Light-induced denaturation of bacteriorhodopsin solubilized by octyl-ß-glucoside

Yuri Mukai1,3, Naoki Kamo1 and Shigeki Mitaku2

1 Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060–0812 and 2 Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184–8588, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
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The structural stability of bacteriorhodopsin (bR) solubilized by octyl-ß-glucoside was studied by measuring the denaturation kinetics under visible light irradiation and in the dark. The denaturation of bR solubilized by 50 mM octyl-ß-glucoside was very slow at room temperature when it was left in the dark. However, its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation and in the dark could be well described by a single decay constant. The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light irradiation was 12.5 kcal/mol, which was much smaller than the corresponding value in the dark, 26.2 kcal/mol. These results strongly suggest that some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D crystalline state. Comparing the denaturation behavior in the 2D crystalline state and that in the octyl-ß-glucoside-solubilized state, our findings suggest that protein–protein interaction contributes to the stability of this protein.

Keywords: activation energy/bacteriorhodopsin/kinetics/light-induced denaturation/octyl-ß-glucoside/protein–protein interaction/solubilization


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteriorhodopsin (bR) is a typical intrinsic membrane protein with seven transmembrane helices, whose biological function is a light-driven proton pump (Bogomolni et al., 1976Go). BR has several advantages over other membrane proteins for studying the physical mechanisms responsible for the structural stability of a membrane protein: (1) the 3D structure of bR has already been determined at high resolution, with reference to which various factors contributing to the stability of this protein may be discussed in terms of local interactions within the protein structure (Henderson et al., 1990Go; Grigorieff et al., 1996Go; Kimura et al., 1997Go; Heberle et al., 1998Go; Luecke et al., 1998Go); (2) bR is a small membrane protein with no S–S bonds and a single molecule of bR performs its full function (Khorana et al., 1979Go; Kouyama et al., 1988Go), therefore, it is the simplest of the membrane proteins already characterized in terms of 3D structure; (3) most parts of bR are buried in the membrane and the contribution of soluble domains to the stability of the molecular structure is considered to be small (Ohno et al., 1977Go; Fukuda and Kouyama, 1992Go).

In fact, hypotheses concerning the mechanisms of structure formation and the functional mechanisms of bR have been proposed based on the results of regeneration, denaturation and structural analyses (Heyn and Dencher, 1982Go; London and Khorana, 1982Go; Oesterhelt, 1982Go). Regeneration of bR from its fragments in the ground state showed that the tertiary structure of bR is established by interactions between transmembrane helices which do not involve bond formation (Huang et al., 1981Go; London and Khorana, 1982Go; Popot et al., 1987Go). Engelman et al. (1990) proposed a two-stage mechanism of structure formation based on the observation that the 3D structure of bR is spontaneously re-established from preformed transmembrane fragments in the membrane.

Alcohol denaturation experiments revealed that a C-terminal segment in addition to retinal serves to stabilize the structure of bR to some extent (Mitaku et al., 1988Go, 1995Go). Moreover, it was also shown that the native structure of bR was preserved even when purple membrane was suspended in pure hexane, but complete denaturation occurred upon addition of alcohol. These observations strongly suggested that polar interactions stabilize inter-helix binding, which may be disrupted by the hydroxyl group of an alcohol (Mitaku et al., 1988Go, 1995Go). This has been successfully incorporated into a method for the determination of the positioning and orientation of transmembrane helices in the bR molecule (Suwa et al., 1995Go).

Recent structural analyses have led to the conclusion that bR undergoes a conformational switch between closed and open states through its photocycle (Kamikubo et al., 1997Go; Lanyi, 1997Go). This conformational change in bR also provides information about the mechanism of structure formation. The photocycle is coupled with changes in the electrical state of essential residues, Asp85, Asp96 and Lys216, due to protonation and deprotonation. The position and tilting of helix F have been shown to change in the photointermediate state M in which all of the essential residues are neutralized (Brown et al., 1997Go; Lanyi, 1997Go). It seems reasonable to assume that the variation of charge distribution affects the electrostatic contribution to the binding between transmembrane helices, leading to a change in protein stability. However, the photointermediate states have not been studied from the viewpoint of structural stability as yet, which is closely coupled to its biological function.

