Direct transmembraneous reconstitution of bacteriorhodopsin into planar phospholipid bilayers

Biochimica et Biophysica A cta, 1183 (1993) 171-179 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00

171

BBABIO 43916

Direct transmembraneous reconstitution of bacteriorhodopsin into planar phospholipid bilayers Eiro Muneyuki a,,, Mineo Ikematsu b Masahiro Iseki b Yukihiro Sugiyama b Atsuo Mizukami b, Koki Ohno c, Masasuke Yoshida a and Hajime Hirata d a Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227, Kanagawa (Japan), b Tsukuba Research Center, Sanyo Electric Co., Ltd. Tsukuba, lbaraki 305 (Japan), c Department of Physics, University of Occupational and Environmental Health, Kitakyushu 807, Fukuoka (Japan) and d Faculty of Science, Himeji Institute of Technology, Kanaiji 1479-1 Akoh, Hyogo (Japan) (Received 18 February 1993) (Revised manuscript received 29 May 1993)

Key words: Bacteriorhodopsin; Phospholipid bilayer; Reconstitution; Transmembrane; Ion transport; Membrane protein

A new method of incorporating bacteriorhodopsin molecules into planar lipid bilayers was developed and compared with a conventional system where purple membranes were adsorbed onto planar bilayers. By the new method, purple membrane sheets were first solubilized with a detergent and the solubilized fraction was directly added to an aqueous phase of a preformed planar bilayer membranes. Then, the solubilized bacteriorhodopsin molecules were spontaneously incorporated into the planar bilayers. Upon illumination, a steady state electric current was generated and the magnitude of the current was hardly affected by the presence of an uncoupler, FCCP. Furthermore, when bacteriorhodopsin molecules were incorporated into planar bilayers in high temperature range, a transient capacitive peak current was mostly eliminated by successive perfusions with a buffer at room temperature. These properties are in contrast with those of conventional method and strongly indicate that the bacteriorhodopsin molecules are transmembraneously reconstituted into planar bilayer membranes by the new method. The new method will prove useful in quantitative investigation of the properties of active ion transport.

Introduction

Transporting proteins in biological membranes play several essential roles in living organisms. These include energy transduction, information processing, uptake of nutrients, secretion, and so on. In order to characterize these vectorial functions of membrane proteins, it is essential to carry out purification and reconstitution of them into some artificial membrane systems. Among the reconstitution methods of membrane proteins, the system of proteoliposomes has been widely used [1-4]. It has several advantages such as relatively simple experimental procedures, high sensitivity of measurements when combined with the use of radio isotopes, and high reproducibility. However, in many cases, it is rather difficult to achieve constant mem-

* Corresponding author. Fax: + 81 45 922 5179. Abbreviations: FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; A570, absorbance at 570 nm.

brane potential or ionic concentration gradient across the membrane and time resolution is usually not so good. On the other hand, in the case of planar bilayer system, it is possible to control the membrane potential or solute composition of both sides of the membrane [5,6]. These properties are ideal for the study of energy transduction. However, the techniques of incorporating membrane proteins are rather difficult and planar bilayer system has been applied mainly for the research on ion channels. As for ion pumps, the pioneering studies using planar membrane system were achieved by Drachev et al. [7,8]. In their studies, proteoliposomes containing ion pumps were fused onto black membranes and their electrogenic activity was directly demonstrated. Since then, similar systems have been used for the studies on many ion pumps [9-18]. Such systems can provide high time resolution and suitable for the studies on fast stages of electrogenic processes [19-24]. However, judging from the effects of ionophores and a transient capacitive response of the electric current generated by ion pumps, it was concluded that the proteoliposomes and the black membranes were capacitively coupled and the ion pumps

