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The binding site of retinaldehyde in cattle rhodopsin
Biochimica et Biophysica Acta, 303 (1973) 189-193
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA Report BBA 31152 T h e binding site o f r e t i n a l d e h y d e in cattle r h o d o p s i n *
W.J. DE GRIP, S.L. BONTINGand F.J.M. DAEMEN Department of Biochemistry, University of Ni]megen, Geert Grooteplein Noord 21, Ni/megen (The Netherlands)
(Received January 8th, 1973)
SUMMARY The binding site of the chromophoric group in native rhodopsin was identified by means of a 2-fold chemical modification of this vertebrate visual pigment. More than 50 of the 52 primary amino groups (including the chromophore carrying group) present per rhodopsin molecule were amidinated with methylacetimidate. Under these conditions 70 % of the rhodopsin remained spectrally intact with a regeneration capacity of 70 %. The remaining amino groups were labeled with dansylchloride under conditions leading to removal of the chromophore. Subsequent hydrolysis and quantitative thin.layer chromatography revealed the presence o f 1.4 e-amino lysine groups, 0.5 ethanolamine residue and less than 0.1 serine residue per rhodopsin. In membranes illuminated prior to amidination only 0.4 e-amino lysine group could afterwards be dansylated. This clearly demonstrates that in rhodopsin the chromophoric group is bound to the e-amino group of a lysine residue of opsin.
The binding site of the chromophoric group, 11-cis retinaldehyde, in the vertebrate rod visual pigment, rhodopsin, has been the subject of much investigation. The nature of the link seems fairly well established now. Morton and Pitt 1 several years ago presented evidence for a protonated aldimine bond ( ' C = ~ - ) , which has recently been HH confirmed by laser Raman scattering 2 . This rather labile link in synthetic retinylidene aldimines is readily hydrolysed by water, converted to an oxime by hydroxylamine or reduced to a stable secondary amine by NaBH4. In rhodopsin, however, it withstands such treatments and appears to be shielded in some way. Bownds and Wald 3 and Akhtar et al. 4 *Biochemicalaspects of the visual process, XXI.
190
BBA REPORT
showed that this shielding effect is abolished by illumination, since simultaneous treatment with NaBH4 results in stabilisation of the retinaldehyde binding. Subsequent hydrolysis of the reduced preparation yields e-retinylaminolysine, showing that in the photoproduct metarhodopsin II the chromophore is bound via an aldimine link to an e-amino group of a lysine residue of the protein. However, this does not necessarily prove that this is also the binding site in native rhodopsin, since model studies by Daemen e t al. s show that at physiological temperature and pH the retinaldehyde moiety rapidly migrates from one amino group to another and that this process can also take place upon illumination of rhodopsin preparations or under denaturing conditions. The following provides an example of this. The conclusion of Poncelot e t aL 6 that phosphatidylethanolamine is the binding site of the chromophore in native cattle rhodopsin, which had already been questioned by Anderson e t al. 7 and Zorn 8 , was proved to be invalid by the enzymatic delipidation studies of Borggreven e t al. 9 ,~o and to be due to artifactual transiminization by Daemen e t aL s Daemen e t aL s obtained indications for the involvement of an e-amino lysine group in the binding of the chromophore in native rhodopsin. Further support for this binding site has recently been reported by Fager e t aL 11, who succeeded in reduction in the dark of rhodopsin in Emulphogene at pH 4 - 5 with the more lipophilic sodium cyanoborohydride (NaCNBH3). Subsequent hydrolysis gave e-retinylaminolysine, in a yield of up to 20 % of the original amount of rhodopsin. In the present paper we report that which we consider to be conclusive evidence for this binding site of the chromophore in rhodopsin. In the course of studies concerning the influence of chemical modification on various photoreceptor membrane properties, we succeeded in obtaining almost complete (97 + 1%) amidination of primary amino groups with methylacetimidate x2 . Nil
Membr,ane--N÷H3~-HCO--C// \¢~
~
H ,.NH 2,~ / . / Membr, a n e - - N --~-~C
+ CH3OH
\CH3
