1 Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido 0600812 and 2 Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 1848588, Japan
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Abstract |
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Keywords: activation energy/bacteriorhodopsin/kinetics/light-induced denaturation/octyl-ß-glucoside/proteinprotein interaction/solubilization
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Introduction |
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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, 1982; London and Khorana, 1982
; Oesterhelt, 1982
). 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., 1981
; London and Khorana, 1982
; Popot et al., 1987
). 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., 1988, 1995
). 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., 1988
, 1995
). 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., 1995
).
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., 1997; Lanyi, 1997
). 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., 1997
; Lanyi, 1997
). 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 proteinprotein 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., 1997). 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 proteinprotein 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 proteinprotein 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 proteinprotein interaction is lost.
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Materials and methods |
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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., 1997). 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., 1997). 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 3048°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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Results |
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Discussion |
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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, 1980). 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 proteinprotein 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 proteinprotein interaction may be important.
Previously, we have found that in the temperature range 8086°C, which is just above the melting-point of the 2D crystalline structure (Jackson and Sturtevant, 1978; Hiraki et al., 1981
; Kahn et al., 1992
), denaturation of bR occurred in the dark and furthermore under visible light irradiation the speed of denaturation was increased (Etoh et al., 1997
). 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 proteinprotein interaction was lost.
Therefore, the stabilizing effect of proteinprotein interaction on the solubilized bR molecules and its photointermediates is the most plausible mechanism. As shown in Figure 4, 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:
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The total denaturation rate constant k3 can be described by two terms:
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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 proteinprotein 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 proteinprotein interaction was essential for the stability of bR and the importance of the interaction would be applicable to many other membrane proteins.
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Acknowledgments |
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Notes |
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References |
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Received April 5, 1999; revised May 31, 1999; accepted June 8, 1999.