J. Mol. Biol. (1992) 224, 527-528
Crystallization of the BSOO-820 Light-harvesting Complex from Rhodopseudomonas acidophila Strain 7750 N. Guthriel, G. MacDermottl, R. J. Cogdel12, A. A. Freerl, N. W. Isaac9 A. M. Hawthornthwaite2, E. Halloren2 and J. G. Lindsay3 Departments University
of ‘Chemistry, 2Botany and 3Biochemistry of Glasgow, Glasgow G12 SQQ, Scotland
(Received 18 November 1991; accepted 12 December 1991) The
BSOO-820
light-harvesting complex, an integral membrane protein, from acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 1218 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820a,, B800-820az, B800-820P1 and BSOO-82Ofi,). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000. Rhodopseudomonas
Keywords:
crystals; membranes; protein; light-harvesting;
Reaction centre and light-harvesting antenna complexes are the major pigment-binding apoproteins of the photosynthetic apparatus of the purple non-sulphur bacteria. The three-dimensional structures of two types of bacterial reaction centres have been determined (Deisenhofer et al., 1985; Michel et al., 1986; Allen et al., 1987). In Rhodopseudomonas they are localized in the intraacidophita cytoplasmic membranes. The light-harvesting complexes gather light energy and funnel it to the reaction centre, where a charge-separated state occurs. Reaction centres are always surrounded by a constant number of core antenna complexes. Some light-harvesting species also have additional complexes, whose number, type and expression are dependent on environmental conditions such as temperature or light intensity (Zuber, 1985). These complexes are named in terms of their absorbance in the near infra-red, thus the B800-820 complex absorbs at 800 and 820 nm. The 7750 strain of Rps. acidophila always produces the B880 core complex and also produces either the B800-820 or the B800-850 light-harvesting complex, or a mixture of both complexes, depending on the temperature and light intensity. In the 7750 strain the B800-820 complex is produced specifically in response to temperatures of typically 24”C, whereas in Rps. acidophila 10050, the only peripheral antenna complex produced is the B800-850 (Papiz et al., 1989). The B800-820 complex from Rps. acidophila strain 7750 is formed from the aggregation of four low molecular weight, pigment-binding apoproteins, namely the B800-820a,, B800-820a2, B800-8208,
and B800-820Pz. The primary structures of three of these apoproteins have been determined (Brunisholz et al., 1987). The B800-820a, has 53 amino acid residues with a molecular weight of 5567 daltons, the B800-820p1 has 43 amino acid residues and a molecular weight of 4721 daltons, and the B800-820& has 41 amino acid residues and a molecular weight of 4520 daltons. The B800-820a, is as yet unsequenced. The stoichiometric ratio of bacteriochlorophyll alcarotenoids is 2 : 1 (Evans, 1989). Cells from Rps. acidophila 7750 were grown photosynthetically at 24°C on Pfennigs medium (Pfennig, 1969). These conditions yield the B880 core complex and also the B800-820 lightharvesting complex. The cells were centrifuged at 2800 revs/min for 100 minutes at 4°C using a COOLSPIN centrifuge, and the resulting pellets were retained and combined using 50 ml of 61 MKC1 in 20 mM-Mes buffer (pH 6.8). The combined pellets were then centrifuged at 12,000 revs/min using an MSE18 centrifuge. Again, the pellets were retained and homogenized in 50 ml of 20 mMTris. HCl (pH 8.0). The broken cells were then centrifuged at 12,000 revs/min for 20 minutes at 4°C and the pellet retained, while the supernatant was spun at 45,000 revs/min for one hour. The pellet formed from the high speed spin was combined with the low-speed spin and the absorbance at 800 nm was adjusted to 50 with 20 mM-Tris. HCl (pH 8.0). The diluted membrane solution was then made 1 y0 (v/v) with lauryl dimethyl N-amide oxide (LDAO), by stirring at room temperature for ten minutes. 527