As much as 70 + 5 % of the rhodopsin remains intact under these conditions, while only 1.9 + 0.4 (n = 3) primary amino groups per rhodopsin molecule including the chromophore binding one is left. Displacement of the chromophore, labeling and identification of the remaining amino groups, therefore, permits definite identification of the chromophore binding site in rhodopsin. For all experiments enriched cattle rod outer segment membrane preparations 13 were used. The extent of amidination was calculated by determination of the primary amino groups left after amidination with trinitrobenzene sulfonic acid 12'14. Optimal (97 %) amidination was obtained by five successive treatments of methylacetimidate (20-fold excess) of the membrane suspension in phosphate buffer (pH 7.5, 30 min, 20 °C). The resulting preparation gave 70 + 6 % regeneration after illumination and subsequent dark incubation with excess 11-cis retinaldehyde, while only 3 % of amidinated opsin is capable of regeneration 12. Dansylation of untreated as well as amidinated preparations was performed by treatment with 0.2 % dansylchloride in 1:1 acetone-0.2 M NaHCOa (pH 9,
BBA REPORT
191
Fig. 1. Thin-layer chromatogram (silica gel G) of hydrolysates of dansylated rod outer segment membrane preparations and reference substances. Solvent system: chloroform-methanol-aceticacid (15:4:1, by vol.). Excitation wavelength, 360 rim. The four spots at the left side represent reference substances. O-Dansyltyrosineis contaminated with the blue fluorescingdansylic acid (weak upper spot). "Fheother dansyl derivativesproduce yellow fluorescent spots. Rh, untreated membrane preparation; RhCH, membranes delipidated with phospholipase C and extracted with hexane; RhCHAS, membranes delipidated by treatments with phospholipase C~hexane, phospholipase A and serum albumin; Rh97%Am, extensivelyamidinated membranes.
30 min, 37 °C), in which solvent the chromophore is removed. The resulting dansylated preparations were washed free from excess dansylic acid, lyophilized, hydrolysed (6.7 M HC1, 18 h, 110 °C) and subjected to thin-layer chromatography (silica gel G) (Fig. 1). Since the primary amino groups in the rod outer segment membrane are provided only by e-aminolysine residues of the proteins* and ethanolamine and serine residues from phospho lipids ~2, untreated membranes yield the dansyl derivatives of these three amino compounds in addition to the blue fluorescing dansylic acid, a hydrolysis artifact, and O-dansyltyrosine. After extensive amidination the dansylserine spot has nearly completely disappeared, indicating nearly complete amidination of the serine amino group. Likewise, only a small amount of dansylethanolamine is left and the quantity of e-dansyllysine has greatly been reduced. After extraction of the spots with methanol-conc. NHa (95: 5, by vol.), the amounts of dansyl derivatives were determined by quantitative fluorimetric analysis. For untreated preparations the results thus obtained show, after correction for the losses during hydrolysis and extraction, good agreement with the values calculated from amino acid analysis (Table I, Columns 2 and 3). Similar analysis of extensively amidinated preparations show that only the e-aminolysine group is still present in sufficient amount to account for the binding of the chromophore (Table I, Column 4). This is further underlined by the fact *In contrast to Reporter and Reedis , we do not detect any methylated basic amino groups (lysine, arginine, histidine) in our membrane preparations (de Grip et al., to be published).
16.3 + 0.6 28.0 +_ 0.5 8.5 + 0.5
15.8 + 0.9 29 + 1 8 + 1
1.4 + 0.3 0.5 + 0.2 < 0.1
0.4 + 0.2 0.5 + 0.3 0.6 + 0.2
0.2 0.3 0.1
(n=l)
*For comparison with the dark amidination experiments, the n u m b e r of a m i n o c o m p o u n d s left after amidination in the light is calculated relati~'e to the a m o u n t of rhodopsin remaining in m e m b r a n e s amidinated in the dark, i.e. 70 % o f the a m o u n t present in the untreated membranes.
e-Aminolysine (Lipid)ethanolamine (Lipid)serine
(n=2)
Illuminated* + NADPH
(n=3)
(n=7)
Illuminated*
Dark
Dansylation procedure
Amino acid analysis (n=3)
After amidination, dansylation procedure
Un trea ted
Primary a m i n o groups, including the c h r o m o p h o r e binding group, per mole of rhodopsin present before a n d after five amidination cycles of rod outer segment m e m b r a n e preparations in darkness (Column 4), after previous illumination ( C o l u m n 5) or after previous illumination and reduction o f the retinaldehyde by addition o f NADPH (last column). T h e amine c o m p o u n d s were quantitatively identified v/a labeling with dansylchloride 12. n = n u m b e r o f experiments.
