- Email: [email protected]
[2] Preparation and properties of monomeric bacteriorhodopsin
[2]
BACTERIORHODOPSIN
M O N O M E R S IN D E T E R G E N T S
5
surfaces. From the second through the fourth day, an additional 500 W of incandescent light is added. Pigmentation develops on the fourth day and cells are harvested on'the seventh or eighth day. It is not necessary to sterilize the large flasks, but if, as is likely to happen, the laboratory becomes contaminated with a halophilic organism, one must take care to harvest just at full pigmentation and not let the culture overgrow. Isolation of the Purple M e m b r a n e All centrifugations except the firstarc at 4 °. The cellsuspension is centrifuged at 600 g for 2 min to remove coarse material. The cells are then pelleted by centrifuging 20 min at 5000 g. The pelletis slurriedin residual supernatant fluid,a small fraction of a milligram (for each literof cells)of DNasc are added to each centrifuge bottle, the viscous slurry is suspended in D20, and the contents of allthe bottles are poured into a conical flask. Final volume should be about 50 ml/liter of harvest. The suspension is stirred vigorously (magnetic stirringbar) for 2-3 hr at room temperature. The suspension is then centrifuged at 40,000 g for 15 min. The supernatant is discarded and the purple sediment resuspcndcd in DzO. Hard, g u m m y pelleted material should not bc resuspended. The cycle of stirring and centrifuging (40,000 g for 30 min) is repeated two or three times (stir I-2 hr)14. If the final pure suspension is to be stored for any length of time, add a littlesalt to assure stability.The yield will bc about 15 mg/litcr of culture. Purple membrane labeled with ~3C or ~SN can bc generated in a similar manner using suitably labeled algae. ~4 One can intersperse with a washing with E D T A - D 2 0 buffer to minimize trace metal impurities[(D. B. Kell and A. M. Griffiths,Photobiochem. Photobiophys. 2, 105 (1981))].
[2] P r e p a r a t i o n
and Properties of Monomeric Bacteriorhodopsin
By N O R B E R T A . D E N C H E R a n d M A A R T E N P . H E Y N
The bacteriorhodopsin (BR) molecules in the purple membrane (PM) are organized into a two-dimensional hexagonal lattice of trimers that are surrounded by about 30 lipid molecules. In order to study the structure and function of isolated BR molecules it is advantageous to solubilize the PM into BR monomers. ~-4 These BR monomers can be reconstituted with M. P. Heyn, P. J. Bauer, and N. A. D e n c h e r , Biochem. Biophys. Res. Commun. 67, 897 (1975). METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-lg1988-4
6
BACTERIORHODOPSIN
[2]
exogenous phospholipids to form vesicles 5"6 and planar bilayer membranes, z They can also be reassembled into two- and three-dimensional crystals, s'9 To date Triton X-100 and octyl-fl-D-glucoside are the only detergents that fulfil the essential criteria for this purpose: They solubilize the PM to the state of monomers without significantly affecting the spectral and functional properties and can be removed again if desired, z,a Other detergents tested--Ammonyx LO, 1°,~1 cetyltrimethylammonium bromide, 1° cholate, ~z deoxycholate, ~2 digitonin, 1° dodecyl trimethylammonium bromide, la Emulphogene,~° and Tween 801°--either denature BR or do not yield monomers. Preparation of BR Monomers A PM sample is considered to be solubilized if it fails to sediment when subjected to 200,000 g for 45 rain. The percentage solubilization is determined by dividing the absorbance of the supernatant after centrifugation by the absorbance before centrffugation. The rate and extent of BR monomer formation during solubilization can be measured by monitoring the disappearance of the exciton coupling effects in the visible circular dichroism spectrum. 1,2,14 For Triton X-100 and octyl glucoside these two parameters are not only a function of the detergent to BR ratio but are also strongly dependent on pH, ionic strength, and temperature. Whereas at pH 6.9 (25 mM phosphate buffer) complete solubilization is reached in about 20 hr at 20° with a Triton X-100 to BR ratio of 6.2 (w/w), at pH 5.0 (100 mM acetate buffer) it takes 48 hr to reach approximately 60%. 