Báo cáo khoa học: Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity An infrared spectroscopic study docx

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity An infrared spectroscopic study Richard W. Hendler 1 , Steven M. Barnett 2 , Swetlana Dracheva 1 , Salil Bose 1 and Ira W. Levin 2 1 Laboratory of Cell Biology, National Heart, Lung, and Blood Institute and 2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0510, USA Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow) 66, 1623–1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic envi- ronment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane a-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton- treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein a-helical amide I mode at 1660 cm )1 , reflecting the motional char- acteristics of the transmembrane helices, and the lipid- induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photo- cycle M-state intermediates. Keywords: enzyme control; kinetics; lipid–protein inter- actions; membrane protein structure. Previous studies, summarized in [1], demonstrate the importance of specific membrane lipids and amino-acid residues in the cytoplasmic loop regions of bacteriorhodo- psin for the normal operation of the bacteriorhodopsin photocyle. Specifically, the extensive damage to the normal photocycle caused by brief exposure of purple membrane to dilute Triton X-100 is repaired completely by the addition of squalene and phosphatidylglycerophosphate methyl ester lipids extracted from purple membrane [2]. This reconstitu- tion requires charge-screening by either high-salt concen- trations or titration of a group with an apparent pK of 5 [3,4]. Although phosphatidylglycerophosphate methyl ester alone completely restores the M-slow (M s ) fi BR photo- cycle pathway, squalene is required to re-establish the M-fast (M f ) fi O fi BR pathway [2,5]. The pK  5 titration implicates the involvement of peripheral acidic amino acids of bacteriorhodopsin near the membrane surface, namely, Asp36, Asp38, Asp102, and Asp104 within the cytoplasmic loop region (R. W. Hendler & S. Bose, unpublished work). These observations indicate that M f activity requires the site of the trimers to be in a membrane region containing squalene, the most hydrophobic lipid in the purple membrane, in close proximity to the four aspartates. However, trimers located in a membrane region containing polar lipid in the absence of squalene produce M s activity. This heterogeneous distribution of lipids within the membrane results in the formation of microdomains. As the only difference between M f -andM s -eliciting trimers is the presence of a hydrophobic environment for the charged acidic amino acids, M s photocycles can be converted into M f photocycles by providing a hydrophobic environment (R. W. Hendler & S. Bose, unpublished results). On the basis of the above considerations, we proposed a mechanism for the control of bacteriorhodopsin photo- cycles through interactions involving squalene, charged lipids, and the four acidic amino acids in the cytoplasmic loop region (R. W. Hendler & S. Bose, unpublished results). Thus, in the absence of squalene, electrostatic repulsive forces at the negatively charged membrane surface under the loop region containing the charged acidic amino acids should produce a strain limiting the mobility of both the amino-acid-containing loops and the attached transmem- brane a-helices. These interactions would then lead to the Correspondence to I. W. Levin, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0510, USA or R. W. Hendler, Laboratory of Cell Biology, National Heart, Lung, and Blood Institute. Abbreviations: BR, ground state of bacteriorhodopsin; M f , M-fast intermediate which decays through the O intermediate; M s , M-slow intermediate which decays directly to bacteriorhodopsin. (Received 13 September 2002, revised 20 January 2003, accepted 22 January 2003) Eur. J. Biochem. 