TABLE I
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193
that in similar preparations illuminated before amidination, the number of e-amino lysine groups available for dansylation is clearly reduced (Table I, Column 5), the difference being roughly equivalent to the amount of rhodopsJ,n present in the membranes which are amidinated in the dark. The retinaldehyde "14berated" upon illumination partly remains covalently attached to the membrane via randomly formed aldimine links, which explains why nearly complete amidination may be accomplished only after previous reduction of the liberated retinaldehyde to retinol (Table I, last column). Thus, we have shown that the free amino groups of non-illuminated rod outer segment membranes can be almost completely blocked without major spectral damage to rhodopsin. Upon subsequent denaturation of the modified protein involving release of the chromophore, one further e-amino group of lysine becomes free. Blocking of free amino groups in illuminated rod outer segment membranes under the same conditions causes modification of one more amino group and in this case no additional amino group appears upon denaturation of the protein. These data yield, in our opinion, incontrovertible proof that the chromophoric group in native rhodopsin is bound to an e-amino group of a lysine residue. We should like to acknowledge the skilful technical assistance of Mrs A. Valenteyn Temmink and Mr G. van de Laar. Amino acid analyses were performed by Mr M.G.J. Buys. Financial support was received from the Netherlands Foundation for Chemical Research (S.O.N.). REFERENCES 1 Morton, R.A. and Pitt, G.A.J..(1957) in Fortschr. Chem. Org. Naturstoffe, Vol. 14, pp. 244-316, Springer-Verlag, Wien 2 Rimai, L., Kilponen, R.G. and Gill, D. (1970) Biochem. Biophys. Res. Commun. 41,492-497 3 Bownds, D. and Wald, G. (1965) Nature 205,254-257 4 Akhtar, M., Blosse, P.T. and Dewhurst, P.B. (1964) Life Sci. 4, 1221-1226 5 Daemen, F.J.M., Jansen, P.A.A. and Bonting, S.L. (1971)Arch. Biochem. Biophys. 145,300-309 6 Poincelot, R.P., MiUar, P.G., Kimbel, Jr, R.L. and Abrahamson, E.W. (1970)Biochemistry 9, 1809-1825 7 Anderson, R.E., Hoffman, R.T. and Hall, M.O. (1971) Nature 229, 249-250 8 Zorn, M. (1971)Biochim. Biophys. Acta 245,216-220 9 Borggreven,J.M.P.M., Rotmans, J.P., Bonting, S.L. and Daemen, F.J.M. (1971)Arch. Biochem. Biophys. 145,290-299 10 Borggreven,J.M.P.M., Daemen, F.J.M. and Bonting, S.L. (1972)Arch. Biochem. Biophys. 151, 1-7 11 Fager, R.A., Segnowski, Ph. and Abrahamson, E.W. (1972)Biochem. Biophys. Res. Commun. 47, 1244-1247 12 de Grip, W.J., Bonting, S.L. and Daemen, F.J.M. Biochim. Biophys. Acta, submitted* 13 de Grip, W.J., Daemen, F.J.M. and Bonting, S.L. (1972) Vision Res. 12, 1697-1707 14 Habeeb, A.F.S.A. (1966)Anal. Biochem. 14, 328-336 15 Reporter, M. and Reed, D.W. (1972)Nat. New Biol. 239, 201-203
*Presented in preliminary form at the Association for Research in Vision and Ophthalmology Meeting, 24-28 April 1972, Sarasota, Fla., U.S.A. and at the Symposium on Biochemistry and Physiology of
Visual Pigments, 27-30 August 1972, Bochum, Germany.
