The bacteriorhodopsin proton pump: Effect of crosslinkings of lysine residues

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 260, No. 2, February 1, pp. 725-731,1988

The Bacteriorhodopsin RUDOLF *Institute

T6TH-BOCONADI,* NASHMUTDIN

Proton Pump: Effect of Crosslinkings of Lysine Residues

STEFKA G. TANEVA,? ALEKSANDR V. KISELEV,* G. ABDULAEV,$ AND LAJOS KESZTHELYI**l

of Biophysics, Biological Research Center, Szeged, Hungary, tCentra1 Laboratory Sofia, Bulgaria, and $%emyakin Institute of Bioorganic Chemistry, Academy of Sciences of USSR, Moscow, USSR

of Biophysics,

Received June 23,1987, and in revised form October 1,1987

All six available lysine residues in bacteriorhodopsin were amidinated with dimethyl-3,3’-dithiobispropionimidate, which is a crosslinking agent. The photocycle was studied by measuring light absorption and electric signals. The data show an essential change in the photocycle: instead of single components, the rise of the signal due to the M intermediate can be decomposed into two components, and the decay into three. The life-times and the intensities of these components and in general the proton pumping activity of bacteriorhodopsin depend only negligibly upon pH. Changes upon removing the crosslinks are not significantly different from those in the crosslinked samples. The lysine residues therefore may not be considered of primary importance in proton translocation. 0 1988 Academic Press, Inc. Chemical modification of the amino acid side chains of bacteriorhodopsin (bR) has been extensively applied to obtain information on the structure of bR2 and the role of amino acid residues in proton pumping. Previous reports indicate the involvement of certain amino acid residues (Tyr, Glu, Asp) in light-induced proton transport across the purple membrane (l-lo). In order to clarify the functional role of lysine residues of bR, modifications with dansyl chloride (11) and monofunctional and bifunctional imidoesters (9, 12), acetylation of the t-amino groups of Lys-30, -40, and -41(13), and methylation of lysine residues (14) have been used. In general these modifications changed the time constants of the photocycle; however, the proton pumping activity was not altered significantly. In this study, a depleted amidination of all six available lysine residues with di-

methyl-3,3’-dithiobispropionimidate (Fig. 1) was performed and the effect of this modification on the photocycle of bR and on the kinetics of the charge transport was investigated over a broad pH range. The reduction of the S-S bond in the modifying reagent permitted us to check the influence of crosslinking on the chromoprotein properties. MATERIALS

AND METHODS

PM fragments from the ET 1001 strain of Halobachalo&urn were isolated by the procedure of Oesterhelt and Stoeckenius (15). PM (10 mg/ml protein) in 0.5 M borate buffer (pH 9.5) was treated with dimethyl-3,3’-dithiobispropionimidate dihydrodichloride (Pierce, Rockford, IL) diluted in the same buffer. The reagent was added six times in portions of 20 mg every second hour. The reaction mixture was stirred throughout. Excess reagent was removed by washing (several times) or dialysis (24 h). Amino acid analysis was carried out on a Durrum (D-500) analyzer. Polyacrylamide gel electrophoresis of the modified bR-s was performed according to (16). Bands were visualized using Coomassie brilt&urn

r To whom correspondence should be addressed. ’ Abbreviations used: bR, bacteriorhodopsin, PM, purple membrane. 725

0003-9861/W $3.00 Copyright All rights

0 1988 by Academic Press. Inc. of reproduction in any form reserved.

726

T6TH-BOCONADI

ET AL.

liant blue R-250 (Bio-Rad) and quantitated by densitometry. Flash-induced absorbance changes and electric signals were measured using the apparatus described in Ref. (1’7). The orientation of PM fragments was preserved in gel slabs as described in (18). pH was adjusted by addition of HCl and NaOH in small quantities to the soaking medium. The data were taken at room temperature (22~24’C). FIG. 2. Amino acid analysis of native (I) and modified (II) bR. The absorbance A of the column eluate was monitored at 570 nm; A is proportional to the amino acid content.