270, 1920–1925 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03547.x slower kinetic forms characteristic of the M state turnover. To examine further these relationships, we investigated the effects of purple membrane lipids on both the M turnover time constants and the flexibility of the bacteriorhodopsin transmembrane a-helices. Materials and methods Purple membranes were extracted from the ET1001 strain of Halobacterium salinarum as described previously [3]. Then 200 lL 1% Triton X-100 was added to a mixture of 100 lL purple membranes (5 mg bacteriorhodopsin per mL suspension) and 1700 lL50m M potassium phosphate (pH 7.2). The suspension was immediately centrifuged at 4 °C in a Beckman TL-100 centrifuge at 200 000 g (Triton exposure time  7 min), and the pellet was washed three times by resuspension in 3 mL water and centrifugation. Purple membrane lipids were extracted as described previ- ously [6,7] and resuspended to a stock concentration of 4mgÆmL )1 . Reconstitution with lipid was performed by mixing 10 mol of previously extracted native purple mem- brane lipid per mol bacteriorhodopsin in the presence of 0–4 M NaCl [3]. This is the lipid concentration present in native purple membrane. As described previously [3], salt was removed from the reconstituted preparation by succes- sive centrifugations in dilute buffer. Determinations of the wavelength of maximum absorbance were performed on a Cary 14DS spectrophotometer. Kinetic bacteriorhodopsin photocycle data, after an actinic light flash, were obtained and analyzed as previously described [8,9]. Infrared spectroscopic measurements were obtained throughfilmscastat35°ContoaBaF 2 window from 75 lL purple membrane suspension (5 mgÆmL )1 ). Meas- urements were performed at 0.5 cm )1 resolution on a Bomem DA3 spectrometer equipped with a mercury cadmium telluride detector under either vacuum or a nitrogen purge; spectra were similar for both instrumental conditions. Neutron scattering studies have demonstrated that the average mean square displacements of molecular vibrational modes in partially dried purple membrane films are unchanged from fully hydrated systems [10]. Direct measurements [11] for the number of water molecules per bacteriorhodopsin molecule in our preparations yielded values close to 300, which is close to the value of 340 determined by neutron diffraction analysis [10]. While other low-frequency, anharmonic large-amplitude membrane motions have been observed to precede the protein conformational changes during the photocycle [12], the behavior of various internal modes, such as the amide I and amide II vibrations, provides a direct indication of the dynamic properties of the transmembrane a-helices within the bilayer assembly, as we have observed in variable- temperature infrared-spectroscopic studies (unpublished work). Vibrational spectroscopic bandwidths are functions of dynamic parameters derived from intermolecular and intramolecular motions. Band shapes are often analyzed in the context of statistical mechanical theories of irreversible processes. Interactive forces between the system and its surrounding medium influence the vibrational relaxations of the molecular assemblies under consideration. The duration of the re-equilibration processes that define the lifetimes of the upper or excited vibrational levels leads to increments in the observed bandwidths. When molecules absorb radi- ation, band broadening occurs from the small differences in the environment that the molecular assembly encounters as a consequence of its mobility; that is, the system experiences inhomogeneous broadening effects. Additional discussion of band profiles and reorientation effects can be found in references [13–15]. Spectral curve-fitting procedures Subtle protein motional changes, reflected specifically by perturbations in the amide I spectral region, are most easily and systematically monitored through curve-fitting methods applied to the 1720–1480 cm )1 spectral interval, the region comprised primarily of the protein amide I and II vibrational modes. Curve fitting of the infrared spectra of perturbed purple membrane assemblies was performed with a Bomem Grams/386. Briefly, the amide I and II envelope of the infrared spectrum of purple membrane assemblies was represented by seven curves initially located at 1660 cm )1 (representing the amide I modes of the a-helices), 1680 cm )1 (the amide I of b-turn structures), 1640 cm )1 (the amide I of random coil and b-sheet structures), 1545 cm )1 (the amide II of a-helical A mode), and 1520 cm )1 (the amide II of a- helical E 1 mode), with two smaller features at 1620 and 1585 cm )1 . For all spectra fitted in this manner, the correlation coefficient was greater than 0.99, with the residuals being equivalent to the noise, indicating that these seven curves provide an excellent approximation to the data. Results