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA Report BBA 31152 T h e binding site o f r e t i n a l d e h y d e in cattle r h o d o p s i n *
W.J. DE GRIP, S.L. BONTINGand F.J.M. DAEMEN Department of Biochemistry, University of Ni]megen, Geert Grooteplein Noord 21, Ni/megen (The Netherlands)
(Received January 8th, 1973)
SUMMARY The binding site of the chromophoric group in native rhodopsin was identified by means of a 2-fold chemical modification of this vertebrate visual pigment. More than 50 of the 52 primary amino groups (including the chromophore carrying group) present per rhodopsin molecule were amidinated with methylacetimidate. Under these conditions 70 % of the rhodopsin remained spectrally intact with a regeneration capacity of 70 %. The remaining amino groups were labeled with dansylchloride under conditions leading to removal of the chromophore. Subsequent hydrolysis and quantitative thin.layer chromatography revealed the presence o f 1.4 e-amino lysine groups, 0.5 ethanolamine residue and less than 0.1 serine residue per rhodopsin. In membranes illuminated prior to amidination only 0.4 e-amino lysine group could afterwards be dansylated. This clearly demonstrates that in rhodopsin the chromophoric group is bound to the e-amino group of a lysine residue of opsin.
The binding site of the chromophoric group, 11-cis retinaldehyde, in the vertebrate rod visual pigment, rhodopsin, has been the subject of much investigation. The nature of the link seems fairly well established now. Morton and Pitt 1 several years ago presented evidence for a protonated aldimine bond ( ' C = ~ - ) , which has recently been HH confirmed by laser Raman scattering 2 . This rather labile link in synthetic retinylidene aldimines is readily hydrolysed by water, converted to an oxime by hydroxylamine or reduced to a stable secondary amine by NaBH4. In rhodopsin, however, it withstands such treatments and appears to be shielded in some way. Bownds and Wald 3 and Akhtar et al. 4 *Biochemicalaspects of the visual process, XXI.
190
BBA REPORT
showed that this shielding effect is abolished by illumination, since simultaneous treatment with NaBH4 results in stabilisation of the retinaldehyde binding. Subsequent hydrolysis of the reduced preparation yields e-retinylaminolysine, showing that in the photoproduct metarhodopsin II the chromophore is bound via an aldimine link to an e-amino group of a lysine residue of the protein. However, this does not necessarily prove that this is also the binding site in native rhodopsin, since model studies by Daemen e t al. s show that at physiological temperature and pH the retinaldehyde moiety rapidly migrates from one amino group to another and that this process can also take place upon illumination of rhodopsin preparations or under denaturing conditions. The following provides an example of this. The conclusion of Poncelot e t aL 6 that phosphatidylethanolamine is the binding site of the chromophore in native cattle rhodopsin, which had already been questioned by Anderson e t al. 7 and Zorn 8 , was proved to be invalid by the enzymatic delipidation studies of Borggreven e t al. 9 ,~o and to be due to artifactual transiminization by Daemen e t aL s Daemen e t aL s obtained indications for the involvement of an e-amino lysine group in the binding of the chromophore in native rhodopsin. Further support for this binding site has recently been reported by Fager e t aL 11, who succeeded in reduction in the dark of rhodopsin in Emulphogene at pH 4 - 5 with the more lipophilic sodium cyanoborohydride (NaCNBH3). Subsequent hydrolysis gave e-retinylaminolysine, in a yield of up to 20 % of the original amount of rhodopsin. In the present paper we report that which we consider to be conclusive evidence for this binding site of the chromophore in rhodopsin. In the course of studies concerning the influence of chemical modification on various photoreceptor membrane properties, we succeeded in obtaining almost complete (97 + 1%) amidination of primary amino groups with methylacetimidate x2 . Nil
Membr,ane--N÷H3~-HCO--C// \¢~
~
H ,.NH 2,~ / . / Membr, a n e - - N --~-~C
+ CH3OH
\CH3
