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Methylation of the Active-Site Lysine of Rhodopsin? Colin Longstaff and Robert R. Rando* Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 021 15 Received June 20. 1985
ABSTRACT: Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 1 1-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated a t 4 OC. Methylation under these conditions leads to the incorporation of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of e-dimethyl1ysine:cmonomethyllysine:lysinebeing approximately 5:4: 1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a, , ,A = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site €-lysine monomethylated rhodopsin with a A,,, = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin. Thus, the active site of rhodopsin can accommodate a methyl group attached to the active-site lysine, and the , , A of this pigment supports the hypothesis that the Schiff base of rhodopsin bears a full formal positive charge. Furthermore, since this new pigment can be bleached, it must mean that deprotonation of the Schiff base must not be obligate for this conversion to proceed. With this methyl reporter group attached to the active-site lysine of rhodopsin, spectroscopic and biochemical experiments on the role of charge and charge movement in the mechanism of action of rhodopsin can be performed.
R h o d o p s i n is an integral rod outer segment disk protein containing 11-cis-retinal bound to active-site lysine-296 via a protonated, or partially protonated, Schiff base (Hargrave et al., 1983). Photolysis of rhodopsin results in the isomerization of the 11-cis chromophore to its all-trans congener, with the subsequent hydrolysis of the Schiff base linkage, to form the protein opsin and all-trans-retinal (Wald, 1968). One of the spectroscopically identifiable intermediates on the way to This work was supported by U S . Public Health Service Research Grant EY 03624 from the National Institutes of Health. * Correspondence should be addressed to this author.
0006-2960/85/0424-8137$01.50/0
all-trans-retinal formation, probably metarhodopsin 11, is responsible for initiating the cascade of biochemical events which leads to the hyperpolarization of the rod outer segment, and hence to visual signal transduction (Parkes et al., 1979; Calhoon et al., 1981). It should be noted that metarhodopsin I1 may be comprised of several distinct conformers. The biochemical events affected by the photochemical activation of rhodopsin are now at least partially understood. Activated rhodopsin (metarhodopsin 11) catalyzes the exchange of GTP for GDP in a G protein (GTPase), also called transducin, which can then in turn activate a phosphodiesterase specific for cGMP (Shinozawa et al., 1979; Fung et al., 1981). There
0 1985 American Chemical Society
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is now good evidence to show that cGMP opens the plasma membrane sodium channels and hence its hydrolysis would, of course, cause them to close (Fesenko et al., 1985). It has been noted that the initial biochemical events in vision, notably the rhodopsin-catalyzed exchange of GDP for GTP, are shared by many drug/receptor complexes and, indeed, notable sequence homology exists within the family of G proteins (Hurley et al., 1984). An important question that arises in determining the mechanism of the metarhodopsin I1 mediated activation of the GTPase is that of the nature of the activated rhodopsin complex. Although this question is multifaceted, one aspect of it concerns the structure of the chromophore as it relates to the mechanism of the activation process. Of great interest here is how the photochemical isomerization of the chromophore leads to the energization of rhodopsin. Much of the light energy captured by rhodopsin is transformed into the chemical potential energy of the holoprotein (Cooper, 1979). The role of the potentially charged Schiff base in this energization step and in visual transduction is likely to be considerable. Charge movement is probably the key to the conversion of light energy into chemical potential energy by rhodopsin (Honig et al., 1979a). Furthermore, at the critical metarhodopsin I metarhodopsin I1 conversion, a proton is taken up from the medium at the same time that spectroscopicevidence suggests that the Schiff base is either deprotonated or hydrolyzed (Cooper & Converse, 1976; Doukas et al., 1978). In addition, the protonated Schiff base is thought to be of paramount importance in determining the positions of maximal absorption for the rod and cone pigments (Honig et al., 1979a). It should also be noted that the bacterial counterpart of mammalian rhodopsin, bacteriorhodopsin, may pump protons via its protonated Schiff base (Stoeckenius et al., 1979). For these reasons, it was considered important to place a reporter group at the active-site lysine of rhodopsin, which would allow one to assess the importance of charge and charge movement during bleaching, signal transduction, and regeneration. To these ends, we have prepared active-site lysine-methylated rhodopsin and report here on some of its biochemical properties.