Effect of NaCl concentration on the reconstitution of native purple membranes The extent of normal bacteriorhodopsin photocycle activity, generated in Triton-treated purple membranes by reconsti- tution with native phytanyl chain lipids, is dependent on NaCl concentration [3]. As infrared spectroscopy provides an effective approach for detailing changes in integral membrane protein structure [16,17] in both native and perturbed purple membrane systems [18], we examined the vibrational spectra of Triton-treated purple membranes reconstituted in various concentrations of NaCl (0–4 M )to elucidate more specifically the protein structural changes that correlate with the recovery of native bacteriorhodopsin photocycle activity. The effect of NaCl concentration on structural reorgani- zations in the bacteriorhodopsin protein on reconstitution of Triton-treated purple membrane with native lipids was monitored through changes in infrared spectra in the amide I and II regions at  1660 cm )1 and  1545 cm )1 , respectively. Figure 1 displays the infrared spectra from 1710 to 1490 cm )1 (normalized to the intensity of the amide I mode at 1660 cm )1 ) of native purple membrane (solid line), purple membrane after mild exposure to Triton (0.1% Triton, 7 min; dashed line), and Triton-treated purple membrane reconstituted in the absence of NaCl (dotted line) and with 2 M NaCl in phosphate buffer (dash- dot line). The decrease in half-width of the amide I mode at 1660 cm )1 after exposure to Triton suggests decreased a-helical conformational flexibility (bacteriorhodopsin is Ó FEBS 2003 Control of bacteriorhodopsin conformational flexibility and photocycle activity (Eur. J. Biochem. 270)1921 composed of  65% a-helical structure [18]); that is, the amide I mode monitors primarily the dynamics of the protein’s transmembrane segments in contrast with the loop regions. Increases in the width of the amide I mode are observed in variable-temperature infrared spectroscopic studies of the purple membrane system (unpublished observations). In these studies, however, a decrease in peak width of the amide I vibrational mode is accompanied by a decrease in the intensity and bandwidth of the amide II mode on the release of retinal induced by either heat or light (S. Barnett & I. W. Levin, unpublished work). These small decreases in the amide I and amide II peak parameters were observed in the present study and in previous communica- tions [19,20] on lipid reconstitution in the absence of NaCl (Fig. 1, dotted line). Recovery of these parameters to levels near those observed in native purple membrane are now observed on reconstitution in 2 M NaCl (Fig. 1, dash-dot line). Examination of the lineshape features of an infrared- active spectroscopic feature, such as the peak heights and bandwidths, provides insights into the molecular dynamics of the ensemble [21]. In particular, to define more explicitly the structural alterations in bacteriorhodopsin after expo- sure to Triton and subsequent lipid reconstitution, the amide I and II regions of the infrared spectra in both the native and perturbed purple membrane systems were fitted to seven mixed Gaussian–Lorentian functions. Figure 2 displays the infrared spectrum of native purple membrane from 1720 to 1480 cm )1 (top curve) fitted to the seven deconvoluted curves. The amide I region is composed of a predominant feature centered at 1660.3 cm )1 with a half- width (Dm 1/2 )of30.9±0.5cm )1 (mean ± SEM from at least eight independent measurements used on all native and treated purple membrane preparations), assigned to the a-helices of bacteriorhodopsin, as well as, in part, curves typical of b-turn (1685 cm )1 ) and either random coil or b-sheet (1638 cm )1 ) structures. The frequencies and relative intensities of the spectroscopic features that comprise the amide I region predict that bacteriorhodopsin is composed of  65% a-helical structure, in agreement with previous infrared spectroscopic studies of bacteriorhodopsin secon- dary structure [18]; the curves displayed in Fig. 2 represent the only combination that provided an a-helical composi- tion of greater than 50%. Table 1 lists the deconvoluted full width at half heights of the a-helical amide I mode at Fig. 1. Infrared spectra from 1710 to 1490 cm -1 of native purple membrane (solid line), purple membrane exposed briefly to Triton (dashed line), and purple membrane reconstituted with purple