, Volume 181, Issue 2, pp 204–211 | Cite as

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

  • H. Paulsen
  • U. Rümler
  • W. Rüdiger


A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding.

Key words

Antenna complex Expression plasmids Light-harvesting chlorophyll a/b-binding protein Pigment-protein assembly Pisum (light-harvesting complex) Thylakoid biogenesis 



circular dichrosim




isopropyl thiogalactoside


lithium dodecyl sulfate


light-harvesting complex associated with photosystem II


light-harvesting chlorophyll a/b-binding protein


LHCP derivatives overproduced in bacteria


precursor of light-harvesting Chl a/b-binding protein


sodium dodecyl sulfate




Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anderson, J., Waldron, J.C., Thorne, S.W. (1978) Chlorophyll-protein complexes of spinach and barley thylakoids. FEBS Lett.92, 227–233CrossRefGoogle Scholar
  2. Bennett, J. (1983) Regulation of photosynthesis by reversible, phosphorylation of the light-harvesting chlorophyll a/b protein. Biochem. J.212, 1–13PubMedGoogle Scholar
  3. Britton, G., Goodwin, T.W. (1971) Biosynthesis of carotenoids. Methods Enzymol.18C, 654–701Google Scholar
  4. Buctow, D.E., Chen, H., Erdös, G., Yi, L.S.H. (1988) Regulation and expression of the multigene family coding light-harvesting chlorophyll a/b-binding proteins of photosystem II. Photosynth. Res.18, 61–97CrossRefGoogle Scholar
  5. Bujard, H., Gentz, R., Lanzer, M., Stueber, D., Mueller, M., Ibrahimi, I., Haeuptle, M.T., Dobberstein, B. (1987) A T5 promoter-based transcription-translation system for the analysis of protein in vivo and in vitro. Methods Enzymol.155, 416–433PubMedGoogle Scholar
  6. Butler, P.J.G., Kühlbrandt, W. (1988) Determination of the aggregate size in detergent solution of the light-harvesting chlorophyll a/b-protein complex from chloroplast membranes. Proc. Natl. Acad. Sci. USA85, 3797–3801PubMedGoogle Scholar
  7. Cashmore, A.R. (1984) Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide. Proc. Natl. Acad. Sci. USA81, 2960–2964PubMedGoogle Scholar
  8. Chitnis, P.R., Thornber, J.P. (1988) The major light-harvesting complex of photosystem II: aspects of its molecular and cell biology. Photosynth. Res.16, 41–43CrossRefGoogle Scholar
  9. Chitnis, P.R., Harel, E., Kohorn, B.D., Tobin, E.M., Thornber, J.P. (1986) Assembly of the precursor and processed light-harvesting chlorophyll a/b protein ofLemna into the light-harvesting complex II of barley etiochloroplasts. J. Cell Biol102, 982–988CrossRefPubMedGoogle Scholar
  10. Chitnis, P.R., Nechushtai, R., Thornber, J.P. (1987) Insertion of the precursor of the light-harvesting chlorophyll a/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor. Plant Mol. Biol.10, 3–11CrossRefGoogle Scholar
  11. Cline, K. (1988) Light-harvesting chlorophyll a/b protein. Membrane insertion, proteolytic processing, assembly into LHCII, and localization to appressed membranes occurs in chloroplast lysates. Plant Physiol.86, 1120–1126PubMedGoogle Scholar
  12. Davies, B.H. (1965) Analysis of carotinoid pigments. In: Chemistry and biochemistry of plant pigments, pp. 489–532. Goodwin, T.W., ed. Academic Press, New YorkGoogle Scholar
  13. Ellis, J. (1987) Proteins, as molecular chaperones. Nature328, 378–379CrossRefPubMedGoogle Scholar
  14. Goodwin, T.W. (1980) The biochemistry of the carotinoids. Chapman and Hall, New YorkGoogle Scholar
  15. Gülen, D., Knox, R., Breton, J. (1986) Optical effects of sodium dodecyl sulfate treatment of the isolated light harvesting complex of higher plants. Photosynth. Res.9, 13–20CrossRefGoogle Scholar