As much as 70 + 5 % of the rhodopsin remains intact under these conditions, while only 1.9 + 0.4 (n = 3) primary amino groups per rhodopsin molecule including the chromophore binding one is left. Displacement of the chromophore, labeling and identification of the remaining amino groups, therefore, permits definite identification of the chromophore binding site in rhodopsin. For all experiments enriched cattle rod outer segment membrane preparations 13 were used. The extent of amidination was calculated by determination of the primary amino groups left after amidination with trinitrobenzene sulfonic acid 12'14. Optimal (97 %) amidination was obtained by five successive treatments of methylacetimidate (20-fold excess) of the membrane suspension in phosphate buffer (pH 7.5, 30 min, 20 °C). The resulting preparation gave 70 + 6 % regeneration after illumination and subsequent dark incubation with excess 11-cis retinaldehyde, while only 3 % of amidinated opsin is capable of regeneration 12. Dansylation of untreated as well as amidinated preparations was performed by treatment with 0.2 % dansylchloride in 1:1 acetone-0.2 M NaHCOa (pH 9,
BBA REPORT
191
Fig. 1. Thin-layer chromatogram (silica gel G) of hydrolysates of dansylated rod outer segment membrane preparations and reference substances. Solvent system: chloroform-methanol-aceticacid (15:4:1, by vol.). Excitation wavelength, 360 rim. The four spots at the left side represent reference substances. O-Dansyltyrosineis contaminated with the blue fluorescingdansylic acid (weak upper spot). "Fheother dansyl derivativesproduce yellow fluorescent spots. Rh, untreated membrane preparation; RhCH, membranes delipidated with phospholipase C and extracted with hexane; RhCHAS, membranes delipidated by treatments with phospholipase C~hexane, phospholipase A and serum albumin; Rh97%Am, extensivelyamidinated membranes.
30 min, 37 °C), in which solvent the chromophore is removed. The resulting dansylated preparations were washed free from excess dansylic acid, lyophilized, hydrolysed (6.7 M HC1, 18 h, 110 °C) and subjected to thin-layer chromatography (silica gel G) (Fig. 1). Since the primary amino groups in the rod outer segment membrane are provided only by e-aminolysine residues of the proteins* and ethanolamine and serine residues from phospho lipids ~2, untreated membranes yield the dansyl derivatives of these three amino compounds in addition to the blue fluorescing dansylic acid, a hydrolysis artifact, and O-dansyltyrosine. After extensive amidination the dansylserine spot has nearly completely disappeared, indicating nearly complete amidination of the serine amino group. Likewise, only a small amount of dansylethanolamine is left and the quantity of e-dansyllysine has greatly been reduced. After extraction of the spots with methanol-conc. NHa (95: 5, by vol.), the amounts of dansyl derivatives were determined by quantitative fluorimetric analysis. For untreated preparations the results thus obtained show, after correction for the losses during hydrolysis and extraction, good agreement with the values calculated from amino acid analysis (Table I, Columns 2 and 3). Similar analysis of extensively amidinated preparations show that only the e-aminolysine group is still present in sufficient amount to account for the binding of the chromophore (Table I, Column 4). This is further underlined by the fact *In contrast to Reporter and Reedis , we do not detect any methylated basic amino groups (lysine, arginine, histidine) in our membrane preparations (de Grip et al., to be published).
16.3 + 0.6 28.0 +_ 0.5 8.5 + 0.5
15.8 + 0.9 29 + 1 8 + 1
1.4 + 0.3 0.5 + 0.2 < 0.1
0.4 + 0.2 0.5 + 0.3 0.6 + 0.2
0.2 0.3 0.1
(n=l)
*For comparison with the dark amidination experiments, the n u m b e r of a m i n o c o m p o u n d s left after amidination in the light is calculated relati~'e to the a m o u n t of rhodopsin remaining in m e m b r a n e s amidinated in the dark, i.e. 70 % o f the a m o u n t present in the untreated membranes.
e-Aminolysine (Lipid)ethanolamine (Lipid)serine
(n=2)
Illuminated* + NADPH
(n=3)
(n=7)
Illuminated*
Dark
Dansylation procedure
Amino acid analysis (n=3)
After amidination, dansylation procedure
Un trea ted
Primary a m i n o groups, including the c h r o m o p h o r e binding group, per mole of rhodopsin present before a n d after five amidination cycles of rod outer segment m e m b r a n e preparations in darkness (Column 4), after previous illumination ( C o l u m n 5) or after previous illumination and reduction o f the retinaldehyde by addition o f NADPH (last column). T h e amine c o m p o u n d s were quantitatively identified v/a labeling with dansylchloride 12. n = n u m b e r o f experiments.
