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. 1994 Aug 1;13(15):3423–3429. doi: 10.1002/j.1460-2075.1994.tb06647.x

Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex.

S Hobe 1, S Prytulla 1, W Kühlbrandt 1, H Paulsen 1
PMCID: PMC395244  PMID: 8062818

Abstract

The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions. Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen K. D., Staehelin L. A. Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach. Plant Physiol. 1992 Nov;100(3):1517–1526. doi: 10.1104/pp.100.3.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amos L. A., Henderson R., Unwin P. N. Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol. 1982;39(3):183–231. doi: 10.1016/0079-6107(83)90017-2. [DOI] [PubMed] [Google Scholar]
  3. Andersson L. A., Loehr T. M., Cotton T. M., Simpson D. J., Smith K. M. Spectroscopic analysis of chlorophyll model complexes: methyl ester ClFe(III)pheophorbides. Biochim Biophys Acta. 1989 May 8;974(2):163–179. doi: 10.1016/s0005-2728(89)80369-x. [DOI] [PubMed] [Google Scholar]
  4. Bassi R., Pineau B., Dainese P., Marquardt J. Carotenoid-binding proteins of photosystem II. Eur J Biochem. 1993 Mar 1;212(2):297–303. doi: 10.1111/j.1432-1033.1993.tb17662.x. [DOI] [PubMed] [Google Scholar]
  5. Burke J. J., Ditto C. L., Arntzen C. J. Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts. Arch Biochem Biophys. 1978 Apr 15;187(1):252–263. doi: 10.1016/0003-9861(78)90031-0. [DOI] [PubMed] [Google Scholar]
  6. Cashmore A. R. Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide. Proc Natl Acad Sci U S A. 1984 May;81(10):2960–2964. doi: 10.1073/pnas.81.10.2960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eisele J. L., Rosenbusch J. P. In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation. J Biol Chem. 1990 Jun 25;265(18):10217–10220. [PubMed] [Google Scholar]
  8. Huang K. S., Bayley H., Liao M. J., London E., Khorana H. G. Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments. J Biol Chem. 1981 Apr 25;256(8):3802–3809. [PubMed] [Google Scholar]
  9. Kühlbrandt W., Wang D. N., Fujiyoshi Y. Atomic model of plant light-harvesting complex by electron crystallography. Nature. 1994 Feb 17;367(6464):614–621. doi: 10.1038/367614a0. [DOI] [PubMed] [Google Scholar]
  10. Kühlbrandt W., Wang D. N. Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature. 1991 Mar 14;350(6314):130–134. doi: 10.1038/350130a0. [DOI] [PubMed] [Google Scholar]
  11. London E., Khorana H. G. Denaturation and renaturation of bacteriorhodopsin in detergents and lipid-detergent mixtures. J Biol Chem. 1982 Jun 25;257(12):7003–7011. [PubMed] [Google Scholar]
  12. Mullet J. E., Klein R. R., Grossman A. R. Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates. Eur J Biochem. 1986 Mar 3;155(2):331–338. doi: 10.1111/j.1432-1033.1986.tb09495.x. [DOI] [PubMed] [Google Scholar]
  13. Nussberger S., Dörr K., Wang D. N., Kühlbrandt W. Lipid-protein interactions in crystals of plant light-harvesting complex. J Mol Biol. 1993 Nov 20;234(2):347–356. doi: 10.1006/jmbi.1993.1591. [DOI] [PubMed] [Google Scholar]
  14. Parkes-Loach P. S., Sprinkle J. R., Loach P. A. Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated alpha- and beta-polypeptides and bacteriochlorophyll a. Biochemistry. 1988 Apr 19;27(8):2718–2727. doi: 10.1021/bi00408a011. [DOI] [PubMed] [Google Scholar]
  15. Paulsen H., Finkenzeller B., Kühlein N. Pigments induce folding of light-harvesting chlorophyll a/b-binding protein. Eur J Biochem. 1993 Aug 1;215(3):809–816. doi: 10.1111/j.1432-1033.1993.tb18096.x. [DOI] [PubMed] [Google Scholar]
  16. Paulsen H., Hobe S. Pigment-binding properties of mutant light-harvesting chlorophyll-a/b-binding protein. Eur J Biochem. 1992 Apr 1;205(1):71–76. doi: 10.1111/j.1432-1033.1992.tb16752.x. [DOI] [PubMed] [Google Scholar]
  17. Paulsen H., Kuttkat A. Pigment complexes of light-harvesting chlorophyll a/b binding protein are stabilized by a segment in the carboxyterminal hydrophilic domain of the protein. Photochem Photobiol. 1993 Jan;57(1):139–142. doi: 10.1111/j.1751-1097.1993.tb02269.x. [DOI] [PubMed] [Google Scholar]
  18. Peter G. F., Thornber J. P. Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins. J Biol Chem. 1991 Sep 5;266(25):16745–16754. [PubMed] [Google Scholar]
  19. Plumley F. G., Schmidt G. W. Reconstitution of chlorophyll a/b light-harvesting complexes: Xanthophyll-dependent assembly and energy transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):146–150. doi: 10.1073/pnas.84.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Popot J. L., Trewhella J., Engelman D. M. Reformation of crystalline purple membrane from purified bacteriorhodopsin fragments. EMBO J. 1986 Nov;5(11):3039–3044. doi: 10.1002/j.1460-2075.1986.tb04603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ryrie I. J., Anderson J. M., Goodchild D. J. The role of the light-harvesting chlorophyll a/b protein complex in chloroplast membrane stacking. Cation-induced aggregation of reconstituted proteoliposomes. Eur J Biochem. 1980 Jun;107(2):345–354. doi: 10.1111/j.1432-1033.1980.tb06035.x. [DOI] [PubMed] [Google Scholar]
  22. Shipley G. G., Green J. P., Nichols B. W. The phase behavior of monogalactosyl, digalactosyl, and sulphoquinovosyl diglycerides. Biochim Biophys Acta. 1973 Jul 18;311(4):531–544. doi: 10.1016/0005-2736(73)90128-4. [DOI] [PubMed] [Google Scholar]
  23. Trewhella J., Popot J. L., Zaccaï G., Engelman D. M. Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction. EMBO J. 1986 Nov;5(11):3045–3049. doi: 10.1002/j.1460-2075.1986.tb04604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wang D. N., Kühlbrandt W. High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media. J Mol Biol. 1991 Feb 20;217(4):691–699. doi: 10.1016/0022-2836(91)90526-c. [DOI] [PubMed] [Google Scholar]

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