172 did not penetrate the planar bilayers in these systems [12,24,25]. Due to their complicated structure, there were some difficulties in quantitative analyses for steady-state kinetics. Therefore, a method for transmembraneous reconstitution of ion pumps has been desired. In fact, attempts for transmembraneous incorporation of ion pumps have been already described [26-29]. Bamberg et al. compared different methods systematically [26] and Braun et al. provided strong indication of transmembraneous incorporation of bacteriorhodopsin [27]. Our previous method [28,29] was used to analyze steady-state kinetics of FoFFATPase [30] and the same method was applied to sarcoplasmic Ca2+-ATPase by another laboratory [31]. All of these previous methods used ion pumps in native membrane fragments or vesicles as starting materials for reconstitution. However, the effects of ionophores have never been examined in these studies and the possibility that adsorbed vesicles or membrane fragments contributed to the steady-state current could not be excluded. In the present paper, we describe a new method of incorporating bacteriorhodopsin molecules into planar lipid bilayers and compared it with a conventional system where purple membrane sheets adsorbed onto planar membranes. By the new method, bacteriorhodopsin molecules solubilized with a detergent were directly incorporated into a preformed planar bilayer and the steady-state current did not increase, even in the presence of a proton conductor, FCCP. Furthermore, the capacitive current was eliminated by perfusion of a buffer. These properties are the strongest indication of transmembraneous reconstitution of ion pumps so far obtained. Materials

and

Formation of planar bilayers and electrical measurements Nominally solvent-free planar bilayers were formed by the method of Montal and Mueller [34] at a hole (diameter, 0.2 mm) in a Teflon film (25 /~.m thick) separating two Teflon chambers set in a shield box as described previously [29]. Internal volume of each ~,

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sheets were isolated from Halobacterium halobium strain ET-1001 according to the procedure described by Oesterhelt and Stoeckenius [32]. Purple membranes were solubilized with Triton X-100 and subjected to gel filtration as described by Bayley et al. [33]. Briefly, purple membrane pellet containing 25 mg of protein was homogenized with 1.3 ml of 5% Triton X-100, 0.1 M sodium acetate buffer (pH 5.0) and gently stirred at room temperature in the dark for 3 days. Then, it was centrifuged at 100 000 × g for 30 min at 4°C and supernatant was collected. The solubilized bacteriorhodopsin was applied on a column of Bio-Gel A-05m (1.8 × 84 cm) pre-equilibrated with a buffer containing 0.25% deoxycholic acid, 0.15 M NaC1, 0.025% NAN3, and 10 mM Tris-HC1 (pH 8.0) and eluted at a flow rate of 5 m l / h at 4°C in the dark. Triton X-100 was confirmed to be separated from bacteriorhodopsin by measuring the absorbance of fractions at 280 nm and 570 nm. Peak fractions of

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solubilized bacteriorhodopsin were collected and stored at - 8 0 ° C until use.

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Fig. 1. Generation of electric current by directly reconstituted bacteriorhodopsin a n d e f f e c t of FCCP. (A) 40 ~1 of solubilizedbacteriorhodopsin fraction (A570 = 0.2) was added to the c/s compartment and incubated while stirring. After 2 h, excessbacteriorhodopsinwas removed by perfusion. Upon illumination, some 40 fA of electric current was induced. At that time membrane resistance was 240 GO. (B-D) Effect of FCCP. FCCP dissolved in methanol was successively added to the c/s compartment of the same membrane as (A). The membrane resistance was 99 GO for (B), 20 GI2 for (C) and 1.6 GO for (D), respectively.

173 chamber was 1.5 ml. The number of the holes in the Teflon film was increased up to six to obtain larger electric response. The Teflon chambers were set in a temperature controlling jacket connected to a water bath circulator. Aqueous solution in the chamber was stirred with a small magnetic stirrer. The temperature of the solution in the chambers was calibrated as a function of the temperature of circulating water in separate experiments. The short circuit electric current was measured with a patch/whole cell clamp amplifier (Type CEZ-2300, Nihon Koden) or a laboratory-made I - V converter which contained a Burr-Brown OPA104CM operational amplifier with a 10GO feedback resistor. The signals were filtered with a multifunction decade filter (E-3201A NF Electric Instruments) and distributed to a monitoring oscilloscope or a strip chart recorder. The side of the chamber connected to the amplifier was defined as trans and the other side was defined as c/s. The light for excitation of bacteriorhodopsin was generated by a 150 W slide projector lamp set outside the shield box and lead through a light guide made of poly(methyl methacrylate). The content of the Teflon chamber was perfused using a peristatic pump.