TABLE I
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to
BBA REPORT
193
that in similar preparations illuminated before amidination, the number of e-amino lysine groups available for dansylation is clearly reduced (Table I, Column 5), the difference being roughly equivalent to the amount of rhodopsJ,n present in the membranes which are amidinated in the dark. The retinaldehyde "14berated" upon illumination partly remains covalently attached to the membrane via randomly formed aldimine links, which explains why nearly complete amidination may be accomplished only after previous reduction of the liberated retinaldehyde to retinol (Table I, last column). Thus, we have shown that the free amino groups of non-illuminated rod outer segment membranes can be almost completely blocked without major spectral damage to rhodopsin. Upon subsequent denaturation of the modified protein involving release of the chromophore, one further e-amino group of lysine becomes free. Blocking of free amino groups in illuminated rod outer segment membranes under the same conditions causes modification of one more amino group and in this case no additional amino group appears upon denaturation of the protein. These data yield, in our opinion, incontrovertible proof that the chromophoric group in native rhodopsin is bound to an e-amino group of a lysine residue. We should like to acknowledge the skilful technical assistance of Mrs A. Valenteyn Temmink and Mr G. van de Laar. Amino acid analyses were performed by Mr M.G.J. Buys. Financial support was received from the Netherlands Foundation for Chemical Research (S.O.N.). REFERENCES 1 Morton, R.A. and Pitt, G.A.J..(1957) in Fortschr. Chem. Org. Naturstoffe, Vol. 14, pp. 244-316, Springer-Verlag, Wien 2 Rimai, L., Kilponen, R.G. and Gill, D. (1970) Biochem. Biophys. Res. Commun. 41,492-497 3 Bownds, D. and Wald, G. (1965) Nature 205,254-257 4 Akhtar, M., Blosse, P.T. and Dewhurst, P.B. (1964) Life Sci. 4, 1221-1226 5 Daemen, F.J.M., Jansen, P.A.A. and Bonting, S.L. (1971)Arch. Biochem. Biophys. 145,300-309 6 Poincelot, R.P., MiUar, P.G., Kimbel, Jr, R.L. and Abrahamson, E.W. (1970)Biochemistry 9, 1809-1825 7 Anderson, R.E., Hoffman, R.T. and Hall, M.O. (1971) Nature 229, 249-250 8 Zorn, M. (1971)Biochim. Biophys. Acta 245,216-220 9 Borggreven,J.M.P.M., Rotmans, J.P., Bonting, S.L. and Daemen, F.J.M. (1971)Arch. Biochem. Biophys. 145,290-299 10 Borggreven,J.M.P.M., Daemen, F.J.M. and Bonting, S.L. (1972)Arch. Biochem. Biophys. 151, 1-7 11 Fager, R.A., Segnowski, Ph. and Abrahamson, E.W. (1972)Biochem. Biophys. Res. Commun. 47, 1244-1247 12 de Grip, W.J., Bonting, S.L. and Daemen, F.J.M. Biochim. Biophys. Acta, submitted* 13 de Grip, W.J., Daemen, F.J.M. and Bonting, S.L. (1972) Vision Res. 12, 1697-1707 14 Habeeb, A.F.S.A. (1966)Anal. Biochem. 14, 328-336 15 Reporter, M. and Reed, D.W. (1972)Nat. New Biol. 239, 201-203
*Presented in preliminary form at the Association for Research in Vision and Ophthalmology Meeting, 24-28 April 1972, Sarasota, Fla., U.S.A. and at the Symposium on Biochemistry and Physiology of
Visual Pigments, 27-30 August 1972, Bochum, Germany.