RESULTS

Modi$cation of E-NH* Gmups of Lgsine Residues of bR The reaction scheme of the t-NH2 groups of the lysine residues of bR molecule with the bifunctional reagent is shown in Fig. 1. The modifying reagent was used for the following reasons: (i) Amidination of lysine residues is highly selective and does not influence the activity of protein molecules; (ii) the degree of modification could be satisfactorily determined since the reaction products are stable toward acid hydrolysis; and (iii) since the pK of the amidinated lysine residues is about 12, it is very unlikely that the modified product takes part in proton transfer across the membrane. If the entire amount (100 mg) of the modifying agent was added to the PM suspension in one portion the modified derivate had the same absorbance maximum as native bR (570 nm) but this treatment resulted in a partial denaturation of the protein. The AB0/As70 ratio was 4 for the modified protein, whereas it was 1.8 for native bR. When the reagent was added in small portions no alteration of the 570 nm absorbance and a small denaturation of the chromoprotein (A280/A570 = 2.2) was ob-

. P @NH-C-CHFCHFS-S-CH~CH~C-NH-

served. Since it is =20% of denaturated bR, its effect is neglected in the analysis. Densitometry of the absorption peaks of the eluate of the amino acid analyzer (Fig. 2) showed a ratio 5.4 of the area of the lysine peaks (native to modified) from which the number of modified lysines is 5.7 of the total 7 lysines. Evidently, one lysine residue (the Schiff-base Lys) was not modified. Figure 3a is an electrophoretogram of the modified bacteriorhodopsin. About 40% of the protein molecules was found to be in monomeric state, while the rest form dimers, trimers, and higher order oligomers. After reduction of the S-S bond with 3% mercaptoethanol for 2 h at pH 8.0 the quantity of monomeric chromoprotein was increased to 90% (Fig. 3b). In other experiments the quantity of monomeric

h

a

b

$42 l

2CH30H

FIG. 1. Reaction scheme for the modification of the c-NH, group of lysine.

FIG. 3. Polyacrylamide gel electrophoresis of bacteriorhodopsin (a) crosslinked and (b) reduced with mercaptoethanol.

CROSSLINKING

OF

LYSINES

bacteriorhodopsin before and after reduction fell in the ranges 30-40% and 60-90%) respectively. We assume that the disulfide bond of the modifying reagent was reduced with the same efficiency for the inter- and intramolecular crosslinks because the lysine residues (except for the one at the chromophore binding site) seem to be located on the membrane surface (19), and therefore should be equally acceptable to mercaptoethanol. Thus, by comparing the photochemical and electrogenic properties of the modified chromoprotein before and after reduction it was possible to elucidate the effect of the crosslinking and assess whether conformational changes caused by the crosslinks influence the known bacteriorhodopsin functions. Absorption and Electric Response Signals in Modified bRs The absorption changes and electric response signals were measured on the same sample of gel over a broad pH range (3.8 -9.0). The purple membranes used in this experiment were not treated with mercaptoethanol. In Fig. 4 the absorption

Y

A A = 1 ‘I.

FIG. 4. Light absorption = 6.7, room temperature, immobilized in gel.

L

8Oms

changes modified

at X = 400 nm, pH bR oriented and

IN

BACTERIORHODOPSIN

727

change at X = 400 nm and pH 6.7 is shown with two different time resolutions. The fast rising signal and the slow falling one are assigned to the rise and decay of the M intermediate. To obtain the decompositions in these figures other data taken with different time bases were used also. It is seen that the M rise has two components with 71 = 22 bs (47%) and 72 = 80 ps (53%). The decay has three components with 71 = 11.7 ms (30%), 72 = 55 ms (50%), and 73 = 260 ms (20%). The absorption measurements at other pH values were not so detailed. The general trend, however, was the same: the M rise always had two components (TV = 15-25 ps, 72 = 70-80 ps), with an intensity ratio of 50-50%; the M decay behaved similarly at all pH values: it had three components with 71 = 8-11 ms (20-30%), 72 = 40-50 ms (x50%) and 73 = 160-260 ms (20-30%). Figure 5 contains the electric response at different time and amplitude scales for the selected pH = 6.7. It starts with a large negative signal (related to the proton motion) which appears rapidly and decays with the time constant of the electrical circuit. It is assigned to the bR-K transition (17-20). The second small negative signal corresponding to the K-L transition is not resolved in the present experiment. The first positive signal decays with TV = 22 ps but it falls below the zero level. This feature appears clearly in Fig. 5b and is much more pronounced in Fig. 5c. It was determined that this rather small IWgatiVe Component appears with Tl = 22 PLSand decays with T,,,~ = 140 ps. A long living positive signal with a small amplitude follows with 71 = 10.4 ms and 72 = 53 ms. The signals at other pH values were similar except that the negative component appeared only in the pH range 5.2-7.8. By comparing the life-times of the optical and electrical signals it is seen that the 80-ps component of the M rise and the 200-ms component of the M decay do not have electrical counterparts, while the T,,~ = 140 PUSelectric signal has no counterpart in absorption.