In this work, we studied the light-induced denaturation of bR which had been solubilized by octyl-ß-glucoside, focusing on two questions concerning the structural stability of this protein. The first question was whether bR in the membrane is stabilized by protein–protein interaction. Previously, we have found that bR in purple membrane shows light-induced denaturation at temperatures above 80°C, at which the 2D crystalline lattice melts (Etoh et al., 1997Go). Comparison of the denaturation behavior of bR in purple membrane and that of bR in the solubilized state in this work is expected to provide information on the role of protein–protein interaction in its stabilization in the membrane. The second question concerns the changes in stability associated with the conformational changes that occur through the photocycle. Although the stability of bR in functional intermediate states cannot be measured in equilibrium, the phenomenon of light-induced denaturation indicates that there is a decrease in the stability of bR in some of its photointermediate states.

The results obtained in this work show that denaturation of solubilized bR occurs even at temperatures around room temperature, which is much lower than the temperature (about 80°C) required to induce denaturation of bR in purple membrane. This change in denaturation temperature together with the large decrease in activation energy upon solubilization of bR is considered to be partly due to the lack of protein–protein interaction in the solubilized state. Furthermore, the critical temperature and the activation energy for denaturation were much lower under visible light irradiation than in the dark, indicating that bR is destabilized in the course of the photocycle under conditions where protein–protein interaction is lost.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Purple membrane of Halobacterium salinarium, strain R1M1, was prepared according to the method established by Oesterhelt and Stoeckenius (1974). The purple membrane was suspended in 10 mM Tris–HCl buffer (pH 7.0).

Octyl-ß-glucoside (50 mM), purchased from Wako Chemical, was added to the suspension of purple membrane. The octyl-ß-glucoside concentration was set based on the results of a preliminary experiment examining the dependence of bR solubilization on detergent concentration (Mukai et al., 1997Go). After incubation for 2 h at 37°C, the sample was centrifuged at 105 000 g for 60 min at 4°C. The absorption spectra of the supernatant after the centrifugation showed the absorption peak at 560 nm corresponding to the native state bR. The circular dichroism (CD) spectra of the supernatant did not show the exciton coupling. These results indicated that the sample was solubilized bR judging from the two criteria for the solubilization of purple membrane by Dencher and Heyn (1982): (1) failure of sedimentation by the ultracentrifugation and (2) disappearance of the exciton coupling. The concentration of the solubilized bR was ~2.5 µM.

Denaturation was monitored using a diode-array spectrophotometer (HP8452A, Hewlett-Packard) with a xenon lamp, as reported previously (Etoh et al., 1997Go). The temperature of the cell holder was controlled by a micro-cooling circulator (RTE-8, Neslab). In experiments examining the light-induced denaturation kinetics in the temperature range 30–48°C, the absorption spectra were successively measured at intervals of 150 s for about 1 h after the start of visible light irradiation. Actinic constant illumination was provided through a sharp cut-off filter (>520 nm) to excite bR in the ground state. We also examined the denaturation kinetics of the same sample in the dark as a control.


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Figure 1Go shows the absorption spectra of solubilized bR at 37°C under visible light irradiation (A) and in the dark (B). The spectra were successively measured at 150 s intervals. As shown in Figure 1AGo, under irradiation, the absorption peak at 560 nm decreased monotonically with time and a concomitant increase in a peak at about 380 nm was observed. After 1 h, under the irradiation, the absorption at 560 nm corresponding to the native state completely disappeared and this peak did not reappear upon subsequent incubation at 4°C in the dark for 24 h, suggesting that the change in the absorption spectra was due to the denaturation of bR. Because the absorption band at 560 nm was not observed even when excess retinal was added to the sample, the spectral change must be due to irreversible denaturation of this protein. On the other hand, a very small change in the absorption spectra was observed under the same conditions in the dark. Although the small decrease in the spectral band at 560 nm did not recover after incubation at 4°C for 24 h, the effect of visible light irradiation was significant, clearly indicating that it induced the denaturation of octyl-ß-glucoside-solubilized bR.