0022-2836/92/060527-02
$03.00/O
crystallography
0
1992 Academic
Press Limited
528
N. Quthrie
This solution was then centrifuged at 12,000 revs/ min for ten minutes at 4°C to remove any unsolubilized material (Cogdell et aE., 1983). A Whatman DE52 anion exchange column was used to separate the core and light-harvesting complexes. The column was pre-equilibrated with 20 mr\l-Tris . HCl (pH 8.0) and then loaded with the membrane solution. Elution of the light-harvesting complexes took place using increasing concentrations of NaCl (50, 100, 150, 250 mM, all plus 61 y. (v/v) LDAO), with 1 ml fractions assayed spectrophotomerically using the ratio of the absorbance of bacteriochlorophyll at 800 nm to the absorbance of aromatic amino acids at 270 nm as a measurement of purity. After two columns, ratios of 2 : 1 or greater were pooled. A 1 m Sephacryl S-200 column was used to further purify the complex and ratios of 3 : 1 or greater were pooled. The complex was then concentrated on a mini DE52 column (poured in a Pasteur pipette), washed with four column volumes of Tris. HCl (pH 80) to remove free LDAO, and the remaining detergent exchanged wit’h one column volume of 1 y. (w/v) /I-octyl glucoside. The pure complex was then eluted using 350 rnN-NaCI in 1 y. (w/v) p-octyl glucoside and 20 m&r-Tris * HCl (pH %O). Crystals were grown using the sitting drop method. A 25 ~1 drop containing approximately 8.0 mg protein/ml, 1.0 M-di-potassium hydrogen orthophosphate and 2.5 */* (w/v) benzamidine hydrochloride in Tris . HCl at pH 9.0, was equilibrated by vapour diffusion against an 8 ml well of 2.1 M-ammonium sulphate in Tris . HCl (pH 9.0). Crystals grew at 18 “C as tabular plates to a size of 69 mm x 94 mm x 0.2 mm over a period of six weeks. The crystals were soaked in 2 M-di-potassium hydrogen orthophosphate at pH 9.5 for 30 minutes before mounting in 1 mm glass capillary tubes. Diffraction data were collected on a Siemens area detector mounted on a rotating anode source, operating at 50 kV and 80 mA. The crystal diffracted to a resolution limit of 5 A (1 A = 0.1 nm), and data were collected over a rotation range of 108.75” with a step size of 0.25”. The previously unknown cell dimensions were obtained by the automatic indexing procedure (Howard et al., 1985), which hexagonal dimensions cell gave a with a= b = 121.8 A and c = 283.1 A. The data were integrated using the XDS program (Kabsch, 1988a,b), giving: R symm= i
CIh,i
’
a value of 0.066 for the space group R32. This is a similar cell and space group to that of the B800-850 antenna complex from Rps. acidophila 10050 (Papiz et al., 1989; a= b = 121.1 A, c = 296.7 A and space group R32). The computed V, value is 2.2 A3/Da, assuming a molecular mass of 92,000 daltons in the asymmetric unit. This falls within the range reported by 1Matthews (1977), and gives a solvent composition of 44 %. The close similarity in the unit cell and space
Edited
et al. group of these protein crystals and crystals of the B800-850 antenna complex suggests a similar asymmetric unit in both cases. Papiz et al. (l9S9) propose an asymmetric unit containing six &,/P subunits, 18 bacteriochlorophyll and nine carotenoid molecules for the B800-850 complex. The asymmetric unit of the B800-820 light-harvesting complex would then be a trimer of molecular assemblies, with each assembly consisting of a, aaPl /IZ proteins, six bacteriochlorophyll and three carotenoid molecules. This work Initiative.