2 At pH 5.0 in the same buffer and 25° complete solubilization could be ob2 lq. A. Dencher and M. P. Heyn, FEBS Lett. 96, 322 (1978). a N. A. Dcncher and M. P. Heyn, in "Energetics and Structure of Halophilic Microorganisms" (S. R. Caplan and M. Ginzburg, eds.), p. 233. Elsevier/North-Holland Biomedical Press, Amsterdam, 1978. * R. Casadio, H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, Biochim. Biophys. Acta 590, 13 (1980). 5 R. J. Cherry, U. Miiller, R. Henderson, and M. P. Heyn, J. Mol. Biol. 121, 283 (1978). 6 N. A. Dencher and M. P. Heyn, FEBS Lett. 108, 307 (1979). 7 E. Bamberg, N. A. Dencher, A. Fahr, and M. P. Heyn, Proc. Natl. Acad. Sci. U.S.A. 78, 7502 (1981). 8 H. Michel, D. Oesterhelt, and R. Henderson, Proc. Natl. Acad. Sci. U.S.A. 77, 338 (1980). 9 H. Michel and D. Oesterhelt, Proc. Natl. Acad. Sci. U.S.A. 77, 1283 (1980). l0 Norbert A. Dencher and M. P. Heyn, unpublished observation. 11 B. Becher, F. Tokunaga, and T. G. Ebrey, Biochemistry 17, 2293 (1978). 12 S. -B. Hwang and W. Stoeckenius, J. Memb. Biol. 33, 325 (1977). la M. Happe and P. Overath, Biochem. Biophys. Res. Commun. 72, 1504 (1976). 14 B. Becher and T. G. Ebrey, Biochem. Biophys. Res. Commun. 69, 1 (1976).
[9.]
BACTERIORHODOPSIN
M O N O M E R S IN D E T E R G E N T S
7
tained within 48 hr by increasing the detergent-to-BR ratio to 35. 4 A similar pH dependence is observed for octyl glucoside. With this detergent (41 raM) in 25 mM phosphate buffer, pH 6.9, no solubilization occurred during 48 hr in samples containing in addition sodium chloride in a concentration higher than 140 mM. Even in the presence of 15 mM NaC1 the solubilization rate is approximately three times smaller than that in the pure buffer system. 2 In general, the solubilization rate is similar for both detergents. In the case of octyl glucoside, however, differences in the rate and extent can be observed, depending on the quality of this detergent. Solubilization of BR in Triton X-IO0. PM suspended in 25 mM phosphate buffer, pH 6.9, is mixed with a 10% (w/w) solution of Triton X-100 (available from Packard Instrument Co., Inc., Sigma) in the same buffer to give a detergent-to-BR ratio by weight of about 4. The buffer volume is chosen in such a way that the Triton X-100 concentration lies between 0.2 and 0.5% (w/w). After sonication for 20 sec in a water bath sonifier the sample is kept for about 30 hr in the dark at room temperature (approx. 20°). Subsequently the sample is centrifuged at 200,000 g for 45 rain to remove any nonsolubilized material (usually less than 2%). Prior to this step the buffer may be changed by dialysis for 12 hr (e.g., 100 mM sodium acetate, pH 5.0, containing 0.2% Triton X-100). The detergent concentration should be above the critical micelle concentration (0.017% 15) and can be increased up to 2%. Titration experiments show that a minimum ratio of 1.8--2.0 mg Triton X-100/mg PM is required for successful solubilization. 5 In order to increase the solubilization rate, higher detergent-to-BR ratios can be used. Solubilization o f BR in Octyl-fl-o-glucoside. Octyl glucoside can be synthesized following published procedures 16-1a or purchased from various companies (e.g., Calbiochem-Behring Corp., Riedel-de-Haen). Optimum conditions for the solubilization of BR are obtained with 40 mM octyl glucoside (~- 1.2% (w/w), MW -- 294.4) and a detergent-to-protein ratio by weight of 20 in low ionic strength buffer of neutral pH (e.g., 25 mM phosphate buffer, pH 6.9). Following 20-sec sonication, the sample is allowed to stand in the dark at room temperature for 20-30 hr. After this time 85-95% of the PM is solubilized to BR monomers. Depending on the quality of the detergent used it is sometimes necessary to increase the octyl glucoside concentration up to 100 m M or possible to decrease the detergent-to-BR ratio below l0 at a final concentration is p. W. Holloway and J. T. Katz, Biochemistry 11, 3689 (1972). 16 C. R. Noller and W. C. Rockwell, J. Am. Chem. Soc. 60, 2076 (1938). 17 C. Baron and T. E. Thompson, Biochim. Biophys. Acta 382, 276 (1975). is j. F. W. Keana and R. B. Roman, Memb. Biochem. 1, 323 (1978). 19 j. T. Lin, S. Riedel, and R. Kinne, Biochim. Biophys. Acta 557, 179 (1979).