T6TH-BOCONADI

OSmVL 1oJLs

b

d

FIG. 5. Electric signals after light absorption. ples are the same as those in Fig. 4.

Sam-

The magnitude for an electric signal (1’7, 20) of rate constant ki is NQFc& V,(t) = 7 R kie-‘@,

ET

AL.

sured. That may be the reason the 30-ps and the 200-ms components of the electric signal are not seen in the presence of their faster counterparts (22 ps and 50 ms, respectively). The sum of the areas of the electric signals ZAi represents the pumping activity of bR (20). In Fig. 6 the total area related to the area of the first negative signal (-ZAi/A1) is shown for all pH values measured. The pumping activity is negligible at pH 3.2-3.8 in agreement with previous results that low pH inhibits proton pumping (21, 22). In the pH range 5.2-9.8 the areas scatter around a mean value of x35. The experimental tendency of increase or decrease with pH is not observed when we use the mean value of the total area, which is 25% smaller than in normal bR. One contribution to the loss is well understood. The area of the negative signal following the first positive signal represents a charge motion in the negative direction. The L intermediate branches to an M’ intermediate from which the protons cannot go in the forward direction but fall back with life-time T,,,~ = 140 ps to the bR ground state. From the area, this branching is 15% at pH 6.7, and it changes as shown in Fig. 6 (where -A,,,/A1 is given with respect to pH). A second contribution to the loss may be apparent. The 200-ms component found in

-?$

60-

PI

10-

where N is the number of molecules excited by the laser flash, Q is the elementary charge, F is a normalizing constant of order 1, di is the distance the proton moves in the ith step of its path, R is the measuring resistance, and D is the distance between the electrodes that detect the signal. (Equation [l] is valid if ki << l/RC where C is the capacitance of the measuring circuit.) The amplitude of the signal is proportional to ki, which means that a longer living component is poorly mea-

200.

.

. a,' ’

.

a.

l

. ? 3

:

: 5

:

:

7

:

:

9

:

pH

FIG. 6. pH dependence of the sum of the areas of the electric signals, normalized to the first component.

CROSSLINKING

OF

LYSINES

the optical signal was found not to have an electrical equivalent, but it may simply be undetectable because of its very small amplitude. The intensity of this component in absorption is 2.5 times smaller than that of the 50-ms component, and the small ki implies a further 4- to 5-fold decrease in the amplitude of the electrical component. A signal with a IO-fold overall smaller amplitude than that of the 50-ms component is not measurable by the present sensitivity. The probable existence of this long living component was checked by a different experiment. In a former study (23) it was shown that illuminating the bR in the M form by a blue flash resulted in the protons turning back and producing a short lived negative signal. The amplitude of this signal was proportional to the number of protons to be translocated. This “blue effect” was used to monitor whether protons were still in the M form for long times after excitation. The result, shown in Fig. 7, clearly demonstrates that negative signals from backward driven protons do exist even in the case of the 200-ms component. Eflect of the Reduction

of the S-S Bonds

Absorption and electric signals from two samples with 30 and 60% (after mer-

FIG. 7. Dependence of the amplitude of the fast negative signal produced by blue-flash excitation upon the time elapsed after starting the photocycle.