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MATERIALS AND METHODS Materials. Dodecyl maltoside and methyl a-mannopyranoside were obtained from Behring Diagnostics. Pyridine/borane was a product of Aldrich Chemical Co. [3H]Formaldehyde and [ I4C]formaldehyde were obtained from New England Nuclear. N'-Methyllysine and Ne-dimethyllysine were obtained from Vega Chemicals, Tucson, AZ. Ammonyx LO was provided by Onyx Chemical Co. alltran~-[~H]Retinol was a product of Amersham Corp. Thinlayer chromatography (TLC) plastic sheets of silica gel 60 (without fluorescent indicator), 0.2-mm thickness, were products of E. Merck, West Germany. Sep-Pak cartridges were obtained from Waters Associates. Concanavalin ASepharose 4B, o-phthalaldehyde, and 2-mercaptoethanol were obtained from Sigma Chemical Co. Hydroxylapatite, DNA grade, was a product of Bio-Rad Laboratories. Scintillation fluid (Hydrofluor) was obtained from National Diagnostics. All other chemicals and solvents used were of the highest grade commercially available. Preparation of Proteins. Rod outer segments and solubilized rhodopsin were prepared as described previously (Calhoon & Rando, 1985). Unless otherwise stated, rhodopsin modifications were carried out in a buffer of 10 mM 1,4-piperazinediethanesulfonic acid (PIPES), pH 6.5, containing 6 mM dodecyl maltoside. All procedures involving the handling of
nonbleached rhodopsin were conducted under dim red light. Rhodopsin and modified rhodopsins were stored at -70 OC between experiments. Methylation Procedures. Detergent-solubilized rhodopsin was methylated prior to bleaching to produce permethylated rhodopsin in which >95% of all available, Le., non-active-site, lysines were dimethylated. This was accomplished by carrying out two rounds of methylation using 2 mM formaldehyde and 20 mM pyridine/borane. A stock solution of 0.2 M formaldehyde was prepared by hydrolyzing paraformaldehyde, and this was stored at 4 "C and used over a period of several months (Jentoft & Dearborn, 1983). Stock solutions of pyridine/borane (2 M) were made up in 2-propanol and stored at -70 OC in aliquots of 1-2 mL. Rhodopsin concentrations varied between 10 and 40 pM, on the basis of the absorbance at 500 nm using t = 40000 M-I cm-' (Applebury et al., 1974). Each round of methylation of rhodopsin was allowed to proceed for 20-24 h at 20-25 OC, whereupon modified protein was isolated from the reaction mixture by using a desalting column of G-25 Sephadex. Methylation of the active-site lysine was carried out following bleaching of the permethylated rhodopsin under strong white light, in the presence of 10 mM hydroxylamine. Bleached protein was isolated on a small column of concanavalin A-Sepharose 4B (ca. 1-mL gel for each 100 nmol of protein) and subsequently eluted with 0.5 M methyl a-mannopyranoside. All reactions and manipulations of the bleached protein were performed at 4 OC because opsin was found to be much more susceptible to thermal denaturation than rhodopsin. As above, reagent concentrations were 2 mM formaldehyde and 20 mM pyridine/borane. Protein concentrations were routinely 3-15 pM, on the basis of the absorbance at 278 nm, using t = 64000 M-' cm-' (Applebury et al., 1974). For greater levels of active-site methylation, reactions were allowed to proceed for 48 h, and a further addition of both reagents at 24 h was made (final concentrations 4 mM formaldehyde and 40 mM pyridine/borane). On completion of this step, modified protein was isolated by using a desalting column of G-25 Sephadex, and modified rhodopsin was regenerated by adding ca. a 5-fold excess of 11-cis-retinal in 2-propanol [volume of 2-propanol added being