8
BACTERIORHODOPSIN
[2]
of 40 mM. Furthermore, it is occasionally advantageous to terminate the solubilization after a shorter period (e.g., after 12 hr) and to use only the supernatant obtained after centrifugation for subsequent experiments. This avoids possible contamination of the sample with denatured BR. Properties of Solubilized B R M o n o m e r s Various experimental approaches show that B R is solubilized in both detergents to the state of monomers. The exciton coupling effects in the visible C D spcctrurn disappear and are replaced in the case of Triton XI00 by a small positive C D monomer band centered at about 560 rim.1,2,~4 Gel filtrationexperiments give values of 28 - 5 A 2 and approximately 50 A 4 for the Stokes radius of the BR-lipid-octyl glucosidc and B R lipid-Triton X-100 complex, respectively, excluding the possibility that the rniccllescontain more than onc B R molecule. This conclusion is confirmed by the determination of the molecular weight of B R in the mixed Triton-lipid- B R micellcs.2° The Triton X-100-solubilizcd B R preparations arc stable for scveral days when stored in the dark at room temperature and show no evidence for chromophorc loss. B R solubilizcd in octyl glucosidc can be stored at 4 ° for some days; at room temperature, however, progressive chromophorc loss occurs after about 2 days. It is advantageous to store the solubilized samples at low pH, e.g., at p H 5.0. In terms of a number of criteria,the propcrtics of B R in the solubilizcd form differonly slightlyfrom those in the native membrane. The secondary structure of Triton X-100 and octyl glucosidc-solubilizcd BR, determined from the circulardichroism at 208 and 222 nm, contains about 70% a-helix and is thus quite similar to that of B R in the PM. 2"2°APart from a decrease in thc extinction coefficient and a small bluc shiftof the visible absorption band the absorption spectrum of rnonomcric B R is unchanged. The phenomenon of light-dark adaptation can stillbc observed in the solubilizcd statc; however, the extent of light adaptation is less than that in PM. 2,4 Thc absorption m a x i m u m occurs at 552 n m in the light-adapted state, shifting to 546 n m upon dark adaptation (0.2% Triton X-100, p H 6.9, 20°). The photocyclc of Triton X-100-solubilized B R is qualitatively the same as in the PM. Whereas the half-time of the decay of the M4~2 intermediate remains unchanged, the formation is about three times faster. In octyl glucoside-solubilized BR, however, the cycle seems to bc slightly different.2 Recently it was shown that illumination of Triton X100-solubilizcd B R results in a reversible hydrolysis of the retinal aldi20 j. A. Reynolds and W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 74, 2803 (1977).