is supported
by the
SERC
Membrane
References Allen, J. P., Feher, G., Yeates, T. O.? Komiya, II. B Rees, D. C. (1987). Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc. Nat. Acad. Sci., U.S.A. 84. 5730-5734. Brunisholz, R. A., Bissig, I., Miederer; E., Suter, F. & Zuber, H. (1987). Structural studies on the lightharvesting polypeptides of Rhodopseudomonas acidophz’la. In Progress in Photosynthesis Research (Biggins, J., ed.), vol. 2> sect. 1; 13-16, Martinus Nijhoff, The Hague. Cogdell, R. J., Durant, I.; Valentine, J., Lindsay, J. G. & Schmidt, K. (1983). The isolation and partial characterisation of the light-harvesting pigment-protein complement from Rhodopseudormonas acidophila strain 10050. Biochim. Biophys. Acta, 722, 427-455. Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel, H. (1985). Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 A resolut,ion. Nature (London), 318, 618-624. Evans: M. B. (1989). The structure and function of the light-harvesting antenna complexes from purple photosynthetic bacteria. Ph.D. thesis, Glasgow University. Howard, A. J., Nielsen, C. & Xuong, Tu’g, H. (19S5). Software for a diffractometer with multiwire area detector. Methods Enzymol. 114, 211-237. Kabsch, W. (1988a). Automatic indexing of rotation 21, 67-71. diffraction patterns. J. Appl. Crystallogr. Kabsch, W. (19883). Automatic indexing of rotation diffraction patterns. J. Appl. Crystallogr. 21; 916-924. Matthews, B. W. (1977). In Proteins (Keurath, R, $ Hill; R. L., eds), voi. 3, pp. 4033590, Academic Press, London. Michef, H., Epp, 0. & Deisenhofer, J. (1986). Pigmentprotein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis. EMBO J. 5, 2445-245 I. Papiz, M. Z., Hawthornthwaite, A. M., Cogdell, R. J., Wooley, K. J., Wightman, P. A., Ferguson, L. A. & Lindsay, J. G. (1989). Crystallisation and characterisation of two crystal forms of the B800-850 lightharvesting complex from RhodopseudomorLas acidophila strain 10050. J. Mol. Biol. 209, 833-835. Pfennig? N. (1969). Rhodopseudomonas acidophila, sp. 3.: a new species of the budding purple non-sulfur bacteria,. J. Bacterial. 99. 597-602. Zuber, H. (1985). Structure and function of lightharvesting complexes and their polypeptides. Photochem. Pfwtobiol. 42, 821-844.
by A. Klug
Crystallization of the BSOO-820 Light-harvesting Complex from Rhodopseudomonas acidophila Strain 7750 N. Guthriel, G. MacDermottl, R. J. Cogdel12, A. A. Freerl, N. W. Isaac9 A. M. Hawthornthwaite2, E. Halloren2 and J. G. Lindsay3 Departments University
of ‘Chemistry, 2Botany and 3Biochemistry of Glasgow, Glasgow G12 SQQ, Scotland
(Received 18 November 1991; accepted 12 December 1991) The
BSOO-820
light-harvesting complex, an integral membrane protein, from acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 1218 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820a,, B800-820az, B800-820P1 and BSOO-82Ofi,). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000. Rhodopseudomonas
Keywords:
crystals; membranes; protein; light-harvesting;
Reaction centre and light-harvesting antenna complexes are the major pigment-binding apoproteins of the photosynthetic apparatus of the purple non-sulphur bacteria. The three-dimensional structures of two types of bacterial reaction centres have been determined (Deisenhofer et al., 1985; Michel et al., 1986; Allen et al., 1987). In Rhodopseudomonas they are localized in the intraacidophita cytoplasmic membranes. The light-harvesting complexes gather light energy and funnel it to the reaction centre, where a charge-separated state occurs. Reaction centres are always surrounded by a constant number of core antenna complexes. Some light-harvesting species also have additional complexes, whose number, type and expression are dependent on environmental conditions such as temperature or light intensity (Zuber, 1985). These complexes are named in terms of their absorbance in the near infra-red, thus the B800-820 complex absorbs at 800 and 