Anion-dependent transition of two acidic forms of bacteriorhodopsin

190

Biodtimica et Biophysica Acta, 976 (1989) 190-195 Elsevier

BBABIO 43052

Anion-dependent transition of two acidic forms of bacteriorhodopsin Alexander L. Drachev 1, Lel A. Drachev 1, Andrey D. Kaulen 1, Lyubov V. Khitrina 1, Vladimir P. Skulachev i Gerasim P. Lepnev 2 and Lina N. Chekulaeva 3 I A, N. Belozersky LaboratoO' of Molecular Biology and Bioorganic Chemistry. Moscow State University, Moscow. " l.xtboratorv of Glass Electrochemisto,, Institute of Chemistry, Department of Chemistry, Leningrad State University, Petrodvorets, Leningrad and ~ Institute of Biological Physics, U.S.S.R. A caden 0, of Sciences, Pushchmo, Moscow Region ( U. S. S. R.)

(Received 25 May 1989)

Key words: Bacteriorhodopsin;Hill plot; Harned's rule; Chloride ion activity; Blue acidic form; Purple acidic form The effects of H + and CI- concentrations upon bacteriorhodopsin have been studied. In a suspension of bacteriorhodopsin sheets it is shown that the transition of the blue (BR6os) to the purple (BRs6 5) acidic form is caused by an increase in the concentration of CI - rather than of H +. The transition as a function of pCi is linear in Hill plots, with a slope close to 3. The Hill coefficient for the pCI dependence of bacteriorhodopsin, solubilized in laurylsucrose, lies between 2 and 3. The data are consistent with the assumption that the cooperativity of the interconversion of two acidic forms of bacteriorhodopsin is due to the reversible binding of three chloride anions by a bacteriorhodopsin molecule.

Introduction Bacteriorhodopsin is the only protein of purple membrane sheets isolated from the extreme halophile Halobacterium halobium. Together with specific phospholipids, bacteriorhodopsin trimers form a hexagonal lattice. At neutral pH, the light-adapted bacteriorhodopsin has an absorption maximum at 568 nm, which shifts to 558 nm after the pigment has been kept in darkness [1,2]. A pH lowering results in the reversible conversion of bacteriorhodopsin to the blue acidic form with a maximum at 605 nm: BR568 ( B R s s 8 ) ~ BR605 [1-9]. The p K of this conversion decreases with the increase in the cation concentration [3-5]. The efficiency of the effect of cations increases with the cationic charge [5,10-12]. The properties of the blue acid deionized form of bacteriorhodopsin are widely studied [12-17]. Incorporation of bacteriorhodopsin into proteoliposomes results in a substantial decrease in the sensitivity of the pH transition to cations [3,4]. An effect of the lipid environment on the cationic sensitivity was also described [18,19]. The possible mechanisms of these interrelations were discussed [6,7,12,13,18-22].

Abbreviation: BR, bacteriorhodopsin. Correspondence: V.P. Skulachev, A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, U.S.S.R.

It was shown that the cationic effect on the apparent pK of transition takes place only through a change of the surface potential [13,18,19]. Further acidification of the mixture was shown to cause the conversion of the blue acidic form into the purple acidic one: BR605~--BR565 [2-4,7,8]. Initially, this transition was viewed as pH-dependent [3,4,8]. It was subsequently found, however, that it was due to an increase in anion concentration [7,23]. The p r o t e i n / anion stoichiometry of such an effect remained uncertain. The aim of the present work was to carry out a quantitative analysis of the anion dependence of the BR605 ~ BR565 transition. Materials and Methods

Bacteriorhodopsin preparations Bacteriorhodopsin was isolated according to Oesterhelt and Stoeckenius [1,24] from the 353-P strain of H. halobium. Purple membranes were solubilized in 2% or 5% laurylsucrose according to the procedures described in Ref. 25. Optical measurements The fast kinetics of optical changes in response to the laser flash was measured in a suspension of purple membranes (approx. 8 . 3 - 1 0 -6 M bacteriorhodopsin) [4,26,27]. The photochemical cycle was excited with a

0005-2728/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)