Incorporation of bacteriorhodopsin into planar bilayers Asolectin (type IV-S, Sigma) was used for the formation of the planar bilayers without further purification. Before forming planar bilayer membranes, the holes on the Teflon film were pre-treated with 0.15-0.3 /zl of a solution containing hexadecane, chloroform and methanol (1:33:66). The aqueous solution filled in the Teflon chambers contained 20 mM Tris-maleate,

100 mM NaCI, and 2 mM MgSO4 (pH 7.0). Before starting experiments, it was confirmed that the electric resistance of the planar bilayers was more than 200Gg2 per one hole by applying + 35 mV of voltage. Leaky membranes with a resistance less than 200 GO per hole were not used. Solubilized bacteriorhodopsin was incorporated into the planar bilayers as follows. An aliquot (30-60/xl) of the solubilized bacteriorhodopsin obtained as above (A570 = 0.2-1) was directly added to the aqueous phase of the c/s side of the pre-formed planar bilayer. Then it was incubated at a desired temperature for 20-40 min while stirring. After positive photoresponse was detected, the remainder of the bacteriorhodopsin was removed by perfusion and further manipulations such as repeated perfusion or cooling was carried out. Details are given in the legends. As a control, purple membrane sheets were adsorbed onto planar bilayer and photoresponse was examined. In this case, approximately 300/zl of purple membrane suspended in distilled w a t e r (A570 = approximately 3) was added to the c/s side of the aqueous phase. After 15 min of stirring, 40/.d of 1 M CaCI 2 was added and kept on stirring for another 30 min. Then, photoresponse was checked and excess purple membranes were removed by perfusion. Results

Generation of electric current by bacteriorhodopsin incorporated into planar bilayers Fig. 1A shows a typical photoelectric response of solubilized bacteriorhodopsin after incorporation into

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Fig. 2. Generation of electric current by purple membrane adsorbed planar bilayer. Six holes were made in a teflon film separating two chambers. (.4,) 300/zl of purple membrane suspended in distilled w a t e r ( A 5 7 0 = 3) was added to the c/s side of the planar bilayer and incubated for 15 min while stirring. Then 40 p,l of 1 M CaCI 2 was added and incubated for another 30 min. After excess purple membranes were removed by perfusion, the electric resistance of the membrane was 113 GO. (B-D) Effect of FCCP. FCCP dissolved in methanol was successively added to the same membrane as (A). The membrane resistance was 5.5 GO for (B), 1.4 GO for (C),and 0.5 GO for (D), respectively.

174 planar bilayers at room temperature. The result was obtained simply by adding the solubilized bacteriorhodopsin to the c/s side of the planar bilayer as described in Materials and Methods and the figure legend. The electric current corresponded to the movement of positive charges from the c/s chamber to the trans chamber. In this case, although some capacitive peak current was included, the steady-state level was unaffected by a proton conductor, FCCP (Fig. 1B-D). This means that almost all the bacteriorhodopsin molecules reconstituted by the present method penetrate the planar bilayer. As a comparison, photoresponse generated by purple membrane sheets adhering to the planar bilayer is shown in Fig. 2. Large capacitive peak current preceded the slowly decreasing steady-state current. FCCP greatly increased the steady-state level by decreasing the membrane resistance (Fig. 2B-D). The relationship between the concentration of FCCP, the membrane resistance, and the amplitude of steady-state current is shown in Fig. 3 for both directly reconstituted bacteriorhodopsin and purple membrane sheets attached onto the planar bilayer. In both cases, FCCP decreased the membrane resistance to similar extents. However, there was a significant difference in the influences on the amplitude of steady-state current between them. It is clear that in the case of purple membrane sheets attached onto the membrane, the steady-state current increased even more than 20 fold when FCCP was added to saturating level. On the other hand, in the case of directly reconstituted system, the steady-state current was almost insensitive to the decrease of membrane resistance. Sometimes, the steady-state current even slightly decreased as FCCP concentration was increased in direct incorporation. A large amount of FCCP may shunt the protonic current across the planar bilayer in this case.

