 are of special interest. They could be assigned to hydrogen-out-of-plane (HOOP) vibrations of the retinal (i.e., the hydrogens move perpendicular to the polyene plane), and the high intensities were attributed to twists around single bonds of the polyene.3°,31 This means that the chromophore, already isomerized from 11-cis to all-trans, could not yet adopt a relaxed planar configuration owing to steric interaction with the protein, which is rigid at low temperature. A similar effect was observed for the 80 K photoproduct of bacteriorhodopsin, the K intermediate, 32and interpreted in a similar way. A peculiarity in the bathorhodopsin spectrum is the anomalous decoupling of the 1I- and 12-HOOP vibrations, causing the band at 920 cm-~; it was attributed to a specific interaction with a negatively charged residue or dipole of the protein. In recent illustrative applications of RR spectroscopy, demonstrating the selectivity of the method, the visual pigments in the retina of toad, anglefish, gecko, and bullfrog have been investigated) 3 In this case, the 27 j. j. L6pez-Garriga, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7241 (1986). 28 j. j. L6pez-Garriga, S. Hanton, G. T. Babcock, and J. F. Harrison, J. Am. Chem. Soc. 108, 7251 (1986). G. Eyring and R. A. Mathies, Proc. Natl, Acad. Sci. U.S.A. 76, 33 (1979). 3o G. Eyring, B. Curry, A. Broek, J. Lugtenburg, and R. Mathies, Biochemistry 21, 384 (1982). 3~ I. Palings, E. M. M. Van den Berg, J. Lugtenburg, and R. A. Mathies, Biochemistry 28, 1498 (1989). 32 M. Braiman and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 79, 403 (1982). 33 B. A. Barry and R. A. Mathies, Biochemistry 26, 59 (1987).
[12]
RESONANCE RAMAN AND INFRARED STUDIES OF RETINAL PROTEINS
I
