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Retinal migration during dark reduction of bacteriorhodopsin ( cyanoborohydride / Lys-41 / proton pump / reductive amination / Schiff base )
| Content Provider | Semantic Scholar |
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| Author | Wolber, Paul K. St, Walther S. T. O. E. C. K. E. N. I. U. |
| Abstract | When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, =45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the e-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine Eamino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin. The determination of the primary protein (1, 2) and structural gene (3) sequences of bacteriorhodopsin (bR), the lightdriven proton pump from the purple membrane (pm) of Halobacterium halobium, has led to a reinvestigation of the retinal-binding site by chemical reduction and peptide analysis. This resulted in a reassignment for the binding site from the E-amino group of Lys-41 (4) to the E-amino group of Lys-216 (5-9). Additional chemical modification (10) and resonance Raman (11) studies have demonstrated conclusively that the binding site does not change during the photoreaction cycle as originally postulated by Ovchinnikov's group (12). The reason for the original misassignment of the binding site by Bridgen and Walker is obvious in retrospect (7). However, the same arguments cannot explain the more recent observations of reductive retinal linkage to Lys-40/41 (7, 9, 12). Lemke and Oesterhelt (6) consider these to be in error due to a previously unnoticed proteolytic cleavage of bR by NaBH4. We show here that this explanation does not hold, at least for our results, and that under some conditions of reduction, two-thirds of the retinal are indeed found bound to Lys-40/41. More important, we have previously argued that while binding to Lys-40/41 is a preparation artifact, it might still be used to obtain important information about the tertiary structure of bR. We have, therefore, repeated and extended our earlier experiments. Unfortunately, the new observations considerably weaken the structural argument, but they demonstrate an unusually high reactivity of Lys-40/41 for Schiff base formation and/or reduction and are of considerable interest for the interpretation of experiments trying to identify the two additional retinal pigments recently discovered in H. halobium (13-15). MATERIALS AND METHODS Materials. Bovine serum albumin (fraction V, essentially fatty acid free), 2-(N-cyclohexylamino)ethanesulfonic acid (Ches), chymotrypsin (treated with N-tosyl-L-lysine chloromethyl ketone), all-trans retinol, all-trans retinal, LH-20, and LH-60 were purchased from Sigma and used without further purification. Sodium cyanoborohydride (NaBH3CN, Alfa recrystallized grade) was stored in a desiccator, under argon. Formic acid (Aldrich, 95-97%) was distilled at reduced pressure before use. All-trans retinal, tritiated at the aldehyde carbon (78.2 mCi/mmol; 1 Ci = 37 GBq), was synthesized from all-trans [15-3H]retinol (New England Nuclear, 14.3 Ci/mol), after 1:100 dilution with unlabeled retinol, by MnO2 oxidation in CH2Cl2 (5), and purified by preparative thin-layer chromatography on silica gel G (Analtech, Newark, DE) with 15% acetone in petroleum ether. Retinal stock solutions in ethanol were stored under argon, in blackened containers, at -120TC. Purple membrane was prepared from H. halobium (JW-3 strain; formerly ET1001) by standard procedures (16). The pm was stored suspended in 4 M NaCl solution at 4°C. Repenerated, Chymotryptically Cut, [3H]Retinal-Labeled pm ( H-RG-CT-pm). RG-CT-pm was prepared essentially as described (7, 17), except that hydroxylamine-bleached pm was cut at a concentration of 0.125 mg/ml, in the presence of 50 mM CaCl2, with 10 ,ug of chymotrypsin per ml. The product was washed with 75 mM EDTA at pH 7 to remove membrane-bound Ca2+. Aliquots of stock solutions of all-trans [15-3H]retinal (0.53 mM in ethanol) were concentrated by evaporation with a stream of argon in the dark prior to regeneration of bleached, cleaved pm (CT-pm). Reduction of RG-CT-pm. Eight milligrams of 3H-RG-CTpm in 20 ml of 4 M NaCl was dark adapted for at least 1 hr under argon. Argon-saturated 200 mM Ches (pH 10) containing 20% (wt/vol) NaBH3CN was centrifuged (15,000 rpm, 20 min, Sorvall SS-34 rotor) to remove solid contaminants, and 20 ml was added to the 3H-RG-CT-pm suspension under dim red light. The reaction mixture was placed in a black beaker, under argon, for 6 days, with magnetic stirring. In some cases, the beaker also held a control mixture, which contained all reaction components except NaBH3CN. The reduced product was washed three times with distilled water and stored under argon, in the dark, at 4°C. Reduction After pH Jump. Two-hundred fifty micrograms of 3H-RG-CT-pm in 263 ,A of water was added to 263 ,1 of 0.1 M sodium acetate (pH 5.0) or 263 ,ul of 0.1 M Ches (pH 10.0) and left at room temperature in the dark overnight. Next, 526 ,u of ethanol was added while spinning in a vortex, followed immediately with an addition of 150 ,ul of 1 M HCl. Abbreviations: bR, bacteriorhodopsin; CT, chymotryptically cut; pm, purple membrane; RG, regenerated; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid. *Present address: Advanced Genetic Sciences, Inc., 6701 San Pablo Ave., Oakland, CA 94608. tTo whom reprint requests should be sent. 2303 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2304 Biochemistry: Wolber and Stoeckenius The sample was then placed on ice and, after it had cooled, 100 ,/1 of 20% (wt/vol) NaBH3CN was added. After 15 min, each sample was centrifuged, the supernatants were discarded, and the pellets were prepared for NaDodSO4/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Samples were electrophoresed on 10-20% linear gradient gels (18); the gels were stained, the stained bands were excised and dissolved with H202, and the radioactivity migrating with each band was measured by liquid scintillation counting. Chromatography and Sequence Analysis. Unreduced retinylidene linkages in reduced RG-CT-pm (RD-RG-CT-pm) samples were broken by addition of an equal volume of ethanol to a concentrated (>1 mg/ml) sample, followed by addition of 1 M hydroxylamine (pH 7.0) to a final concentration of 50 mM. The sample was then centrifuged (15,000 rpm, 40 min, Sorval SS-34), and the pellet was suspended in formic acid. LH-60 chromatography to separate the chymotryptic fragments, CT-I and CT-II, and cyanogen bromide cleavage of the fragments were performed as described (7, 19). Column conditions for fractionation of the cyanogen bromide fragments are discussed in the legends of Figs. 2 and 3. High-performance liquid chromatography (HPLC) was performed as described in the legend of Fig. 4. Amino acid sequence analysis was performed with a Beckman 890C sequencer as described (20) in the laboratory of S. C. Rall, Jr. |
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| Language | English |
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| Content Type | Text |
| Resource Type | Article |