[2]
BACTERIORHODOPSIN MONOMERS IN DETERGENTS
9
mine. 21 Furthermore, reconstitution experiments prove that BR monomers can effectively pump protons .6 R e m o v a l of E n d o g e n o u s Phospholipids The action of Triton X-100 and octyl glucoside leads to partial delipidation of BR. Sucrose density gradient centrifugation and gel filtration in the presence of these detergents allow a separation of detergent-lipid and detergent-lipid-protein micelles. When PM solubilized in 5% Triton X100 or 5% octyl glucoside is chromatographed on BioGel A-0.5 m in buffer containing 1% octyl glucoside and 25 mM sodium phosphate at pH 6.9, about 90% of the polar lipids are removed from BR. 22 Solubilization of the PM with Triton X-100 followed by gel filtration in deoxycholate solution results in a removal of at least 99% of endogenous lipids. 22 R e m o v a l of Detergent The detergents can be removed from the solution containing solubilized BR by prolonged dialysis. The low critical micelle concentration of Triton X-100 is a disadvantage in this procedure. Since the critical micelle concentration of octyl glucoside is about 100 times higher (25 mM 23), this detergent can be removed more rapidly. Using the return of the amplitude of the excitation CD effect as a signal to monitor the detergent removal, equilibrium was reached in 40 hr with octyl glucoside as opposed to about 7 days with Triton X-100 (4°, 100 mM acetate buffer, pH 5.0). Most of the effects of solubilization are reversible upon slow dialysis; e.g., the absorption maximum of BR shifts back to its original position and the exciton CD bands reappear with the same specific ellipticity as in the native PM. 2 During dialysis, reassembly occurs and large hexagonal crystalline domains are formed again. 5 A small amount of detergent, however, remains bound to BR. 5 Centrifugation using a detergent-free sucrose gradient and gel filtration provide alternative faster methods to remove the detergents. A rapid removal of the detergents from solubilized BR can be achieved in the time course of minutes by absorption to resins, e.g., Bio-Beads SM-2 (Bio-Rad Labs) 19,z4 or Amberlite XAD-2 (Rohm and Haas Ltd.). 25 For octyl gluco21 A. M. Shkrob, A. V. Rodionov-and Yu. A. Ovchinnikov, Bioorg. Khim. 4, 354 (1978). zz K.-S. Huang, H. Bayley, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 77, 323 (1980). 23 K. Shinoda, T. Yamaguchi, and R. Hod, Bull. Chem. Soc. Jpn. 34, 237 (1961). P. W. Holloway, Anal. Biochem. 53, 304 (1973). 25 p. S. J. Cheetham, Anal. Biochem. 92, 447 (1979).
10
BACTERIORHODOPSlN
[3]
side a final detergent concentration of 0.018% can be reached with BioBeads SM-2, TM i.e., much lower than its critical micelle concentration of 0.74%. Choice of Detergent Both Triton X-100 and octyl glucoside can be considered to be suitable detergents to prepare BR monomers, since no gross structural and functional alterations occur during solubilization. In contrast to Triton X-100, octyl-/3-D-glucoside is a chemically well-defined compound. For spectroscopic investigations octyl glucoside is superior to Triton X-100. The far UV absorbance is much higher for Triton X-100 and its strong fluorescence in the near UV makes fluorescence work in this range exceedingly difficult. The higher critical micelle concentration of octyl glucoside allows more rapid detergent removal and makes it more suitable for reconstitution work. Also its smaller micelle size and weight can be of advantage. Asolectin, diphytanoylphosphatidyicholine, egg lecithin, and phosphatidylserine vesicles can only be prepared with BR solubilized in octyl glucoside. 6,~6 On the other hand, BR is more stable in Triton X-100. And last but not least, Triton X-100 is much cheaper than octyl glucoside. 26M. P. Heyn and N. A. Dencher, this volume, Article [6].