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Fig. 4. Effect of temperature and perfusion on the capacitive current for direct reconstitution system. (A) Relationship between reconstitution temperature and capacitive peak height. " r " plotted in ordinate is defined as a ratio of steady-state photoelectric current to peak one indicating the contribution of bacteriorhodopsin transmembraneously incorporated into planar bilayers. (e), ratio of photoelectric current just after incubation of solubilized bacteriorhodopsin at indicated temperature. ([]), ratio after perfusion at indicated temperature with a buffer at room temperature. (o), ratio after cooling down the buffer temperature to 25°C preceded by perfusion as above. (B) A typical photoresponse of bacteriorhodopsin after reeonstitution at 45°C. Left: before perfusion; Right: after perfusion with the buffer at room temperature for 10 min. The photoelectric response was measured at 25°C. The Teflon film separating two chambers had one hole in this case.

fluidity of the membrane increases and more bacteriorhodopsin molecules can be incorporated easily. In fact, at higher temperature, somewhat more bacteriorhodopsin molecules were incorporated into the planar bilayers. However, reconstitution in high temperature range was effective to eliminate the capacitive peak rather than to increase the steady-state level when perfusion with the buffer at room temperature was followed. The results are summarized in Fig. 4. As shown by filled circles in Fig. 4A, even in the case of direct reconstitution, some peak current preceded to the steady-state current irrespective of the temperature. However, when the content of the c/s chamber was perfused with the buffer at room temperature while the circulating water around the cell was kept hot, the peak current diminished significantly as shown by the open rectangles. Even after the circulating water was cooled down to 25°C, the peak current did not recover (open circles, Fig. 4A). Thus, we could obtain

steady-state photoresponse nearly without capacitive current. A typical response is shown in Fig. 4B. The change in membrane resistance and steady-state curi

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rent before and after the perfusion is shown in Fig. 5. The membrane resistance decreased after perfusion; however, it was still more than several hundreds of GO per hole. As the steady-state current did not increase significantly after perfusion, the effect of perfusion is

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not simply to lower the membrane resistance like FCCP. The effect of FCCP after the temperature control and perfusion is shown in Fig. 6. Again, the steady-state current did not increase by the addition of FCCP while the membrane resistance decreased significantly. These results strongly indicate that bacteriorhodopsin molecules are reconstituted into planar bilayers transmembraneously. We could incorporate the bacteriorhodopsin molecules into planar bilayer membranes by this method using octyl glucoside as a detergent instead of deoxycholate. So far, using deoxycholate or octyl glucoside as the detergent, average amplitude of photocurrents is 303 fA and membrane resistance after incorporation is 400 GO (47 independent experiments). The orientation of the bacteriorhodopsin molecules incorporated into the planar bilayers was examined by using LaC13. It was reported that L a C l 3 inhibited the proton translocation by bacteriorhodopsin only from the cytoplasmic side [8]. Thus, it enabled us to inhibit the proton translocation only in one direction. One of the data of this inhibition experiment is shown in Fig. 7. The extent of inhibition by LaC13 varied from time to time, ranging from 20% to 100% when added to the c/s side. On the other hand, L a C l 3 did not affect the photocurrent at all when added to the trans side (data not shown). These results indicate that most of the bacteriorhodopsin molecules are incorporated into planar bilayers in one direction, facing its cytoplasmic side to the cis side of the planar bilayers. The reason of the variation in the extent of the inhibition is unknown. It seems to depend on the duration of incubation with LaCI 3. Longer incubation tended to give higher extent of inhibition. Braun et al. reported that phosphatidyl serine is necessary for the inhibition by LaCI 3 [27], but we could not improve the data even with phosphatidyl serine in the present study. Discussion

