0031-8655/92 $05.00+0.00 Pergamon Press Ltd

Photochemistry and Photobiology Vol. 56, No. 1, pp. 101-105, 1992 Printed in Great Britain

RESEARCH NOTE 9 , l ~-DIc~s-~~-FLUORORHODOPSIN. CHROMOPHORE PROPERTIES FROM "F-NMR STUDIES LETICIA U. COLMENARES and ROBERT S. H. LIU* Department of Chemistry, University of Hawaii, 2545 The Mall, Honolulu, HI 96822, USA (Received 9 Seprember 1991; accepted 3 December 1991)

Abstract-From a "F-NMR study of 9,11-dicis-12-fluororhodopsinand its photobleached product, we concluded that the initially formed chromophore retained its configuration and the photoproduct corresponded to the two-bond isomerized all-rrans. Upon standing, it slowly isomerized to the 9-cis isomer. The method represents a direct, non-destructive procedure for determining configuration purity of the pigment formed. Its unique fluorine opsin shift value is consistent with the expected different orientation of the fluoro-substituent in a dicis pigment.

INTRODUCTION

Recently, through the use of a series of fluorinated 9-cis and 1 1 4 s isomers of retinal, we successfully demonstrated the possible use of fluorine labels as reporters for structural information related to protein substrate interactions in visual pigments (Colmenares et al., 1991). The method allows one to probe for information in regions immediately beyond that defined by the molecular framework of the chromophore; therefore, it complements the well documented C-NMR (nuclear magnetic resonance)? method (Mollevanger et al., 1987; Smith el al., 1990). The data on fluorine opsin shift (FOS), defined as the difference of chemical shifts between the pigment and the corresponding protonated Schiff base (PSB) (Colmenares et al., 1991), were found to be particularly informative. We now wish to demonstrate that such F-NMR studies can provide additional structural information, especially configurational changes of the polyene chromophore. The system of choice is 9,11-dicis-12-fluororhodopsin. It should be noted that the same pigment in the parent system has been reported; however, its formation was complicated by extensive (> 60%) configuration isomerization of the dicis chromophore during pigment formation (Trehan et a1.,1990).

11-dicis isomer, however, was not isolated previously: IHNMR (CDCI,, 300 MHz) d 1.03 (s); 1.74 (s), 2.00 (s) 2.37 (s), 6.14 (d), 6.29 (d),6.32 (d), 6.54 (d), 6.65 (d,d), 10.15 ( 4 ppm, J7." = 16.0, J,,,.,, = 12.3 Hz, J11.12 = 22.9, Ji4. I5 = 7.1 Hz; I9F-NMR (CDCl,, 283 MHz): -109.2 ppm; (hexane) = 367 nm. Procedures for prepUVNIS: A, aration of samples of the pigment analog for UVNIS and F-NMR studies were the same as those reported by Colmenares et al. (1991). Mefhodr. A Nicolet NT-300 nmr spectrometer, a Jasco 5-600 CD spectrometer and a Perkin-Elmer A-5 recording UVNIS spectrometer were used for this study. Procedure for chromophore extraction was the same as those reported for other fluorinated rhodopsins (Colmenares et al.. 1991). Photobleaching was carried out with a projector lamp (Tungsten) equipped with an Ealing 35-5073 (550 nm) interference filter. RESULTS

The block-averaged IyF-NMR spectra of 9,11dicis-12-fluororhodopsin formed in the absence of any excess retinal and after photobleaching are shown in Fig. l(a). The characteristically broad [- 1 ppm, Colmenares et al. (1991)l pigment signals at -110.8 and -114.7 ppm disappeared upon irradiation with 550 nm light, converting to a new peak at -119.2 ppm, corresponding to that of alltrans-12-fluororetinal. The latter was confirmed by HPLC and UV analyses of the retinal extract derived from the freeze-dried photo-bleached sample. The pigment was also thermally unstable as MATERIALS AND METHODS revealed by the spectra transformed from the proMaterial. 9,11-Dicis-12-fluororetinalwas isolated by preparative high performance liquid chromatography gressive blocks of data acquired during the exper(HPLC) from a synthetic mixture of isomers of 12-fluoro- iment [Fig. l(b)], showing that during the NMR retinal prepared as reported by Liu et al. (1981). The 9, experiment the initial -110.8 signal decreased in intensity with a simultaneous increase of the signal at -114.7 ppm, the latter corresponding to that *Towhom correspondence should be addressed. of 9-cis-12-fluororhodopsin. The unique difference ?Abbreviations: CD, circular dichroism; FOS, fluorine opsin shift; HPLC, high pressure liquid chromatogra- circular dichroism (CD) spectrum (pigment minus phy; NMR, nuclear magnetic resonance; PSB, pro- bleached) of the initial pigment(s) formed from 9, tonated butyl Schiff base. 11-dicis-12-fluororetinal is shown along with those 101