[3] R e c o n s t i t u t i o n o f t h e R e t i n a l P r o t e i n s Bacteriorhodopsin and Halorhodopsin
By DIETER OESTERHELT Reconstitution of the retinal proteins bacteriorhodopsin (BR) and halorhodopsin (HR) from the corresponding apoproteins BO and HO with retinal and retinal analogue compounds is a valuable tool for elucidation of structure-function relationships. This article describes the experimental methods for the preparation of retinal-free membranes and membrane vesicles containing the apoproteins BO or HO, the methods for the reconstitution of BR and HR in membranes, cell vesicles, and cells, and an assay for both chromoproteins. Strains Growth of halobacterial cells is described in Chapter [45] of this volume. The strains ofHalobacterium halobium suited for the experimental
METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181988-4
BACTERIORHODOPSIN
M O N O M E R S IN D E T E R G E N T S
5
surfaces. From the second through the fourth day, an additional 500 W of incandescent light is added. Pigmentation develops on the fourth day and cells are harvested on'the seventh or eighth day. It is not necessary to sterilize the large flasks, but if, as is likely to happen, the laboratory becomes contaminated with a halophilic organism, one must take care to harvest just at full pigmentation and not let the culture overgrow. Isolation of the Purple M e m b r a n e All centrifugations except the firstarc at 4 °. The cellsuspension is centrifuged at 600 g for 2 min to remove coarse material. The cells are then pelleted by centrifuging 20 min at 5000 g. The pelletis slurriedin residual supernatant fluid,a small fraction of a milligram (for each literof cells)of DNasc are added to each centrifuge bottle, the viscous slurry is suspended in D20, and the contents of allthe bottles are poured into a conical flask. Final volume should be about 50 ml/liter of harvest. The suspension is stirred vigorously (magnetic stirringbar) for 2-3 hr at room temperature. The suspension is then centrifuged at 40,000 g for 15 min. The supernatant is discarded and the purple sediment resuspcndcd in DzO. Hard, g u m m y pelleted material should not bc resuspended. The cycle of stirring and centrifuging (40,000 g for 30 min) is repeated two or three times (stir I-2 hr)14. If the final pure suspension is to be stored for any length of time, add a littlesalt to assure stability.The yield will bc about 15 mg/litcr of culture. Purple membrane labeled with ~3C or ~SN can bc generated in a similar manner using suitably labeled algae. ~4 One can intersperse with a washing with E D T A - D 2 0 buffer to minimize trace metal impurities[(D. B. Kell and A. M. Griffiths,Photobiochem. Photobiophys. 2, 105 (1981))].
[2] P r e p a r a t i o n
and Properties of Monomeric Bacteriorhodopsin
By N O R B E R T A . D E N C H E R a n d M A A R T E N P . H E Y N
The bacteriorhodopsin (BR) molecules in the purple membrane (PM) are organized into a two-dimensional hexagonal lattice of trimers that are surrounded by about 30 lipid molecules. In order to study the structure and function of isolated BR molecules it is advantageous to solubilize the PM into BR monomers. ~-4 These BR monomers can be reconstituted with M. P. Heyn, P. J. Bauer, and N. A. D e n c h e r , Biochem. Biophys. Res. Commun. 67, 897 (1975). METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-lg1988-4
6
BACTERIORHODOPSIN
[2]
exogenous phospholipids to form vesicles 5"6 and planar bilayer membranes, z They can also be reassembled into two- and three-dimensional crystals, s'9 To date Triton X-100 and octyl-fl-D-glucoside are the only detergents that fulfil the essential criteria for this purpose: They solubilize the PM to the state of monomers without significantly affecting the spectral and functional properties and can be removed again if desired, z,a Other detergents tested--Ammonyx LO, 1°,~1 cetyltrimethylammonium bromide, 1° cholate, ~z deoxycholate, ~2 digitonin, 1° dodecyl trimethylammonium bromide, la Emulphogene,~° and Tween 801°--either denature BR or do not yield monomers. Preparation of BR Monomers A PM sample is considered to be solubilized if it fails to sediment when subjected to 200,000 g for 45 rain. The percentage solubilization is determined by dividing the absorbance of the supernatant after centrifugation by the absorbance before centrffugation. The rate and extent of BR monomer formation during solubilization can be measured by monitoring the disappearance of the exciton coupling effects in the visible circular dichroism spectrum. 