IN

BACTERIORHODOPSIN

729

captoethanol treatment) monomeric content were measured. In preparing the samples it was observed that the mercaptoethanol treatment decreased the orientability of the PM-s, but nevertheless the electric signals, though smaller, were readily measurable. The data are shown in Table I. We consider the small changes in life-times and amplitudes registered for the optical signals not significant. This is also valid for the electric signals which were measured at three different pH values. A small difference in the areas of the electric signal was found between the two samples. The area data are for three different pH values. The average of these values is 1.3, therefore it is clear that the changes do not reflect the difference by a factor of 2 in monomer content. Because the mercaptoethanol treatment, as was mentioned, reduces not only the intermolecular, which may be followed by determining the monomer content, but also the intramolecular S-S bonds, we may accept that the cutting of inter- and intramolecular crosslinks of lysine residues does not influence the photocycle of bR significantly. DISCUSSION

Previous studies of lysine modified bR-s demonstrated that the proton pumping activity per photocycle is not reduced by the modifications in (9, 11-14). The same is found in this work, using a different modification. Two special features should be emphasized: (a) the pK of the NfHz group is 12 and therefore its participation in proton conduction (24) is excluded. In the case of methylation this exclusion is not so strict (14). (b) It also became possible to study the effect of crosslinking, and it was found not to be significant for the proton pump. The major effects of lysine modification are the increased branching of the photocycle, the remarkable slowing down of the photocycle (seen in both light absorption and electric signals), and the negligible pH dependence in the range 5-9. According to kinetic data the branching should occur before the formation of the L state. We

T6TH-BOCONADI

ET

TABLE COMPARISON

OF PHOTOCYCLE

AL.

I

DATA OF LYSINE-MODIFIED

bR WITH DIFFERENT

MONOMER

CONTENTS

A 30%

monomer

60%

monomer

Life-time

Intensity (%)

Life-time

Intensity (%)

L-M

20 ps 114 ps

68 32

13 /is 80 ps

50 50

M-O

5.7 ms 25 ms 140 ms

22 50 28

5.7 ms 34 ms 127 ms

24 54 22

Transition

B 30%

monomer

60% monomer

PH

bR-K

L-M

M-bR

bR-K

L-M

5.75 6.9 7.85

-12.7 -18.2 -7.5

58 57 53

228 600 192

-7.8 -4.3 -2.8

49 19.4 15.6

Note

(A) absorption

data

(pH

= 6, 2’ = 24°C);

(B) areas

promote the idea that three different bR populations exist after modification. This is similar to the suggestion of two populations in Ref. (25) to explain the pH dependence of spectroscopic data. In Ref. (25) it is assumed that titration changes the protonation state of a tyrosine near the Schiff base. The new charged group influences the height of the barrier and therefore the life-time of the states. The relative intensity of the two components (25) of M formation, and in Ref. (26) also of M decay, changes according to the titration curve of the tyrosine. Our assumption is that the lysine modification produces three different populations of bR with three different internal charge distributions (which is given by the structure of bR). The insensitivity to pH is easily understandable if we take into account the pK value of 12. We should also accept that removing the crosslinking does not alter the relative charge distribution. Modification of carboxyl groups of bR does not decrease the pumping activity (27). The proton conduction model (24,28,

of eiectric

M-bR

30%

60%

ZAi/Al

ZAdAl

100 75 68 signals

in arbitrary

1.1 1.6 1.1 units

(2’ = 24°C).

29) requires a hydrogen bonded chain built up from proton donating and accepting groups formed from amino acid side chains. As carboxyl and lysine groups are seemingly not essential for proton conduction it is thought that a reevaluation of this model is necessary. On the other hand, the suggestion of proton conduction through temporarily opening water channels (30,31) is reconcilable with the above findings. REFERENCES 1. KONISHI, T., AND PACKER, L. (1978) FEBS Zdt. 92,1-4. 2. LEMKE, H.-D., AND OESTERHELT, D. (1981) Eur J. Biochm 115,595-604. 3. ROSENBACH, V., GOLDBERG, R., GILON, C., AND OTTOLENGHI, M. (1982) Photo&em Photcbiol 36,197-201. 4. KATSIJRA, T., LAM, E., PACKER, L., AND SELTZER, S. (1982) Biochem Inf. 5,445-456. 5. OVCHINNIKOV, Yu. A., ABDULAEV,N. G., KISELEV, A. V., NABIEV, I. R., VASILEVA, H. V., AND EFREMOV, R. G. (1986) Bid Membr. 3,325-338. 6. HERZ, I. M., AND PACKER, L. (1981) FEBS Letk 131,158-164.