820 nm. The 7750 strain of Rps. acidophila always produces the B880 core complex and also produces either the B800-820 or the B800-850 light-harvesting complex, or a mixture of both complexes, depending on the temperature and light intensity. In the 7750 strain the B800-820 complex is produced specifically in response to temperatures of typically 24”C, whereas in Rps. acidophila 10050, the only peripheral antenna complex produced is the B800-850 (Papiz et al., 1989). The B800-820 complex from Rps. acidophila strain 7750 is formed from the aggregation of four low molecular weight, pigment-binding apoproteins, namely the B800-820a,, B800-820a2, B800-8208,
and B800-820Pz. The primary structures of three of these apoproteins have been determined (Brunisholz et al., 1987). The B800-820a, has 53 amino acid residues with a molecular weight of 5567 daltons, the B800-820p1 has 43 amino acid residues and a molecular weight of 4721 daltons, and the B800-820& has 41 amino acid residues and a molecular weight of 4520 daltons. The B800-820a, is as yet unsequenced. The stoichiometric ratio of bacteriochlorophyll alcarotenoids is 2 : 1 (Evans, 1989). Cells from Rps. acidophila 7750 were grown photosynthetically at 24°C on Pfennigs medium (Pfennig, 1969). These conditions yield the B880 core complex and also the B800-820 lightharvesting complex. The cells were centrifuged at 2800 revs/min for 100 minutes at 4°C using a COOLSPIN centrifuge, and the resulting pellets were retained and combined using 50 ml of 61 MKC1 in 20 mM-Mes buffer (pH 6.8). The combined pellets were then centrifuged at 12,000 revs/min using an MSE18 centrifuge. Again, the pellets were retained and homogenized in 50 ml of 20 mMTris. HCl (pH 8.0). The broken cells were then centrifuged at 12,000 revs/min for 20 minutes at 4°C and the pellet retained, while the supernatant was spun at 45,000 revs/min for one hour. The pellet formed from the high speed spin was combined with the low-speed spin and the absorbance at 800 nm was adjusted to 50 with 20 mM-Tris. HCl (pH 8.0). The diluted membrane solution was then made 1 y0 (v/v) with lauryl dimethyl N-amide oxide (LDAO), by stirring at room temperature for ten minutes. 527
0022-2836/92/060527-02
$03.00/O
crystallography
0
1992 Academic
Press Limited
528
N. Quthrie
This solution was then centrifuged at 12,000 revs/ min for ten minutes at 4°C to remove any unsolubilized material (Cogdell et aE., 1983). A Whatman DE52 anion exchange column was used to separate the core and light-harvesting complexes. The column was pre-equilibrated with 20 mr\l-Tris . HCl (pH 8.0) and then loaded with the membrane solution. Elution of the light-harvesting complexes took place using increasing concentrations of NaCl (50, 100, 150, 250 mM, all plus 61 y. (v/v) LDAO), with 1 ml fractions assayed spectrophotomerically using the ratio of the absorbance of bacteriochlorophyll at 800 nm to the absorbance of aromatic amino acids at 270 nm as a measurement of purity. After two columns, ratios of 2 : 1 or greater were pooled. A 1 m Sephacryl S-200 column was used to further purify the complex and ratios of 3 : 1 or greater were pooled. The complex was then concentrated on a mini DE52 column (poured in a Pasteur pipette), washed with four column volumes of Tris. HCl (pH 80) to remove free LDAO, and the remaining detergent exchanged wit’h one column volume of 1 y. (w/v) /I-octyl glucoside. The pure complex was then eluted using 350 rnN-NaCI in 1 y. (w/v) p-octyl glucoside and 20 m&r-Tris * HCl (pH %O). Crystals were grown using the sitting drop method. A 25 ~1 drop containing approximately 8.0 mg protein/ml, 1.0 M-di-potassium hydrogen orthophosphate and 2.5 */* (w/v) benzamidine hydrochloride in Tris . HCl at pH 9.0, was equilibrated by vapour diffusion against an 8 ml well of 2.1 M-ammonium sulphate in Tris . HCl (pH 9.0). Crystals grew at 18 “C as tabular plates to a size of 69 mm x 94 mm x 0.2 mm over a period of six weeks. The