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Since the work by Drachev et al. [8], most of the ion pumps reconstituted in planar bilayers did not penetrate the membranes, but capacitively coupled as modeled in Fig. 8A and B [8,12,24]. In this model, an ion pump is approximated by a combination of a battery (Ex), internal resistance (Rx), and a switch (SW). Energization of the ion pump is assumed to correspond to close of the switch for simplicity *. On the basis of this model, the capacitive current is explained as the charging current of the membrane capacitance (C m) and increase of steady-state current by ionophores is explained as a consequence of the decrease of membrane * This is of course a very rough approximation, sufficient only for the present discussion. Our ultimate purpose should be to describe the structure and function of ion pumps at molecular level.

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resistance (Rm). In such a system, as has been already discussed by others [12,24], the apparent reversal potential is an overestimation of the E x. In addition, when the ion translocation is coupled with an enzymatic reaction, the apparent Michaelis constant (K m) obtained from electrical measurement becomes a severe underestimation (see Appendix for details). These drawbacks cannot be relieved even with ionophores, since it decreases the resistance of attached proteoliposomes or membrane sheets (Rf) as well as that of planar bilayer (Rm). Moreover, we cannot obtain the relationship between R m and Rf. Therefore, a method of transmembraneous reconstitution has been desired for quantitative steady-state analyses. The reason that transmembraneous reconstitution could not be achieved so far was that ion pumps in vesicles or large membrane sheets were used as starting materials for reconstitution into planar bilayers. With the use of such large membrane structures, even if some ion pumps could penetrate the membrane, others might remain attached capacitively. On the other hand, when

solubilized ion pump monomers are used for reconstitution, there is essentially no possibility to produce membrane structures capacitively coupled with planar bilayers and transmembraneous reconstitution can be expected. In fact, some ion channels have been reconstituted into planar bilayers directly after solubilization and this is the same principle of detergent dilution method for making proteoliposomes. However, as for ion pumps and planar bilayer system, no attempts in such a way have been reported. Here, we tried this direct reconstitution by solubilization and incubation for bacteriorhodopsin and we could detect distinct photoelectric responses. As we do not know the real relationship between the ion concentration gradient and ionic current, and we cannot directly see the membrane protein embedded in artificial planar bilayer system, the best criteria for transmembraneous reconstitution are: (1) absence of the effects of ionophores on steady-state short circuit current; (2) absence of the capacitive current upon energization. Although there have been many reports which insisted

178 transmembraneous reconstitution of ion pumps [26-29], these criteria have never been examined in any particular case. In the present study, as shown in Fig. 1, FCCP hardly affected the steady-state current. The peak current observed in Fig. 1 may have resulted from some aggregates of bacteriorhodopsin monomers or from some reason other than capacitive coupling of ion pumps and membrane. Another point shown in the present study is an effect of temperature and perfusion. By incubation at high temperature followed by perfusion, the peak current was largely eliminated (Fig. 4) without affecting the FCCP insensitivity (Fig. 6). These features are sufficient for the criteria above and strongly indicate transmembraneous incorporation of bacteriorhodopsin molecules. One disadvantage of the present method may be relatively small amplitude of the steady-state current. However, by the conventional methods, in which FCCP greatly increases steady-state current, we cannot completely exclude the contribution of capacitively coupled ion pumps to the steady-state current through some permeability at planar bilayer-adsorbed membrane junction. In this case, some ambiguity remains as for quantitative analyses on steady-state activity, even if large electric current is obtained. On the other hand, the present method provides steady-state electric current almost insensitive to FCCP. This is an essential advantage of the present method over other methods and will prove useful in quantitative analyses of active transport. The amplitude of the electric current can be increased by making more holes in the Teflon film separating the chambers. Precise measurement of current-voltage relationship, effect of ionic concentration gradient, as well as application of this method to other ion pumps are now in progress.