membrane lipids in solutions without NaCl (dotted line) and with 2 M (dot-dash line) NaCl. Fig. 2. Infrared spectrum of native purple membrane from 1720 to 1480 cm -1 (top curve) and the seven mixed Gaussian–Lorentzian curves used to fit this spectral region. Table 1. Full width at half height of the a-helical amide I modes at 1660 cm -1 (Dm 1/2 ; obtained from deconvoluted spectra; ± 0.5 cm -1 )for different lipid conditions. Conditions Dm 1/2 (cm )1 ) Native 30.9 Triton-exposed 28.5 Reconstituted in absence of NaCl 29.0 Reconstituted in 1 M NaCl 30.4 Reconstituted in 2 M NaCl 31.0 Reconstituted in 4 M NaCl 31.2 1922 R. W. Hendler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 1660 cm )1 (Dm 1/2 ) for all samples in this study. We emphasize the use of this parameter as a measure of bacteriorhodopsin a-helical conformational flexibility, because the 1660 cm )1 feature arises predominantly from a-helical structures [18]. The experimentally observed half-width of the entire envelope comprising the amide I modes in the infrared spectra of the purple membrane decreases  9% (from 48.4 to 44 cm )1 ) on brief exposure to Triton [19]. The curve- fitting procedure used here permits a more accurate evaluation of the specific structural elements affected by Triton exposure. A decrease in intensity of the features corresponding to the b-turn (1685 cm )1 ) and random coil/ b-sheet (1638 cm )1 )structuresisaccompaniedbyan8% decrease in Dm 1/2 (from 30.9 to 28.5 cm )1 ;±0.5cm )1 )on exposure to Triton. The recovery of Dm 1/2 to values observed in native purple membrane on lipid reconstitution into Triton-treated purple membrane occurs as a function of NaCl concentration used during the procedure. Figure 3 displays a plot of Dm 1/2 as a function of the NaCl concentrations used for reconstitution. Reconstitution of Triton-exposed membranes with purple membrane lipids shows a strong dependence on the concentration of NaCl such that, at the highest concentration, the half-width parameter was restored to a value close to that found in native purple membranes, accompanied by a recovery in the b-turn and random coil/b-sheet regions. The observed decrease in Dm 1/2 on exposure to Triton [19], and its recovery on lipid reconstitution in high-saline medium (Fig. 3 and Table 1) presents an opportunity to correlate the structural features altered on lipid perturbation with bacteriorhodop- sin photocycle activity after exposure to Triton. Correlations between the recovery of bacteriorhodopsin photocycle parameters and Dm 1/2 Infrared spectra and bacteriorhodopsin kinetic data were obtained on samples immediately after lipid reconstitution and removal of salt. Correlations between lipid-sensitive bacteriorhodopsin photocycle parameters and Dm 1/2 were performed after reconstitution in the presence of up to 4 M NaCl. Specific parameters describing bacteriorhodopsin structure and photocycle behavior, as noted below, correlate well (compare Figures 3–5) with the recovery of Dm 1/2 in the infrared spectra of reconstituted purple membrane assemblies; other parameters (see below) displayed little or no correlation. The wavelength of maximum absorbance (k max )of protonated retinal Schiff base analogs in solution is 446 nm [22]. Chromophore distortions induced by the surrounding protein surface shift k max to 569 nm in native purple membrane [23]. Exposure to Triton decreases k max to  562 nm [24]; lipid reconstitution in 1 M NaCl restores k max to  566 nm, while in higher NaCl concentrations, this parameter returns to native-like values [3]. Figure 4 displays aplotofk max vs. Dm 1/2 for Triton-treated purple membrane Fig. 3. Plot of the half-width of the a-helical amide I mode (Dm 1/2 )vs. NaCl concentration used for reconstitution. Thelinedrawnistheresult of a second order polynomial fit. Fig. 4. Plot of the wavelength of maximum absorbance for light-adapted purple membrane (k max ) vs. the half-width of the a-helical component of theamideImode(Dm 1/2 ) in bacteriorhodopsin for purple membrane systems reconstituted in the absence of NaCl (Dm 1/2 = 29.2 cm )1 )and in 0.5 M NaCl (Dm 1/2 = 29.6 cm )1 ), 1 M NaCl (Dm 1/2 = 30.4 cm )1 ) and 2 M NaCl (Dm 1/2 = 30.8 cm )1 ). Fig. 5. Plot of the mean M intermediate decay time (j)vs.thehalf- width of the a-helical component of the amide I mode (Dm 1/2 ) in bacte- riorhodopsin. Ó FEBS 2003 Control of bacteriorhodopsin conformational flexibility and photocycle activity (Eur. J. Biochem. 