crystals were soaked in 2 M-di-potassium hydrogen orthophosphate at pH 9.5 for 30 minutes before mounting in 1 mm glass capillary tubes. Diffraction data were collected on a Siemens area detector mounted on a rotating anode source, operating at 50 kV and 80 mA. The crystal diffracted to a resolution limit of 5 A (1 A = 0.1 nm), and data were collected over a rotation range of 108.75” with a step size of 0.25”. The previously unknown cell dimensions were obtained by the automatic indexing procedure (Howard et al., 1985), which hexagonal dimensions cell gave a with a= b = 121.8 A and c = 283.1 A. The data were integrated using the XDS program (Kabsch, 1988a,b), giving: R symm= i
CIh,i
’
a value of 0.066 for the space group R32. This is a similar cell and space group to that of the B800-850 antenna complex from Rps. acidophila 10050 (Papiz et al., 1989; a= b = 121.1 A, c = 296.7 A and space group R32). The computed V, value is 2.2 A3/Da, assuming a molecular mass of 92,000 daltons in the asymmetric unit. This falls within the range reported by 1Matthews (1977), and gives a solvent composition of 44 %. The close similarity in the unit cell and space
Edited
et al. group of these protein crystals and crystals of the B800-850 antenna complex suggests a similar asymmetric unit in both cases. Papiz et al. (l9S9) propose an asymmetric unit containing six &,/P subunits, 18 bacteriochlorophyll and nine carotenoid molecules for the B800-850 complex. The asymmetric unit of the B800-820 light-harvesting complex would then be a trimer of molecular assemblies, with each assembly consisting of a, aaPl /IZ proteins, six bacteriochlorophyll and three carotenoid molecules. This work Initiative.
is supported
by the
SERC
Membrane
References Allen, J. P., Feher, G., Yeates, T. O.? Komiya, II. B Rees, D. C. (1987). Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc. Nat. Acad. Sci., U.S.A. 84. 5730-5734. Brunisholz, R. A., Bissig, I., Miederer; E., Suter, F. & Zuber, H. (1987). Structural studies on the lightharvesting polypeptides of Rhodopseudomonas acidophz’la. In Progress in Photosynthesis Research (Biggins, J., ed.), vol. 2> sect. 1; 13-16, Martinus Nijhoff, The Hague. Cogdell, R. J., Durant, I.; Valentine, J., Lindsay, J. G. & Schmidt, K. (1983). The isolation and partial characterisation of the light-harvesting pigment-protein complement from Rhodopseudormonas acidophila strain 10050. Biochim. Biophys. Acta, 722, 427-455. Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel, H. (1985). Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 A resolut,ion. Nature (London), 318, 618-624. Evans: M. B. (1989). The structure and function of the light-harvesting antenna complexes from purple photosynthetic bacteria. Ph.D. thesis, Glasgow University. Howard, A. J., Nielsen, C. & Xuong, Tu’g, H. (19S5). Software for a diffractometer with multiwire area detector. Methods Enzymol. 114, 211-237. Kabsch, W. (1988a). Automatic indexing of rotation 21, 67-71. diffraction patterns. J. Appl. Crystallogr. Kabsch, W. (19883). Automatic indexing of rotation diffraction patterns. J. Appl. Crystallogr. 21; 916-924. Matthews, B. W. (1977). In Proteins (Keurath, R, $ Hill; R. L., eds), voi. 3, pp. 4033590, Academic Press, London. Michef, H., Epp, 0. & Deisenhofer, J. (1986). Pigmentprotein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis. EMBO J. 5, 2445-245 I. Papiz, M. Z., Hawthornthwaite, A. M., Cogdell, R. J., Wooley, K. J., Wightman, P. A., Ferguson, L. A. & Lindsay, J. G. (1989). Crystallisation and characterisation of two crystal forms of the B800-850 lightharvesting complex from RhodopseudomorLas acidophila strain 10050. J. Mol. Biol. 209, 833-835. Pfennig? N. (1969). Rhodopseudomonas acidophila, sp. 3.: a new species of the budding purple non-sulfur bacteria,. J. Bacterial. 99. 597-602. Zuber, H. (1985). Structure and function of lightharvesting complexes and their polypeptides. Photochem. Pfwtobiol. 42, 821-844.
by A. Klug