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Fig. I. BR605~ BR%s transitions in HCI solutions: (a) estimation by optical density changes at 625 nm: (b) linearization in Hill plots (.462~ (1) or Q (2) values were used for calculations). neodymium Q-switched laser with frequency doublers (X = 532 nm, t~/2 = 15 ns, the green light pulse energy, 80 m J). Bacteriorhodopsin absorption spectra (approx. 10 -7 M) were measured by a Specord UV-Vis spectrophotometer, with the cuvettes placed near the photomultiplier window. To decrease light scattering the diluted suspension of purple m e m b r a n e (½- ~ of the final volume) was rapidly mixed with a solution of the required composition and then shaken vigorously: measurements were performed until large flakes appeared. Bacteriorhodopsin in detergents was likewise,acidified immediately before measurements: the detergent solution (¼ of the final volume) was mixed with an acid-salt solution (½ of the final volume), also before measurements. Circular dichroism spectra were recorded on a Mark III dichrograph from Jobin Ivon. BR605 does not change its extinction and the absorption m a x i m u m during dark and light adaptation [7]. In BR565, such changes are either absent [7] or are very small [8]. Since lowering the p H considerably accelerates dark adaptation [28], at p H 3.8, under normal conditions, purple membranes are dark-adapted. This allowed us to make all the measurements in diffuse light at room temperature.

The Hill method To obtain Hill plots, scattering from absorption spectra was subtracted by the graphical method; a correlation of optical densities at two wavelengths was taken for calculations, as in the procedure described by Renthal and Wallace [29]:

sion of purple m e m b r a n e s in 0.01 M and in 1 M HCI, respectively, and Q ( C ) in the investigated solutions, where C is the total concentration of C I - in the mixture KCI + HC1; X is the isosbestic point for the blue and purple acidic forms (for purple membranes, X = 585 nm); /3 is the portion of BR¢,05 in the bacteriorhodopsin pool * Special experiments demonstrated that the/3 values, computed both from Q and A62.s magnitudes, are plotted into one and the same dependence, but in the former case the dispersion of values is lower (Fig. 1).

Estimation of p l l and pCl In the solutions used in our experiments, the reference electrode potential exerted a significant effect on pH measurements with a p H meter. A p p r o p r i a t e corrections seem to be very complicated [30]. In the present work, we calculated p H and pCl from HCI and KCI concentrations according to the formulas: pH =--Iog(CH,-Ti I. )

pCI = - log( col-. ~'c.t- ) where C H. and Ccl- are respective molar concentrations, and "~'H' and Yc,- are activity coefficients for the proton and C i - anion. The effects of sucrose and laurylsucrose (5%) on the activity coefficients was ne-

* If it is for transition more convenient to calculate the ratio Q' = Ax./Aa,., where both wavelengths are not isosbestic (and transition may be registered for each of them, e.g., AI = 625 nm and A2 = 565 nm), the following formula might be a suitable one:

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193 glected. Control experiments with purple membranes showed the validity of such an approach for sucrose and the absence of its effect on the titration accuracy. T o calculate activity coefficients for HC1 (7~4cl) and KCI (YKa) in the mixtures, we used Harned's rule on linear changes of the logarithm of the activity coefficient in electrolyte isomal mixtures [31]: log "¢FtCl = log "/'HCI(0)-- O~HCItI/KCI log "[KCI = log YKCI(O)- OtKCIm HCI

where mHCI and mKC t are the molalities of HC1 and KC1 in the mixture, ~'HC~0) and "r'KC~10)are the activity coefficients for HCI and KC1 at a concentration for which the molality of each solution is equal to the total molality of HCI and KCI in the mixture (i.e., in an isomolal series, the activity of one electrolyte in the absence of the other), a H C I and aKC~ are the proportionality coefficients in Harned's rule [31]. Two methods were used for obtaining 7H, and 7c~values. Method 1. Activity coefficients for individual ions in HC1 and KCl mixtures were determined as suggested by Robinson and Bates [32]: In Y c t - = .vHct In YHCt + .VKCl In YKCI + 0 . 5 h In a w

h = YKCI hKcI + THCI h n c l .

aw = YncJ aHC"°~+ .VKclaKwc"°) log 7H + = 2 log YHCI -- log 7 c i -

where YHCt and YKcJ are the molal fraction of the electrolytes (YHc~ +YKct = 1), hHc j = 8 and hKc I = 1.9 are the hydrate number of HCI and KCI (taken from Ref. 33), a~vcll°~ and aKwcN~°~ are the water activities in HCI and KC! solutions at concentrations equal to the total molality of the mixture (taken from Ref. 34). Method 2. For HCI solutions, we took the table of CI- activity coefficients from Ref. 35. For mixed solutions of HCI and KC1, activity coefficients were approximated by the linear method: ~CI - =

