May 24, 1991 - Studies by Blatz et al. (36) have shown that the absorption maxima of protonated Schiff bases of retinal in nonpolar solvents undergo a ...
Vol. 266, No. 28, Issue of October 5,pp. 18674-18683,1991 Printed in U.S.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY 3.5 units for neutral single mutants of Asp-85 but only decreases of ~ 1 . units 2 forcorresponding substitutions of Asp-212, relative to thewild type. Substitutionsof Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereasmutants of Asp-212 display minorred shifts orblue shifts. We conclude, therefore, that Asp8 6 is the retinylidene Schiff base counterion in wildtype bacteriorhodopsin. In the mutantAsp-85 + Asnl Asp-212 .-,Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts thefolded structure of wild-type and mutant proteins can be stabilized at low pH in lipidldetergent micelles. The data indicate thatexogenous anionsserve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation orby protonation.
Bacteriorhodopsin (bR)’ is a transmembrane protein that functions as a light-driven proton pump in the purple mem-
brane of Halobacterium halobium (1).The protein contains seven a-helical transmembrane segments and a molecule of all-trans-retinal, which is covalently linked to Lys-216 as a protonated Schiff base (PSB) (Fig. 1).The transport of protons involves a photochemical cycle that consists of at least five transient intermediates (4,5). Recently, site-specific mutagenesis has identified several amino acid residues that are involved in proton translocation. The interactions between Asp-85, Asp-212, Arg-82, and the protonated Schiff base (cf. Fig. 1)were shown to be critically important for the regulation of function and absorption maximum of bR (6-11). In the recently proposed structural model of bR the side chains Of Asp-85 and Asp-212 are approximately equidistant (-4 A) from the PSB(2), and bothresidues appear to be deprotonated in the ground state (7,12).Moreover, the N --., H bond of the chromophore is oriented toward the extracellular side of the membrane (13, 14), allowing an interaction of the PSB with Asp-85 or Asp-212. Thus, either or both aspartate residues are candidatesfor being counterions to thePSB. In aprevious study we showed that replacement of Asp-85 by neutral amino acids leads to a significant reduction in the pK, of the Schiff base to between 7 and 8.2 and suggested that Asp-85 serves as theprimary counterion to the PSB(10). Arg-82 is presumably ionized as well in bR and is likely to interact with Asp85 and/or Asp-212. In addition, there is evidence that Asp85, Asp-212, and Arg-82 are involved in the proton release from the Schiff base in theearly phase of the photocycle (10). Specifically, Asp-85 functions as the proton acceptor during M formation (7, 10, 15-17). Based on action spectra it was proposed that Asp-85 also becomes protonated during the purple to blue transition at low pH (9). In the present work, we have used single and double substitution mutants of Asp-85, Asp-212, and Arg-82 to investigate the counterion environment of the PSBin greater detail (Fig. 1).Our results show that a single carboxylate group (Asp or Glu) at position 85 or 212 fulfills the requirement for folding and subsequent formation of a PSB in bR. Measurements of the pK, values of the SB show large decreases for neutral substitutions of Asp-85 (2-4 orders of magnitude), whereas corresponding mutants of Asp-212 reveal SB pK, values similar to thatof the wild type. Because of the greater effects on the absorption spectrum and on the SB pK, for neutral replacements of Asp-85 relative to Asp-212, we conclude that Asp-85 and the PSB show a stronger electrostatic interaction and that Asp-85 is the retinylidene Schiff base counterion in the ground state of bR. In the absence of an endogenous counterion, as is the case in the double mutant D85N/D212N: exogenous anions can serve as surrogate coun-
* This work was supported in part by Grants GM 28289 and AI 11479 fromthe National Institutes of Health and Grant N00014-82K-0189 from the Office of Naval Research, Department of the Navy (to H. G. K.), and by Grant Sfb 312, TPBl from the Deutsche Forschungsgemeinschaft (to M. P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a postdoctoral fellowship from the Swiss National Science Foundation. ll To whom correspondence should be addressed. ’ The abbreviations used are: bR, bacteriorhodopsin; ebR, bacteri* bR mutants are designated by the wild-type amino acid residue orhodopsin prepared by expression of a synthetic wild-type gene in E. coli; PSB, the protonated Schiff base; SB, the unprotonated Schiff (standard one-letter code) and its position number followed by the base; DA, the dark-adapted form of the chromophore; LA, the light- substituted amino acid residue. Thus, “R82Qsignifies the mutantin adapted form of the chromophore; DMPC, L-a-dimyristoylphospha- which the arginine at position 82 has been replaced by glutamine, tidylcholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l- and “D85N/D212N” signifies the double mutant in which the two aspartates at positions 85 and 212 have been replaced by asparagines. propanesulfonate; SDS, sodium dodecyl sulfate.