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Fig. 1. Absorption spectra of octyl-ß-glucoside-solubilized bacteriorhodopsin at 37°C (A) under visible light irradiation and (B) in the dark. Conditions: pH 7.0, bR 2.5 µM, octyl-ß-glucoside 50 mM.

 
Figure 2AGo and B show the decay curves of the absorbance at 560 nm of solubilized bR in the temperature range 30–48°C under irradiation and in the dark, respectively. Each decay curve was analyzed and a single exponential decay time constant was determined, neglecting the deviation from the single exponential curve in the dead time of several minutes. The denaturation became faster with increasing temperature. The decay time constant under irradiation was much larger than in the dark.



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Fig. 2. Denaturation kinetics of octyl-ß-glucoside-solubilized bacteriorhodopsin in the temperature range 30–48°C (A) under visible light irradiation and (B) in the dark (from the top: 30, 33, 37, 41, 45, 48°C). Conditions: pH 7.0, bR 2.5 mM, octyl-ß-glucoside 50 mM.

 
Figure 3Go shows Arrhenius plots of the decay time constants for denaturation under irradiation and in the dark. The decay time constants in the temperature range 30–48°C were linearly related to the inverse of temperature. The activation energy was estimated to be 12.5 kcal/mol under irradiation and 26.2 kcal/mol in the dark. Thus, considering that the decay constant was several tens of times larger under visible light irradiation and also considering that the activation energy for denaturation was doubled under such conditions, it was concluded that the bR molecule solubilized by octyl-ß-glucoside was more stable in the dark than under visible light irradiation.



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Fig. 3. Arrhenius plot of the kinetics of light-induced denaturation of octyl-ß-glucoside-solubilized bacteriorhodopsin in the temperature range 30–48°C under visible light irradiation and in the dark.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The main findings of the present study can be summarized as follows: (1) the stability of bR became lower when it was solubilized by octyl-ß-glucoside; and (2) solubilized bR showed light-induced denaturation. These findings may be discussed from two aspects: the importance of protein–protein interaction and the low stability of a photointermediate.

There are two possible factors responsible for the low stability of bR solubilized by octyl-ß-glucoside. Octyl-ß-glucoside is well known as a mild detergent often used for solubilization of membrane proteins (Casadio and Stoeckenius, 1980Go). The hydrophobic regions of transmembrane helices in a membrane protein are modified in the course of the evolutionary process to minimize the structural energy in the environment of a lipid membrane. Because detergent micelles provide a hydrophobic environment very similar to that of a lipid bilayer, the molecular structure of a membrane protein will be preserved in the micelle environment. Nevertheless even a mild detergent such as octyl-ß-glucoside may have some destabilizing effect on a membrane protein. Therefore, the low stability may partly be due to the destabilizing effect of octyl-ß-glucoside as a detergent. The other factor is protein–protein interaction in the native purple membrane, as the 2D crystalline array of bR is lost in the present solubilized sample. In general, a crystalline lattice structure is formed by some force of attraction between molecules which confines a molecule at a lattice point. In the case of bR, the force of attraction may reduce structural fluctuations. Although it is difficult to separate quantitatively the contributions of these two factors to the stability of bR in the 2D crystalline state, we propose that protein–protein interaction may be important.

Previously, we have found that in the temperature range 80–86°C, which is just above the melting-point of the 2D crystalline structure (Jackson and Sturtevant, 1978Go; Hiraki et al., 1981Go; Kahn et al., 1992Go), denaturation of bR occurred in the dark and furthermore under visible light irradiation the speed of denaturation was increased (Etoh et al., 1997Go). It should be noted that in the case of native purple membrane in which bR forms a 2D crystalline structure, neither denaturation in the dark nor denaturation under irradiation was observed. In the present work, similar phenomena were observed using a solubilized bR sample in which protein–protein interaction was lost.