  16. Ide, J.P., Klug, D.R., Kühlbrandt, W., Giorgi, L.B., Porter, G. (1987) The state of detergent solubilised light-harvesting chlorophyll-a/b protein complex as monitored by picosecond time-resolved fluorescence and circular dichroism. Biochim. Biophys. Acta893, 34–364Google Scholar
  17. Kohorn, B.D., Tobin, E.M. (1989) A hydrophobic, carboxy-proximal region of a light-harvesting chlorophyll a/b protein is necessary for stable integration into thylakoid membranes. Plant Cell1, 159–166CrossRefPubMedGoogle Scholar
  18. Kohorn, B.D., Harel, E., Chitnis, P.R., Thornber, J.P., Tobin, E. (1986) Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes. J. Cell Biol.102, 972–981CrossRefPubMedGoogle Scholar
  19. Kühlbrandt, W., Downing, K.H. (1989) Two-dimensional structure of plant light-harvesting complex at 3.7 Å resolution by electron crystallography. J. Mol. Biol.207, 823–828CrossRefPubMedGoogle Scholar
  20. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685CrossRefPubMedGoogle Scholar
  21. Lin, S., Knox, R.S. (1988) Applications of a CPII model to excitation transfer experiments on light-harvesting chlorophyll a/b-protein complexes. FEBS Lett.229, 1–15CrossRefGoogle Scholar
  22. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  23. Mattoo, A.K., Edelman, M. (1987) Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein. Proc. Natl. Acad. Sci. USA84, 1497–1501PubMedGoogle Scholar
  24. Nagai, K., Thogersen, H.C. (1987) Synthesis and sequence-specific proteolysis of hybrid proteins produced inE. coli. Methods Enzymol.153, 461–481PubMedCrossRefGoogle Scholar
  25. Peter, G.F., Thornber, J.P. (1988) The antenna components of photosystem. II with emphasis on the major pigment-protein LHCPb. In: Photosynthetic light-harvesting systems, pp. 175–186, Scheer, H., Schneider, S., eds. Walther de Gruyter, BerlinGoogle Scholar
  26. Plumley, F.G., Schmidt, G.W. (1987) Reconstitution of chlorophyll a/b light-harvesting complexes: Xanthophyll-dependent assembly and energy transfer. Proc. Natl. Acad. Sci. USA84, 146–150PubMedGoogle Scholar
  27. Porra, P.J., Thompson, W.A., Kriedemann, P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta975, 384–394Google Scholar
  28. Sauer, K., Scheer, H. (1988) Energy transfer calculations for two C-phycocyanins based on refined X-ray crystal structure coordinates of chromophores. In: Photosynthetic light-harvesting systems, pp. 507–511, Scheer, H., Schneider, S., eds., Walther de Gruyter, BerlinGoogle Scholar
  29. Sayre, R., Cheniae, G.M. (1982) Studies on the regulation of O2 evolution of chloroplasts. Plant Physiol.69, 1084–1095PubMedCrossRefGoogle Scholar
  30. Schmidt, G.W., Bartlett, S.G., Grossman, A.R., Cashmore, A.R., Chua, N.-H. (1982) Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b-protein complex. J. Cell. Biol.91, 468–478CrossRefGoogle Scholar
  31. Thornber, J.P. (1986) Biochemical characterization and structure of pigment-proteins of photosynthetic organisms. In: Encyclopedia of plant physiology, N.S., vol. 2, pp. 98–142, Staehelin, L.A., Arntzen, E.J., eds. Springer, Berlin Heidelberg New YorkGoogle Scholar
  32. Winston, S.E., Fuller, S.A., Hurrell, J.G.R. (1987) Western blotting. In: Current protocols in molecular biology, vol. 2, pp. 10.8.1–10.8.6, Ausubel, F.M., Brent, R., Kingston R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., eds. Wiley, New YorkGoogle Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • H. Paulsen
    • 1
  • U. Rümler
    • 1
  • W. Rüdiger
    • 1
  1. 1.Botanisches Institut III der UniversitätMünchen 19Germany

Personalised recommendations