18674
The Schiff Base Counterion in Bacteriorhodopsin
18675
CYTOPLASMIC SIDE
of 1%DMPC, 1%CHAPS, 0.2% SDS, and 1 mM sodium phosphate, p H 6.0, by theaddition of all-trans-retinal(19).Thekinetics of chromophore formation were measured a t 20 "C in the presence of a >3-fold molar excess of retinal. Under these conditions the regenerationrate is independent of theretinalconcentration (22). The absorbance increase at the , ,X, of the dark-adapted chromophorewas monitored. The effect of p H on the chromophore was studiedin DMPC/CHAPS/SDS mixed micelles, and the pH was adjusted by adding microliter aliquots of 0.1-0.5 M NaOH or 0.1-2 N H,SO,. Extinction coefficients were determined by acid denaturat,ion of each mutant in the dark to give a chromophore with ,X, a t 442 nm (23). The ratio of ahsorption at the A,,, to the absorption at442 nm after acidification to pH 1.9 was compared with thatof wild-type ebR. The extinction coefficient of ebR was assumed tobe 52,000 M" cm" (22). The ,X, values of the chromophores were measured a t 4 "C after dark adaptation followed by light adaptation for 3 min(250-watt projector lamp equipped with a 475-nm long-pass filter). Retinalwas extracted from the chromophores after dark adaptation followed by light adaptation for 3 min, as described previously (23, 24). Effects of Exogenous Salts on the Absorption Spectrum ofbR Mutants-Solutions of sodium salts of halides, perchlorate, and different carboxylicacids (formate,acetate,tartrate,citrate,chloroacetate, dichloroacetate, and trichloroacetate)were prepared a t 2 X (0.1, 1, or 4 M ) concentrations. Thesaltsolutions were titrated with their E X T R A C E L L U L A R SIDE respective acids to give a pH of 6.0 upon dilution to 1 X. The mutants were regeneratedas describedabove and the individual 2 X salt FIG. 1. Diagram of the structure of bacteriorhodopsin. The solutions were then added. In the case of the mutant D85N/D212N drawing is based on the model of Henderson et al. (2) and shows the the chromophorewas directly regenerated in the presence of different seven n-helical transmembrane segments(A-G) and the approximate salts. All measurements were taken at a protein concentration of 6.4 position of the retinylidene chromophore. Retinal is covalently at- p~ in 1% DMPC, 1% CHAPS, 0.2% SDS, and 1 mM sodium phostached to Lys-216 in helix G via a protonated Schiff base linkage. phate at pH6.00 0.05. The Schiff base environment is formed by Asp-212 in helix G and Determination of the pK, of the Schiff Base-The mutant proteins Asp-85 and Arg-82 in helix C. The protonation stateof the groups in (6.4 PM) were prepared as described above in the absence or presence the ground state isshown. An alternative arrangement could include of 2 M NaCI. Titrations were carried out in steps of 0.1-0.3 pH units, water molecules that arelocated in the vicinity of the Schiff base (3). and following complete equilibration (-3 min)pH readings and N T and CT indicate the N- and C-terminal segments of the protein. absorption spectra were recordedfor each point. The pH-induced absorbance changes were determined from difference spectra as follows. A reference spectrum was recorded at a pH where the pigment terionand allow chromophoreregeneration.Thespectral was completely converted to the protonated form. This spectrum was properties of the D85N/D212N mutant are similar to those subtracted from each of the spectra recorded at higher pH values, of the so-called acid-purple form of wild-type bR. The nature and the amountof titrated pigment was determined from the absorbof the anions (halides, perchlorate, and different carboxylic ance increase of the deprotonated SB at 365 nm (for ebR, R82Q, acids) greatly influences the absorption maxima of the mu- D85E, D212A, D212E, D212N, and R82Q/D212N) or 405 nm (for be- D85A, D85N, and D85N/D212N). In the tants. Large spectral shifts are observed for D85A ,X,(, case of R82D/D85R and 405-nmform was incompletely tween 544 and 603 nm), D85E ,X,(, between 560 and 607 R82Q/D85N the transition to the between 494 and 562 nm) in the separated from that to the 365-nmform. Thus, the amountof titrated nm),and D212N (X,, presence of different anions. This indicates that electrostatic pigment was determined from the absorbanceincrease at 382 nm, the isosbestic point of the two species. T o obtain the apparent pK, the interactions between the PSB and its counterions play an absorbancechange LA was plotted versus the pH. The following important role in regulating thewavelength of absorbance in three-parameter curve(25) was then fitted to the points.
*
bacteriorhodopsin.