and ROBERT s. H.LlU LETlClA U. COLMENARES

102

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Figure 1. (a) The block-averaged F-NMR spectrum of pigments from 9,11-dicis-12-fluororetinal and bovine opsin in CHAPS (lower, 5 blocks of 50 000 scans each; total experiment time, 11.53 h), and that after irradiation with orange light (> 540 nm) (lower). The following spectrometer parameters were used: P2 = 40 )IS; D5 = 50 ms; SI = 4096; LB = 80 Hz. (b) spectra corresponding to the five data blocks plotted out separately, the lowest being the first block.

of 9-cis- and 11-cis-12-fluororhodopsin(Fig. 2). As a further corroborative evidence : 0for isomeriz2 . ation to the 9-cis pigment, samples of 9,11-dicis-12fluororhodopsin, its thermally equilibrated pigment (presumably 9-cis-12-fluororhodopsin)and an authentic sample of 9-cis-12-fluororhodopsin were photobleached in parallel. The decrease of pigment absorption, an indication of relative sensitivities toward 550 nm light, are plotted in Fig. 3. It is clear that the thermally equilibrated sample photobleached at a rate intermediate of 9,ll-dicis (slowest) and 9 4 s (relative ratio 1.3:1.0:1.8, from the three curves in Fig. 3). The UVNIS spectra taken during the course of photobleaching (550 nm) of the dicis pigment are shown in Fig. 4*. .

. .

~ . .~. . .~. .~

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Figure 2. Difference CD spectra of "9,11-dicis-12-fluororhodopsin", 9-cis-12-fluororhodopsinand 1 l-cis-12-

*

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Figure 3. Photobleaching of (a) the thermally rearranged pigment, (b) "9,l l-dicis-12-fluororhodopsin",(c) 9-cis-12fluororhodopsin upon simultaneous irradiation with 550 nrn light as monitored by changes of absorption at 510 nm.

The F-chemical shifts of 9,11-dicis-12-fluororhodopsin and the corresponding PSB are shown in Table 1. In it are also listed the corresponding data for the related mono-cis pigments that were reported earlier (Colmenares et al., 1991) and the calculated FOS values of these pigments.

fluororhodopsin.

DISCUSSION

*The presence of an isosbestic point in Fig. 4 should not be taken as sufficient evidence for one-photon-twobond isomerization, especially in view of the fact that the mono-cis pigments have higher relative quantum efficiencies in photobleaching than does the dicis pig ment.

The new isomeric pigment of 12-fluororhodopsin with its unique absorption maximum at 484 nm (see Table l), CD spectrum and F-NMRsignal has provided meaningful information in at least two respects: chromophore orientation and configuration purity.

Research Note

103

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451

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651

Figure 4. UVlVIS spectra of 9,11-dicis-12-fluororhodopsinbefore (curve 1) and after 15, 30, 45, 60, 75, 90, 105, 120, 150 and 210 s (curve 1 1 for the last entry) successive irradiation with 550 nm light. Table 1 . IYF-NMRchemical shifts of PSB, rhodopsin pigments and corresponding FOS

9,ll-dicis-l2-F 11-cis-12-Fll 9-cis-12-F/I 1 1-cis-10-Fll 9 4 s -10-F/(

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-108.7$ ,-107.89: - 120.9$ - 112.25 -119.75

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“Solubilized in CHAPS. +Column 2 minus column 1 . $In CDZCI2. #In CDCI1. IlFrom Colmenares er al. (1991).

arization by a neighboring carboxylate serving the dual function of a counter ion and a second point charge. The value for 9,11-dicis-12-fluororhodopsin is even more abnormal (Table 1) in that it shows a negative FOS, i.e. more shielded than the PSB. At this time the exact nature for the cause of the upfield shift is unclear*, nevertheless simple model construction (Liu and Mirzadegan, 1988) leads to the conclusion that in a dicis chromophore, the 12fluoro-substituent should orient itself in a direction opposite to that in a mono-cis chromophore (such as 9-cis and 11-cis), as shown in Fig. 5. The very different FOS values must then reflect the different environment of the fluoro-substituent for the isomeric chromophores.