CROSSLINKING

OF

LYSINES

7. PACKER, L., TRISTRAM, S., HERZ, I. M., RUSSEL, C., AND BORDERS, C. L. (1979) FEBS Lett. 108, 243-248. 8. KUSCHMITZ, D., ENGELHARD, M., KOHL, K.-D., GERWERT, K., SIEBERT, F., AND HESS, B. (1984) in Short Reports of Third European Bioenerg. Conference, Congress ed., Vol. 3A, pp. 51-52, Hanover. 9. PACKER, L., QUINTANILHA, A. T., CARMELI, C., SULLIVAN, P. D., SCHERRER, P., TRISTRAM, S., HERZ, I., PHEIFHOFER, A., AND MELHORN, R. I. (1981) Photo&m Photobiol. 33,579-586. 10. T~TH-BOCONADI, R., HRISTOVA, S. G., AND KESZTHELYI, L. (1986) FEBS Lett. 195, 164-168. 11. HARRIS, G., RENTHAL, R., TULEY, I., AND ROBINSON, N. (1979) Biochem. Biophys. Res. Commun 91,926-931. 12. KONISHI, T., TRISTRAM, S., AND PACKER, L. (1979)

Photochem, PhotobioL 29,353-358. 13. ABERCROMBIE,

D. M., AND KHORANA,

H. G. (1985)

J. Biol Chem. 261,4875-4880. 14. ABDULAEV, G. A., DENCHER, N. A., DERGACHEV, A. E., FAHR, A., AND KISELEV, A. V. (1984) Biophys. Struct. Me& 10,211-227. 15. OESTERHELT, D., AND STOECKENIUS, W. (1971)

Nature (New Biol.) 233,149-151. 16. ABDULAEV, M. Yu.,

Bioorgan

N. G., KISELEV, AND OVCHINNIKOV,

A. V., FEIGINA, Yu. A. (1977)

Chim. 3,709-710.

17. KESZTHELYI, L., AND ORMOS, 109,189-193.

P. (1980)

FEBSLett

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IN BACTERIORHODOPSIN 18. DI%R, A., HARGITTAI,

P., AND SIMON,

J. (1985)

J.

Biochem Biophys. Methods 10,245-300. 19. OVCHINNIKOV, Yu. A. (1982) FEBS L&t. 148, 179-191. 20. KESZTHELYI, L., AND ORMOS, P. (1983) Bbphys. Cht?m 10,397-405. 21. KIMURA, Y., IKEGAMI, A., AND STOECKENIUS, W. (1984) Photo&em. Photobiol 40,641~646. 22. DRACHEV, L. A., KAULEN, A. D., AND SKULACHEV, V. P. (1978) FEBS I&t. 87,161-167. 23. ORMOS, P., DANCSHhZY, Zs., AND KESZTHELYI, L. (1980) Biophys. J 31,207-214. 24. NAGLE, J. F., AND TRISTRAM-NAGLE, S. (1983) J.

Membr. Biol 74,1-14. 25. HANAMOTO, J. H., DUPUIS, P., AND EL-SAYED, M. (1984) Proc. Natl. Acad. Sci. USA 97, 7083-7087. 26. ORMOS, P., HRISTOVA, S. G., AND KESZTHELYI, L. (1985) Biochim Biophys. Acta 809,181-186. 27. PACKER, L., ROBINSON, A. E., HRABETA, E., HRISTOVA, S. G., T6TH-BOCONhDI, R., AND KESZTHELYI, L. (1987) Biochem Int. 14.977-985. 28. BR~NGER, A., SCHULTEN, Z., AND SCHLJLTEN, K. (1983) 2. Phys. Chem. 136,1-63. 29. MERZ, H., AND ZUNDEL, G. (1981) Biochem. Biophys. Res. Commun 101,540~546. 30. KESZTHELYI, L. (1984) in Information and Energy Transduction in Biological Membranes (Bolis, L., Helmreich, E. J., and Passow, H., Eds.), pp. 57-71, Alan R. Liss, New York. 31. KESZTHELYI, L. (1986) Bioelectrochem Bioenerg. 15,437-445.