1,2,14 For Triton X-100 and octyl glucoside these two parameters are not only a function of the detergent to BR ratio but are also strongly dependent on pH, ionic strength, and temperature. Whereas at pH 6.9 (25 mM phosphate buffer) complete solubilization is reached in about 20 hr at 20° with a Triton X-100 to BR ratio of 6.2 (w/w), at pH 5.0 (100 mM acetate buffer) it takes 48 hr to reach approximately 60%. 2 At pH 5.0 in the same buffer and 25° complete solubilization could be ob2 lq. A. Dencher and M. P. Heyn, FEBS Lett. 96, 322 (1978). a N. A. Dcncher and M. P. Heyn, in "Energetics and Structure of Halophilic Microorganisms" (S. R. Caplan and M. Ginzburg, eds.), p. 233. Elsevier/North-Holland Biomedical Press, Amsterdam, 1978. * R. Casadio, H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, Biochim. Biophys. Acta 590, 13 (1980). 5 R. J. Cherry, U. Miiller, R. Henderson, and M. P. Heyn, J. Mol. Biol. 121, 283 (1978). 6 N. A. Dencher and M. P. Heyn, FEBS Lett. 108, 307 (1979). 7 E. Bamberg, N. A. Dencher, A. Fahr, and M. P. Heyn, Proc. Natl. Acad. Sci. U.S.A. 78, 7502 (1981). 8 H. Michel, D. Oesterhelt, and R. Henderson, Proc. Natl. Acad. Sci. U.S.A. 77, 338 (1980). 9 H. Michel and D. Oesterhelt, Proc. Natl. Acad. Sci. U.S.A. 77, 1283 (1980). l0 Norbert A. Dencher and M. P. Heyn, unpublished observation. 11 B. Becher, F. Tokunaga, and T. G. Ebrey, Biochemistry 17, 2293 (1978). 12 S. -B. Hwang and W. Stoeckenius, J. Memb. Biol. 33, 325 (1977). la M. Happe and P. Overath, Biochem. Biophys. Res. Commun. 72, 1504 (1976). 14 B. Becher and T. G. Ebrey, Biochem. Biophys. Res. Commun. 69, 1 (1976).
[9.]
BACTERIORHODOPSIN
M O N O M E R S IN D E T E R G E N T S
7
tained within 48 hr by increasing the detergent-to-BR ratio to 35. 4 A similar pH dependence is observed for octyl glucoside. With this detergent (41 raM) in 25 mM phosphate buffer, pH 6.9, no solubilization occurred during 48 hr in samples containing in addition sodium chloride in a concentration higher than 140 mM. Even in the presence of 15 mM NaC1 the solubilization rate is approximately three times smaller than that in the pure buffer system. 2 In general, the solubilization rate is similar for both detergents. In the case of octyl glucoside, however, differences in the rate and extent can be observed, depending on the quality of this detergent. Solubilization of BR in Triton X-IO0. PM suspended in 25 mM phosphate buffer, pH 6.9, is mixed with a 10% (w/w) solution of Triton X-100 (available from Packard Instrument Co., Inc., Sigma) in the same buffer to give a detergent-to-BR ratio by weight of about 4. The buffer volume is chosen in such a way that the Triton X-100 concentration lies between 0.2 and 0.5% (w/w). After sonication for 20 sec in a water bath sonifier the sample is kept for about 30 hr in the dark at room temperature (approx. 20°). Subsequently the sample is centrifuged at 200,000 g for 45 rain to remove any nonsolubilized material (usually less than 2%). Prior to this step the buffer may be changed by dialysis for 12 hr (e.g., 100 mM sodium acetate, pH 5.0, containing 0.2% Triton X-100). The detergent concentration should be above the critical micelle concentration (0.017% 15) and can be increased up to 2%. Titration experiments show that a minimum ratio of 1.8--2.0 mg Triton X-100/mg PM is required for successful solubilization. 5 In order to increase the solubilization rate, higher detergent-to-BR ratios can be used. Solubilization o f BR in Octyl-fl-o-glucoside. Octyl glucoside can be synthesized following published procedures 16-1a or purchased from various companies (e.g., Calbiochem-Behring Corp., Riedel-de-Haen). Optimum conditions for the solubilization of BR are obtained with 40 mM octyl glucoside (~- 1.2% (w/w), MW -- 294.4) and a detergent-to-protein ratio by weight of 20 in low ionic strength buffer of neutral pH (e.g., 25 mM phosphate buffer, pH 6.9). Following 20-sec sonication, the sample is allowed to stand in the dark at room temperature for 20-30 hr. After this time 85-95% of the PM is solubilized to BR monomers. Depending on the quality of the detergent used it is sometimes necessary to increase the octyl glucoside concentration up to 100 m M or possible to decrease the detergent-to-BR ratio below l0 at a final concentration is p. W. Holloway and J. T. Katz, Biochemistry 11, 3689 (1972). 16 C. R. Noller and W. C. Rockwell, J. Am. Chem. Soc. 60, 2076 (1938). 17 C. Baron and T. E. Thompson, Biochim. Biophys. Acta 382, 276 (1975). is j. F. W. Keana and R. B. Roman, Memb. Biochem. 1, 323 (1978). 19 j. T. Lin, S. Riedel, and R. Kinne, Biochim. Biophys. Acta 557, 179 (1979).