Chemical shift It was shown earlier that most of the fluororhodopsins demonstrate the same hydrophobic deshielding of the fluorine signal as other protein bound substrates (Gerig, 1988). The average values were found to be 4-8 ppm in the visual pigment analogs (Colmenares et al. 1991). Thus, the values for the 9-cis-12-fluororhodopsin were considered “normal” while those of the 11-cis-12-fluororhodopsin deshielded were believed to be due to depol~~

~

*In collaboration with Dr T. Mirzadegan, we are completing a molecular modelingkomputer graphic study (a 3 D rhodopsin model has recently been reported, (Mirzadegan and Liu, 1991) in order to examine possible roles of individual amino-acid residues contributing to the unusual FOS effects. PAP Ytl-H

Retention of configuration The method of choice for determining the extent of retention of chromophore configuration during pigment formation is denaturation-chromophore extraction-HPLC separation (Pilkiewicz et al., 1977; Maeda etal. , 1978). One drawback of this method is that it is a destructive method requiring a substantial amount of material. Also, occasionally it is complicated by chromophore isomerization during the extraction procedure (which could be minimized by immediate conversion of the chromophore to oximes) (Groenendijk etal., 1980). Thus, in a recent study of pigment isomers formed from dicis retinals, Trehan er al. (1990) concluded that the pigment derived from 9,11-dicis-retinal consisted of a mix-

104

U . COLMENARES and ROBERT S. H. LIU LETICIA

Figure 5 . Models of 9-cis, 1 1 4 s and 9.11-dicis isomers of the tethered retinyl chromophore of 12fluororhodopsin constructed in a manner as described by Liu and Mirzadegan (1988), demonstrating the opposite orientation of the 12-fluoro-substituent in the dicis isomer vs those in the mono-cis. A predicted IS-anti geometry (Liu and Mirzadegan, 1988) as shown in 7,9-dicis-rhodopsin (Loppnow et al., 1990) has been incorporated into the 9,ll-dicis structure.