270)1923 systems reconstituted with native lipids at various NaCl concentrations. The shift in k max on lipid perturbation by Triton originates from twists in the retinal structure and changes in its environment induced by a bacteriorho- dopsin conformational change, which produces altered protein–retinal interactions [25], while the decrease in Dm 1/2 arises from decreased mobility of the bacteriorhodopsin a-helix structures. The linear correlation between the two parameters demonstrates complete recovery to  569 nm as Dm 1/2 recovers to  30.5 cm )1 , approximately the half-width observed in native purple membrane. The recovery of normal bacteriorhodopsin photocycle behavior may also be correlated with Dm 1/2 for kinetic parameters that describe the decay of bacteriorhodop- sin intermediates. The average decay time s of the M 410 intermediate is an important diagnostic parameter. It is the weighted average of time constants for all forms of M present and is influenced by the mix of M-fast and M-slow cycles. A low value results from a preponderance of M-fast cycles, whereas a high value results from a paucity of M-fast cycles. In native purple membrane, the average s is  4 ms, arising from the mix of rapidly decaying species (M f ) with a decay time of  2 ms and the slower component (M s ) with a decay time of  6 ms. Exposure to Triton increases the average s to  70 ms through loss of the M f decay pathway and the generation of new, longer-lived M intermediates (R. W. Hendler & S. Bose, unpublished work). Reconstitution in 1 M NaCl partially recovers the M f pathway and lowers average s to  19 ms, while average s decreases to less than 5 ms on full reconstitution in high saline medium [3]. Figure 5 displays a plot relating s with Dm 1/2 for the reconstituted purple membrane systems. A linear correlation between Dm 1/2 and the average M decay time occurs over a wide range of decay times, illustrating that the conforma- tional flexibility described by Dm 1/2 provides a faithful description of the dynamics of the transmembrane helical segments that relate to M intermediate decay during the bacteriorhodopsin photocycle. The number of bacteriorhodopsin molecules that under- go a photocycle after a brief, high-intensity flash, termed the bacteriorhodopsin turnover, is greatly diminished after Triton exposure, reflecting the decreased ability of actinic light to initiate the bacteriorhodopsin photocycle in the perturbed systems [3]. This turnover may be quantified by either the maximum decrease in absorbance at 569 nm or increased absorbance at 410 nm (representing the M 410 formed) during the photocycle. Specifically, native purple membrane exhibits a change in absorbance at 569 nm of  100 milli-absorbance units for specific conditions des- cribed previously [3]. On exposure to Triton, decreased bacteriorhodopsin turnover results in a decreased change in absorbance of only 66 milli-absorbance units, because fewer bacteriorhodopsin molecules undergo a photocycle for the same conditions. On reconstitution in high-saline medium, the bacteriorhodopsin turnover returns to native-like values as quantified by the return to native values in the absorbance loss at 569 nm during the photocycle. Figure 6 presents the maximum loss in absorbance at 569 nm during the photocycle as a function of Dm 1/2 for the purple membrane systems. The nonlinear recovery of the bacte- riorhodopsin turnover with Dm 1/2 indicates that the struc- tural features that govern bacteriorhodopsin turnover rate involve other considerations than just the mobility of the a-helices. Discussion The data presented here demonstrate definitive correlations between the presence of native purple membrane lipid, the time constants for M-turnover, and the mobility, or motional characteristics, of the bacteriorhodopsin trans- membrane a-helices. We emphasize the ability of infrared spectroscopy to reflect the a-helical conformational flexibi- lity of bacteriorhodopsin in native purple membranes after depletion of lipids by Triton exposure and subsequent stepwise reconstitution in lipid dispersions containing vari- ous concentrations of NaCl which control the extent of lipid rebinding. The intrinsic mobility of the transmembrane a-helices of bacteriorhodopsin in purple membranes is related specifically to the deconvoluted widths of the a-helical amide I mode Dm 1/2 at  1660 cm )1 . On exposure to 0.1% Triton X-100, Dm 1/2 decreases, accompanied by some disruption in the well-ordered purple membrane lattice. Although lipid reconstitution in the absence of NaCl recovers some of the structural parameters affected by Triton exposure [3,19], the presence of NaCl is required for a complete, functionally active system, as demonstrated by correlations between the recovery of specific kinetic bacte- riorhodopsin photocycle parameters and changes in Dm 1/2 (Figs 3–5). Roles of squalene and polar lipid in bacteriorhodopsin function The correlation between the extent of reconstitution with purple membrane lipid (i.e. squalene) and the degree of the M f fi O fi BR photocycle activity and a-helical flexi- ble mobility supports the proposal for bacteriorhodopsin photocycle control being shared among squalene, polar lipids, and acidic amino acids of the cytoplasmic loop region. An extension of this concept accounts for all four Fig. 6. Plot of the absorbance change at 569 nm in photoexcitation (DmOD) vs. the half-width of the a-helical component of the amide I mode (Dm 1/2 ) in bacteriorhodopsin. Thelinedrawnistheresultofa third-order polynomial fit. 1924 R. W. Hendler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 distinct kinetic forms of M present in purple membrane (R. W. Hendler & S. Bose, unpublished results). If we attribute the four kinetic forms to different amounts of modulation of charge repulsion by squalene, the simplest model requires zero, one, two, or three squalenes per monomer. As shown in Table 2, for 10 molecules of wild- type bacteriorhodopsin, this requires three squalenes for each of the four molecules displaying M f activity and two squalenes for each of the six molecules displaying M s activity, yielding a squalene/bacteriorhodopsin ratio of 24 : 10. Similarly, to account for the three forms of bacteriorhodopsin found in a the Triton-treated case listed in Table 2, the ratio would be 5 : 10. Recent redetermina- tions of squalene/bacteriorhodopsin stoichiometries in native purple membrane using NMR procedures raise the originally determined value of 1–2, a value closer to that for the control shown in Table 2 [26]. The type of interaction described in R. W. Hendler & S. Bose (unpublished results) here between a membrane lipid and specific amino-acid residues of an active integral protein such as to influence and control the structure and function of the protein may be a prototype for similar interactions in other membrane-protein systems. References 1. Hendler, R.W., Dracheva, S. & Biochemistry (Moscow) (2001) Importance of lipids for bacteriorhodopsin structure. Photocycle Function 66, 1623–1627. 2. Joshi, M.K., Dracheva, S., Mukhopadyay, A.K., Bose, S. & Hendler, R.W. (1998) Importance of specific native lipids in con- trolling the photocycle of bacteriorhodopsin. Biochemistry 37, 14463–14470. 3. Mukhopadhyay, A.K., Dracheva, S., Bose, S. & Hendler, R.W. 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Casadio, R., Gutowitz, H., Mowery, P., Taylor, M. & Stoeck- enius, W. (1980) Light-dark adaptation of bacteriorhodopsin in Triton-treated purple membrane. Biochim. Biophys. Acta 590, 13–23. 24. Mukhopadhyay, A., Bose, S. & Hendler, R.W. (1994) Membrane- mediated control of the bacteriorhodopsin photocycle. Biochem- istry 33, 10889–10895. 25. Messaoudi, S., Lee, K H., Beaulieu, D., Baribeau, J. & Boucher, F. (1992) Equilibria between multiple spectral forms of bacterio- rhodopsin: effect of delipidation, anesthetics and solvents on their pH-dependence. Biochim. Biophys. Acta 1140, 45–52. 26. Corcelli, A., Lattenanzio, M.T., Mascolo, G., Papadia, P. & Fanizzi, F. (2002) Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extractofthepurplemembrane.J. Lipid Res. 43, 132–140. Table 2. Model for squalene regulation of M-turnover. The values in parentheses are the number of squalenes per bacteriorhodopsin for the kinetic species of M. SQ, Squalene; BR, bacteriorhodopsin. M f (3) M s (2) M 10 (1) M 70 (0) SQ/BR Control 40% 60% – – 2.4 Triton 0% 10% 30% 60% 0.5 Ó FEBS 2003 Control of bacteriorhodopsin conformational flexibility and photocycle activity (Eur. J. Biochem. 270)1925 . Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity An infrared spectroscopic study Richard. cm -1 of native purple membrane (solid line), purple membrane exposed briefly to Triton (dashed line), and purple membrane reconstituted with purple membrane