The apparent reversal potential where energization causes no change in electric c u r r e n t (Eext) can be derived as Eqn. 3, which is larger than the real electromotive force of the ion pump (Ex). This conclusion is the same as previously described by others [12,24].

(Re,)

Eext = 1 +

When the ion translocation is coupled with enzymatic reaction, the number of activated ion pumps (n) is a function of substrate concentration (S). Assuming that the enzymatic reaction follows Michaelis-Menten kinetics, the number, n, can be described as Eqn. 4. S n=a - -

(4)

Km+S

Here, a is a proportional coefficient. When substrate concentration, S, increases, the number of activated ion pumps increases as Eqn. 4. The increase of number n means the decrease of R x by 1/n fold. Thus the intensity of the short circuit electric current when n ion pumps are activated (I n) at a substrate concentration of S can be described as Eqn. 5. In Eqn. 5, it was assumed that the g m value does not depend on the electric potential. As the real K m is multiplied by a factor in [] in the dominator in Eqn. 5 and a is significantly greater than 1 (actually, it roughly corresponds to the number of ion pumps in one adsorbed membrane fragment), an apparent K m value obtained from a measurement of short circuit current would be a significant underestimation of the real K m. aEx'Rf'S Rx(R f + R m)+ aRf'R m ] In =

Appendix

[

Rx(Rf + Rm)

Rx(Rf aEx'Rf IS + R m) + aRf'R m

Km

Ill l+a/_l_l

1

~ Rm q- R f )

(1)

Here we ignored the resistance, R, as it is much smaller than the other resistances. Energization of an ion pump changes the electric current as follows (Eqn. 2). I=

Ee~t[Rx(R m + Rf + R~n) + Rf(R m + R~)] - Ex'R f "R'm Rx[R'(R m + Rf)]+ R " R m ' R f

]

Km[ Rx(~'-~-~-mT~aRf.R m + S

When the conventional membrane fragment adsorbed planar membrane system can be modeled as Fig. 8A and B, the electric current without energization of ion pumps (which is modeled as an open state of the switch SW) is expressed as Eqn. 1. I = Eext ( R ,~---~RRmf+~f--~)

(3)

"E x > E x

(2)

-'

1

(5) +S

'Rx

Previously, we reported the K m of proton translocation by thermophilic FoF~-ATPase obtained from electric measurement [30]. The value agreed well with the K m of ATPase reaction measured in a separate experiment. That means the electric current observed in the previous study was generated mostly by enzymes penetrating the planar bilayer although we cannot completely exclude the possibility that some proteoliposomes still remained adsorbed onto the planar bilayer.

179 The drawbacks in estimating the reversal potential cannot be relieved by addition of ionophores, since they decrease the resistance of the adsorbed membrane fragment (Rf) as well as the resistance of membrane junction ( R m ) . In the case that the K m value obtained from electric measurements is much smaller than that obtained from ATPase reaction, a possibility of error described above should be considered. When the transmembraneously reconstituted ion pump-planar membrane system is modeled as Fig. 8C and D, the apparent reversal potential where energization causes no change in electric c u r r e n t (Eex t) is equal to the real electromotive force of the ion pump (Ex). When the ion translocation is coupled with enzymatic reaction, the magnitude of the short circuit electric current when n ion pumps are activated (I n) at a substrate concentration of S can be described as Eqn. 6.

In

±Is RxJ Km + S

(6)

In this case, the apparent K m value obtained from short circuit electric current measurement will be the same as that of coupled enzymatic reaction. These discussions on the differences between the conventional membrane fragment adsorbed system and transmembraneous reconstitution system hold for the differences between vesicle adsorbed system and transmembraneous reconstitution system. The discussion on the estimation of apparent K m value is also valid for the light intensity dependency of photoelectric current.

Acknowledgement The financial support to E.M. by the Casio Science Promotion Foundation and the Kato Memorial Bioscience Foundation is gratefully acknowledged.

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