8
BACTERIORHODOPSIN
[2]
of 40 mM. Furthermore, it is occasionally advantageous to terminate the solubilization after a shorter period (e.g., after 12 hr) and to use only the supernatant obtained after centrifugation for subsequent experiments. This avoids possible contamination of the sample with denatured BR. Properties of Solubilized B R M o n o m e r s Various experimental approaches show that B R is solubilized in both detergents to the state of monomers. The exciton coupling effects in the visible C D spcctrurn disappear and are replaced in the case of Triton XI00 by a small positive C D monomer band centered at about 560 rim.1,2,~4 Gel filtrationexperiments give values of 28 - 5 A 2 and approximately 50 A 4 for the Stokes radius of the BR-lipid-octyl glucosidc and B R lipid-Triton X-100 complex, respectively, excluding the possibility that the rniccllescontain more than onc B R molecule. This conclusion is confirmed by the determination of the molecular weight of B R in the mixed Triton-lipid- B R micellcs.2° The Triton X-100-solubilizcd B R preparations arc stable for scveral days when stored in the dark at room temperature and show no evidence for chromophorc loss. B R solubilizcd in octyl glucosidc can be stored at 4 ° for some days; at room temperature, however, progressive chromophorc loss occurs after about 2 days. It is advantageous to store the solubilized samples at low pH, e.g., at p H 5.0. In terms of a number of criteria,the propcrtics of B R in the solubilizcd form differonly slightlyfrom those in the native membrane. The secondary structure of Triton X-100 and octyl glucosidc-solubilizcd BR, determined from the circulardichroism at 208 and 222 nm, contains about 70% a-helix and is thus quite similar to that of B R in the PM. 2"2°APart from a decrease in thc extinction coefficient and a small bluc shiftof the visible absorption band the absorption spectrum of rnonomcric B R is unchanged. The phenomenon of light-dark adaptation can stillbc observed in the solubilizcd statc; however, the extent of light adaptation is less than that in PM. 2,4 Thc absorption m a x i m u m occurs at 552 n m in the light-adapted state, shifting to 546 n m upon dark adaptation (0.2% Triton X-100, p H 6.9, 20°). The photocyclc of Triton X-100-solubilized B R is qualitatively the same as in the PM. Whereas the half-time of the decay of the M4~2 intermediate remains unchanged, the formation is about three times faster. In octyl glucoside-solubilized BR, however, the cycle seems to bc slightly different.2 Recently it was shown that illumination of Triton X100-solubilizcd B R results in a reversible hydrolysis of the retinal aldi20 j. A. Reynolds and W. Stoeckenius, Proc. Natl. Acad. Sci. U.S.A. 74, 2803 (1977).