ture of 9-cis-rhodopsin (45%) and 9,l l-dicis-rho- on the chromophore and is also useful as a direct, dopsin (40%). The former was believed to have non-destructive method for determining chromoresulted from prior isomerization of the dicis retinal phore configurational purity. to the 9-cis which combines with opsin at a rate Acknowledgement-The work was supported by a grant faster than that of the dicis. We have demonstrated that "F-NMR can be used from the US Department of Health (DK-17806) and an HFSPO award. LUC's graduate study was supported by as a direct method to examine isomeric purity of a fellowship (1987-1991) from the East-West Center. the pigment chromophore. The detection of a major signal with a F-chemical shift value (- 110.8 ppm) REFERENCES uniquely different from that of 9-cis and 114s pigments (-114.3 and -94.2 ppm respectively) clearly Colmenares, L. U., A. E. Asato, M. Denny, D. Mead, J. P. Zingoni and R. S. H. Liu (1991) NMR studies of suggests minimal isomerization during the initial fluorinated visual pigment analogs. Biophys. Biochem. stage of pigment formation. However, upon standRes. Commun. 179, 1337-1343. ing at room temperature, loss of the hindered 11- Ganapathy, S. and R. S. H . Liu (1990) Photoisomerization of hindered isomers of retinal. Regioselectivity and onecis geometry, similar to but apparently at a slower photon-multiple-bond isomerization. Tetrahedron Lett. rate than the parent system (Trehan et af., 1990), 31, 6957-6960. led to gradual formation of the 9 4 s pigment Gerig, J. T. (1988) Fluorine nuclear magnetic resonance [Fig. l(b)]. It is tempting to conclude that isomerizof fluorinated ligands. Methods Enzyrnof. 177, 3-22. ation originated from the pigment. However, at this Groenendijk, C. W. T., W. J. deGrip and M. Daemen. (1980) Quantitative determination of retinals with comtime it is not possible to rule out the alternative plete retention of their geometric configuration. possibility of isomerization taking place from the Biochim. Biophys. Acta 617, 430-438. free retinal present in a minor equilibrium concen- Loppnow, G. R., M. E. Miley, R. A. Mathies, R. S. tration. H. Liu, H. Kandori, Y. Shichida, Y. Fukada and T. An additional point of interest is that the final Yoshizawa (1990) Structure of the retinal chromophore in 7,9-dicis-rhodopsin. Biochemistry 29, 898543991, all-trans photobleached product corresponds to a two-bond isomerized product. In this case, the near Liu, R . S. H., H. Matsumoto, A. E. Asato, M. Denny, Y. Shichida, T. Yoshizawa and F. Dahlquist (1981) complete retention of configuration of the dicis Synthesis and properties of 12-fluororetinal and 12chromophore in pigment formation, in contrast to fluororhodopsin. A model system for 19FNMR studies of visual pigments. J . Am. Chem. SOC. 103, 7195-7201. the parent 9,ll-dicis-rhodopsin (Trehan et al., 1990), provides an opportunity to investigate in the Liu, R. S. H. and T. Mirzadegan (1988) The shape of a three dimensional binding site of rhodopsin based on near future possible involvement of one-photonmolecular modeling analyses of isomeric and other pig two-bond isomerization in the photobleaching proment analogues. J . Am. Chem. SOC. 110, 8617-8623. cess. The only reported case of multiple-bond iso- Maeda, A., T. Ogurusu, Y. Shichida, F. Tokunaga and T. Yoshizawa (1978) Formation of a 7-cis retinal pigmerization of a visual pigment analog has been ment by irradiating cattle rhodopsin at low temperanegated in a recent reinvestigation of 9,13-dicistures. FEES Lett. 92, 77-80. rhodopsin (Shichida et al., 1988) although such iso- Mirzadegan, T. and R. S. H. Liu (1991) Probing the merization in retinal isomers has been firmly docuvisual pigment rhodopsin and its analogs by molecular modeling analysis and computer graphics. Prog. Retimented (Ganapathy and Liu, 1990). noid Res. 11, 57-74. In conclusion, the study with the 9,ll-dicis p i g L. C. P. J., A. P. M. Kentgens, J . A. ment of 12-fluororetinal has shown that F-NMR is Mollevanger, Pardoen, I. M. L. Courtin, W. S. Veenen, J. Lugtena sensitive method for demonstrating directional burg and W. J. deGrip (1987) High resolution solid property, with respect to the protein cavity, of labels state I3C-nmr studies of carbons C-5 and C-12 of the

Research Note chromophore of bovine rhodopsin: evidence for a 6 4 cis conformation with negative charge perturbation near C12. Eur. J . Biochem. 163, 9-14. Pilkiewicz, P. C., M.J. Pettei, A. P. Yudd and K. Nakanishi (1977) A simple and non-isomerizing procedure for the identification of protein bound retinals. Exp. Eye Res. 24,421-423. Shichida, Y . , K. Nakamura, T. Yoshizawa, A. Trehan, M. Denny and R. S. H. Liu (1988) 9,13-Dicis-Rhodopsin and its one~photon-one-bond isomerization. Biochembtry 21, 649545499.

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Smith, S.,I. Palings, M. Miley, J. Courtin. H.deGroot, J. Lugtenburg, R. Mathies and R. Griffin (1990) Solid state NMR studies of the mechanism of the opsin shift in the visual pigment rhodopsin. Bjochemfitv 29, 8158-8164. K. Trehan, A., R. s. H.Liu, y. Shichida, y. Nakamura and T. Yoshizawa (1990) On retention of chromophore of rhodopsin isomers derived from three dicis retinal isomers. J. Bioorg. Chem. 18, 30-40.

9,11-Dicis-12-fluororhodopsin. Chromophore properties from 19F-NMR studies.

From a 19F-NMR study of 9,11-dicis-12-fluororhodopsin and its photobleached product, we concluded that the initially formed chromophore retained its c...
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