Therefore, the stabilizing effect of protein–protein interaction on the solubilized bR molecules and its photointermediates is the most plausible mechanism. As shown in Figure 4Go, the results of the present study imply that there are two pathways to the denatured state: one is direct denaturation of bR in the ground state in the dark, the decay time constant being designated k1 and the other is denaturation of a photointermediate(s) under irradiation with the decay time constant k2. Under irradiation, not all bR molecules are converted to the intermediates. The time derivative of the concentration of the photointermediate is zero at the photo steady state:

where {alpha}, ß and I are the rate constant of its formation, the decay constant and the light intensity, respectively. The total bR concentration is the sum of the concentration of the ground state bR and of the intermediate(s) PIs:

Consequently, the concentration of the photointermediate(s) is given by



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Fig. 4. Two pathways to the denatured state of octyl-ß-glucoside-solubilized bacteriorhodopsin.

 

The total denaturation rate constant k3 can be described by two terms:


Accordingly,


in which the decay constant k1 in the dark is much smaller than the decay constant k2 under visible light irradiation. Then, our data can be interpreted in terms of Equation 5. Under physiological conditions, bR in the 2D crystalline state is very stable, i.e. k1 = k2 = 0. However, octyl-ß-glucoside-solubilized bR showed the denaturation phenomenon at room temperature, indicating that the decay constants k1 and k2 are finite under those conditions. When k1 << k2, the total denaturation constant is enhanced by the visible light irradiation, which is the case for the solubilized bR. If the onset temperature of denaturation is different between the ground state and photointermediate states, we will have real light-induced denaturation phenomenon, in which bR is stable in the dark (k1 = 0) and denatures under the light irradiation (k2 != 0). Although we have not found such conditions yet, the results indicated that the k2/k1 ratio is much larger than 10.

The remaining question is which photointermediate state is responsible for the enhancement of denaturation under irradiation. Although the most unstable photointermediate has not been identified, we may infer it from the available structural information showing differences in stability among various photointermediates. According to the results of recent structural analyses, bR undergoes structural changes through the photocycle. The structure of the M photointermediate is significantly different from that of bR in the ground state: some of the seven transmembrane helices change position or tilting in the M intermediate. This state has remarkable features also from the viewpoint of physical interactions within the bR molecule. The amino acid residues Asp85, Asp96 and Lys216, which are essential for the light-induced proton pumping function, do not have any charge in this state. It has been established that this change in electrical state is important for the proton pumping function, but the disappearance of charges in the M intermediate should also affect the stability of the molecule, decreasing the electrostatic interaction between helices. Therefore, the most plausible candidate for the denaturation path may be the M photointermediate. A mutant of bR in which Asp96 is replaced by Asn (D96N mutant) has a much longer lifetime than wild-type bR. Experiments on photo-induced denaturation of this mutant may serve to clarify whether the M intermediate plays a key role in the pathway to the denatured state. Such studies are now in progress in our laboratory.

It is concluded that intermolecular interactions are responsible for the high stability of bR in purple membrane. The protein–protein interaction plays an important role not only in the ground state but also in the photointermediate. We should point out that the solubilized sample employed has provided much information on the mechanism of structure formation, although the interaction between detergents and proteins must be considered. It was revealed that protein–protein interaction was essential for the stability of bR and the importance of the interaction would be applicable to many other membrane proteins.


    Acknowledgments
 
The authors thank Dr Masashi Sonoyama (Tokyo University of Agriculture and Technology) for useful discussions. This work was partly supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture (Monbusho).


    Notes
 
3 To whom correspondence should be addressed Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Received April 5, 1999; revised May 31, 1999; accepted June 8, 1999.