EXPERIMENTAL PROCEDURES
Preparation of bR Mutants-The preparation of mutants containingthe single substitutions R82Q, D85A, D85E, D85N, D212A, D212E, or D212N has been reported (6, 8, 10). Genes encoding the double mutants R82Q/D212N, D85E/D212E, D85E/D212N, D85N/ D212E,and D85N/D212N were assembled using restriction fragments of previously constructed pSBO2 vectors that carry the corresponding single mutations (18, 19). In each case a small PuuI-BglII fragment (encoding the mutation at residue 82 or 85) was ligated to a large BglII-PuuI fragment of pSBO2 (encoding the mutation at residue 212). For the double mutants R82Q/D85N and R82D/D85R, ApaI-BglII restriction fragments containing the desired mutations were synthesized and ligated with the small Puul-ApaI fragment and the large BglII-PuuI fragment of wild-type pSB02. The sequences of regions carrying the mutations were verified by direct plasmid sequencing via the dideoxy method (20). The mutant genes were then introduced into the vector pPLl as HindIII-EcoRI fragments (18) and expressed heterologously in Escherichia coli under the controlof the XP,,promoter (€419). All ofthe mutant apoproteinswere extracted from crude E. coli membranes with an organic solvent mixture and purified to apparent homogeneity by DEAE-Trisacryl chromatography (21). Theaverage yield of mutant apoproteins was 40-50 mg/lO g of freeze-dried membranes. Regeneration and Characterization ofbR Mutants-bR-like chromophores were regenerated from the apoproteins (16 p ~in)a solution
=
LAT/ll + 101"'P"
-lJHli
I
The three parameters were: LAT, the total absorbance change of the deprotonated SB; n, the numberof protons involved in the transition; and pK, the midpoint of the titration. RESULTS
I. Effects of Substitutions of Asp-85 and Asp-212 on Chromophore Regeneration Substitutions of Asp-85 or Asp-212 Alter the Kinetics of Chromophore Formation-We have reported previously (6) that substitutionof Asp-85 or Asp-212 by Asn slows down the rate of chromophoreregeneration (tlr2= 41 and31min, respectively; Table I), compared with ebR (tip = 1 min). A similar effect was noticed for the mutant D212E (tIr2= 38 min), whereasfor D85E (tllz= 0.2 min) the ratewas increased over the wild type. Striking differences in the kinetics are also observed with double mutants of Asp-85 and Asp-212 (Fig. 2; Table I). The regeneration process for D85N/D212E ( t 1 / 2 = 190 min) is slower by more than 2 orders of magnitude compared to thewild type. On the other hand D85E/D212N (t1/2 = 0.5 min)and D85E/D212E (tlP2= 2.8 min) reveal relatively normal regeneration times, suggesting that Glu-85
The Schiff Base Counterion in Bacteriorhodopsin
18676
TABLEI Regeneration properties, absorption maxima, and isomer compositionsof the chromophores of Asp-85, Asp-212, and Arg-82 mutants The properties were measured at pH6.0 as described under "Experimental Procedures." Chromophore Regeneration Extinction regeneration ( t B h ) n
Mutation
Absorption maximum extent
Retinal isomer composition
coefficient
DA DA
la-&
min
ebR R82Q D85E D85N D212E D212N R82D/D85R R82Q/D85N R82Q/D212N D85E/D212E D85E/D212N D85N/D212E D85N/D212N
1.0 3.2 0.2 41 38 31 760 73 11
2.8 0.5 190 220
LA
LA
%
M" cm"
nm
84 63 75 84' 74 75 40' 90 75 76 67 81 61'.
52,000 56,200 56,400 54,700' 58,400 41,100 50,700' 52,700 50,400 60,400 53,000 53,100 ND'
551 561 575 580 597 596 595 64594 584 581 560 46548 597 592 583 582 56227 568 584 584 556 544 560 564 558 562
all-tram
9-cis
11-cis
%
60 50 51 36 9 54 16 22 58 6 31 13 15
IS-&
all-trans
%
40 50 49 91 a4 78 42 94 69 a7 85
4 5 1 5 3
5914 5 10 491 2 9
4 3
7 22 12
3 21 23 32 26 34 27 22
97 79 58 63 61 78 73 68 31 50 86
25 43 35 14
Measured at 20 "C with all-trans-retinal.
'The value given is at pH 5.0, since the chromophore exists in an equilibrium of protonated and unprotonated forms near neutral pH (see text). The properties were measured under standard conditions in thepresence of 2 M NaCI. Calculated with t = 52,000 M" cm". The value could not be determined, as no denaturation is observed at pH 1.9 (see text).
C . R020/D05R
A . R82Q/ D85N
0.6
Time (min) FIG. 2. Time course of chromophore regeneration for double mutants of Asp-85, Asp-212, and Arg-82. Themutant apoproteins were regenerated at 20 "C by the addition of excess alltrans-retinal, as described under"Experimental Procedures." The absorbance increase at the, , ,X of the dark-adapted chromophore was recorded. This was replotted after determination of the final absorbance. The corresponding t1p2values are listed in Table I.
can compensate the effect of the Asp-212 substitution in these double mutants. In comparing R82Q/D212N (tllz= 11 min) with R82Q/D85N ( t I l 2= 73 min) we note that chromophore formation in the former double mutant is accelerated about 7-fold. An extremely slow regeneration process is observed for the double mutant R82D/D85R (tl12= 760 min), presumably due to introduction of a positively charged residue near the Schiff base. Chromophore Regeneration Requiresa Carboxylate Group at Position 85 or 212"Studies with the mutants D212N and D85N have shown that a single aspartate residue at either position 85 or 212 is sufficient to form a bR-like chromophore (6). A normal extent of chromophore regeneration (>SO%; Table I) is also observed for the double mutants D85E/D212N and D85N/D212E, which contain a single glutamate residue at position 85 or 212, respectively. However, removal of both aspartate residues in the double mutant D85N/D212N prevented chromophore formation (