)'HCI .y(H(.'l(O)+-|'KCI "YC. K~'I|O}

By using, as yH_Ctc0>and 7 K-cl¢°>, the values of C1- activity coefficients from the table given in Ref. 35 for HCI and KCI solutions, respectively, at a concentration of each equal to the total molality of the mixture, i.e., Harned's rule was assumed relevant to coefficients of individual ions. Results and Discussion

In 0.01 M HCI, bacteriorhodopsin is in the blue form (BR605). Illumination of BR605 was found to induce an electric response composed of the negative phase only

[3,4]. The transition into the blue form is accompanied by the disappearance of intermediate M412 in the photochemical cycle [3,4,14,17], as shown in Fig. 2c, curve 2, instead, an intermediate with a 495 nm differential maximum (Fig. 2d, curve 2=). As assumed by Kobayashi et al. [14], this is an L-type intermediate. It is registered at 412 nm (Fig. 2c and d, curves 2) as well, which makes it necessary to take into account identifying the forms according to photocycles. The rates of formation and relaxation of M412 and of the blue form intermediate differ somewhat. A comparison of their decays is shown in Fig. 2e: curves 2 = and 2, at 495 and 412 nm, respectively, represent the photocycle BR605 intermediate, while curves 1 and 3 represent M412. These differences of the kinetics are not the consequence of the pH-dependence of M412 decay: the pH of suspensions 1 and 3 differs by approx. 1.8, while the pH values for 2 and 3 are fairly close to each other. The transition of BR605 into the purple acidic form (BR565) may be indicated, apart from the shift of the chromophore absorption maximum (Fig. 2a), also by a further increase in the electric response negative phase in the absence of positive phases [3,4] and by the appearance of a bathointermediate with a kinetics corresponding to that of the negative phase of the electric response [3,4] (Fig. 2b, curve 4). As seen from Fig. 2a, the addition of KCI at pH 2 induces the appearance of the purple form. However, this is accompanied by the flash-induced generation of M412 (Fig. 2c, curve 3). Thus, KCI seems to give rise to the neutral purple form of bacteriorhodopsin, since the pK of the transition into the blue form decreases with an increase in the cation concentration [3-5]. On the other hand, the low amplitude of optical changes at 412 nm in the photocycle shows that only a small part of bacteriorhodopsin has transferred into the neutral purple form. Had the transition occurred only between the two forms (BR605 and neutral purple), it could have been expected that at 650 nm curve 3 (Fig. 2b) might have been approximated by a sum of curves 1 and 2 with required contributions. Yet in the microsecond region curve 3 is located higher than are curves 1 and 2. Obviously, curve 3 likewise contains a contribution of type-4 kinetics, characteristic of BR565. We might note that it is difficult to assay the proportion of the KCl-effected transition to BR565 because of the dependence of the rate of formation and relaxation of the bathointermediate on the pH of 0.01-1 M HCI. According to Fischer and Oesterhelt [7], the efficiency of various anions in the BR605 --~ BR565 transition decreases in the following order: F - > C1- > B r > I - > CIO 4 . But since the authors added different salts to the bacteriorhodopsin suspension at p H 2, they actually dealt with two transitions simultaneously: one from the blue to the acidic purple form, and the other from the blue to the neutral purple form.