[2]
BACTERIORHODOPSIN MONOMERS IN DETERGENTS
9
mine. 21 Furthermore, reconstitution experiments prove that BR monomers can effectively pump protons .6 R e m o v a l of E n d o g e n o u s Phospholipids The action of Triton X-100 and octyl glucoside leads to partial delipidation of BR. Sucrose density gradient centrifugation and gel filtration in the presence of these detergents allow a separation of detergent-lipid and detergent-lipid-protein micelles. When PM solubilized in 5% Triton X100 or 5% octyl glucoside is chromatographed on BioGel A-0.5 m in buffer containing 1% octyl glucoside and 25 mM sodium phosphate at pH 6.9, about 90% of the polar lipids are removed from BR. 22 Solubilization of the PM with Triton X-100 followed by gel filtration in deoxycholate solution results in a removal of at least 99% of endogenous lipids. 22 R e m o v a l of Detergent The detergents can be removed from the solution containing solubilized BR by prolonged dialysis. The low critical micelle concentration of Triton X-100 is a disadvantage in this procedure. Since the critical micelle concentration of octyl glucoside is about 100 times higher (25 mM 23), this detergent can be removed more rapidly. Using the return of the amplitude of the excitation CD effect as a signal to monitor the detergent removal, equilibrium was reached in 40 hr with octyl glucoside as opposed to about 7 days with Triton X-100 (4°, 100 mM acetate buffer, pH 5.0). Most of the effects of solubilization are reversible upon slow dialysis; e.g., the absorption maximum of BR shifts back to its original position and the exciton CD bands reappear with the same specific ellipticity as in the native PM. 2 During dialysis, reassembly occurs and large hexagonal crystalline domains are formed again. 5 A small amount of detergent, however, remains bound to BR. 5 Centrifugation using a detergent-free sucrose gradient and gel filtration provide alternative faster methods to remove the detergents. A rapid removal of the detergents from solubilized BR can be achieved in the time course of minutes by absorption to resins, e.g., Bio-Beads SM-2 (Bio-Rad Labs) 19,z4 or Amberlite XAD-2 (Rohm and Haas Ltd.). 25 For octyl gluco21 A. M. Shkrob, A. V. Rodionov-and Yu. A. Ovchinnikov, Bioorg. Khim. 4, 354 (1978). zz K.-S. Huang, H. Bayley, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 77, 323 (1980). 23 K. Shinoda, T. Yamaguchi, and R. Hod, Bull. Chem. Soc. Jpn. 34, 237 (1961). P. W. Holloway, Anal. Biochem. 53, 304 (1973). 25 p. S. J. Cheetham, Anal. Biochem. 92, 447 (1979).
10
BACTERIORHODOPSlN
[3]
side a final detergent concentration of 0.018% can be reached with BioBeads SM-2, TM i.e., much lower than its critical micelle concentration of 0.74%. Choice of Detergent Both Triton X-100 and octyl glucoside can be considered to be suitable detergents to prepare BR monomers, since no gross structural and functional alterations occur during solubilization. In contrast to Triton X-100, octyl-/3-D-glucoside is a chemically well-defined compound. For spectroscopic investigations octyl glucoside is superior to Triton X-100. The far UV absorbance is much higher for Triton X-100 and its strong fluorescence in the near UV makes fluorescence work in this range exceedingly difficult. The higher critical micelle concentration of octyl glucoside allows more rapid detergent removal and makes it more suitable for reconstitution work. Also its smaller micelle size and weight can be of advantage. Asolectin, diphytanoylphosphatidyicholine, egg lecithin, and phosphatidylserine vesicles can only be prepared with BR solubilized in octyl glucoside. 6,~6 On the other hand, BR is more stable in Triton X-100. And last but not least, Triton X-100 is much cheaper than octyl glucoside. 26M. P. Heyn and N. A. Dencher, this volume, Article [6].
[3] R e c o n s t i t u t i o n o f t h e R e t i n a l P r o t e i n s Bacteriorhodopsin and Halorhodopsin
By DIETER OESTERHELT Reconstitution of the retinal proteins bacteriorhodopsin (BR) and halorhodopsin (HR) from the corresponding apoproteins BO and HO with retinal and retinal analogue compounds is a valuable tool for elucidation of structure-function relationships. This article describes the experimental methods for the preparation of retinal-free membranes and membrane vesicles containing the apoproteins BO or HO, the methods for the reconstitution of BR and HR in membranes, cell vesicles, and cells, and an assay for both chromoproteins. Strains Growth of halobacterial cells is described in Chapter [45] of this volume. The strains ofHalobacterium halobium suited for the experimental
METHODS IN ENZYMOLOGY, VOL. 88
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181988-4