194 In experiments shown by Fig. 3, we studied the acidic forms of bacteriorhodopsin at increasing concentration of KCi or of both KCI and HC1 taken at a ratio of 1 : 1. Under these conditions, the hypsochrome shift of the spectrum was due to the formation of the acidic purple form of bacteriorhodopsin as shown by the fast kinetics of the spectral photoresponse. The absence of the neutral purple form at this transition was determined: (1) by the amplitude lower than in 0.01 M HC1 of the short-wave intermediate at 412 nm; and (2) by the lack of O-intermediate contribution at 650 nm (type 2b, curve 1 kinetics), and by the appearance of the bathointermediate characteristic of BR565. To obtain quantitative parameters of the transition, we used Hill plots. In all the systems studied pCl dependences of the transitions coincided (Fig. 3b and c). At the same time, pH dependences proved to be different (Fig. 3a). The addition of KCI to 0.2 M HC1 changed the pH only very slightly. Nevertheless, the BR605 ~ BR565 transition did take place. So this transition is due to a change in the concentration of C1rather than in the concentration of H ÷. As a function of pCl, the transition as linearized in Hill plots, with a slope equal to 2.7 if calculated as described in Materials and Methods (Method 1) (Fig. 3b). 'Real' coefficients for individual ions (Method 2) [35,36] gave a slope of 3.2 (Fig. 3c). Since both methods of pCl- calculations yielded similar results, they appeared to be suitable for evaluation of the activity coefficients of separate ions. It is not clear, however, whether C1- anions are bound as a result of cooperative transition to each molecule of the bacteriorhodopsin triad (one anion per molecule) or each bacteriorhodopsin molecule binds three anions. This question might have been resolved by studying bacteriorhodopsin monomers. Laurylsucrose is the only detergent known to date which makes it possible to investigate conversion of the blue and purple acidic monomers. According to literary data [25,37,38], in laurylsucrose bacteriorhodopsin converts to a monomeric state. As shown in Fig. 4, in 5% laurylsucrose, either in 0.01 M HCI (blue acidic form) or in 1 M HCI (purple acidic form), one could always observe a circular dichroism spectrum with a single positive maximum in the chromophore region only (a standard test for bacteriorhodopsin transition from timers to monomers [25,39-44]). Transition of the blue acidic form of solubilized bacteriorhodopsin to the purple form in HC1 solutions was studied. The Hill coefficients for the pCl dependence of this conversion in 2% and 5% laurylsucrose were found to coincide, equal to 2.2 (Method 1, Fig. 3d) and to 2.6 (Method 2, Fig. 3e). So cooperativity of the transition takes place in the monomeric bacteriorhodopsin too. The transition of the neutral purple form to the blue acidic one is known to be conformational

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[13,18,19]. But then the similar hypsochrome color transition to the acidic purple form ought to be conformational. Bacteriorhodopsin transition to the monomeric form must not change the number of anion binding sites. However, the measured color changes of the forms occur after the binding of the third anion as a result of the change in the protein conformation. Obviously, a theoretical model for the independent binding of each of the three C1- is possible, while the values of binding constants may depend on any changes occurring in bacteriorhodopsin. Some lowering of the Hill coefficient may be due to a 'loosening' of the bacteriorhodopsin structure and a change of the surface potential after the transition from the crystalline lattice in the membrane to the detergent micelles. Thus, one may conclude that in purple sheets the BR6o5 --* BR565 transition is accompanied by a binding of three CI- to one bacteriorhodopsin molecule. Analogous cooperativity (slopes between 3.5 and 4) was also shown for competitive inhibition by MK-473 of CI- transport by halorhodopsin [45]. The similarity of halorhodop~in and acidic bacteriorhodopsin photocycles is of some interest. The photocycle of halorhodopsin in the absence of halogenides contains, as does the photocycle of the halogen-containing from of BR565, only long-wave intermediates with similar kinetics. The photocycles of the Cl-containing form of halorhodopsin and of BR605 have intermediates with differential maxima at 520 nm and 495 nm, respectively, analogous to the L-intermediate in the photocycle of the light-adapted neutral form of bacteriorhodopsin. The specificity of halorhodopsin and bacteriorhodopsin for halogenides is noteworthy. F - is most suitable for BR565 formation. In fact, in the presence of F-, BR565 is formed at pH as high as 4 [3,4]. As to

195 h a l o r h o d o p s i n , F - d o e s n o t c o n v e r t it to t h e h a l o g e n c o n t a i n i n g f o r m [46].

Acknowledgements T h e a u t h o r s o f f e r t h e i r d e e p g r a t i t u d e to D r . N . G . A b d u l a e v a n d D r . V . L . V o e y k o v for the s y n t h e s i s a n d p u r i f i c a t i o n o f l a u r y l s u c r o s e p r e p a r a t i o n s , a n d a l s o to Dr. A . M . A r u t j u n j a n for his h e l p w h e n c i r c u l a r d i c h r o ism s p e c t r a w e r e m e a s u r e d . T h a n k s are a l s o d u e to M r . I.S. K o c h u b e y for his a s s i s t a n c e in p r e p a r i n g t h e E n glish v e r s i o n o f t h e p a p e r .

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