Photosynthesis Research

, Volume 111, Issue 1–2, pp 29–39

Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

  • Yuliya Miloslavina
  • Petar H. Lambrev
  • Tamás Jávorfi
  • Zsuzsanna Várkonyi
  • Václav Karlický
  • Joseph S. Wall
  • Geoffrey Hind
  • Győző Garab
Regular Paper


In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment–pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while “psi-type” CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.


Anisotropic circular dichroism Magnetic circular dichroism Psi-type circular dichroism Thylakoid membranes Grana patches Light-harvesting complexes 



Anisotropic circular dichroism


Photosystem II-enriched grana membrane fragments, isolated according to Berthold, Babcock and Yocum


Circular dichroism


Excitonic circular dichroism


Psi-type circular dichroism




Linear dichroism


Light-harvesting complex of Photosystem II


Magnetic circular dichroism


Photosystem I, II


Scanning transmission electron microscopy


  1. Abdourakhmanov IA, Ganago AO, Erokhin YE, Solovev AA, Chugunov VA (1979) Orientation and linear dichroism of the reaction centers from Rhodopseudomonas-sphaeroides R-26. Biochem Biophys Acta 546:183–186PubMedCrossRefGoogle Scholar
  2. Barzda V, Mustárdy L, Garab G (1994) Size dependency of circular dichroism in macroaggregates of photosynthetic pigment–protein complexes. Biochemistry 33:10837–10841PubMedCrossRefGoogle Scholar
  3. Barzda V, Istokovics A, Simidjiev I, Garab G (1996) Structural flexibility of chiral macroaggregates of light-harvesting chlorophyll a/b pigment–protein complexes. Light-induced reversible structural changes associated with energy dissipation. Biochemistry 35:8981–8985PubMedCrossRefGoogle Scholar
  4. Bloemendal M, van Grondelle R (1993) Linear-dichroism spectroscopy for the study of structural-properties of proteins. Mol Biol Rep 18:49–69PubMedCrossRefGoogle Scholar
  5. Breton J, Vermeglio A (1982) Orientation of photosynthetic pigments in vivo. In: Govindjee R (ed) Photosynthesis. 1: energy conversion by plants and bacteria. Academic Press, New York, pp 153–194Google Scholar
  6. Büchel C, Garab G (1997) Organization of the pigment molecules in the chlorophyll a/c light-harvesting complex of Pleurochloris meiringensis (xanthophyceae). Characterization with circular dichroism and absorbance spectroscopy. J Photochem Photobiol B 37:118–124CrossRefGoogle Scholar
  7. Clayton RK (1980) Photosynthesis: physical mechanism and chemical patterns. Cambridge University Press, CambridgeGoogle Scholar
  8. Cogdell RJ, Scheer H (1985) Circular dichroism of light-harvesting complexes from purple photosynthetic bacteria. Photochem Photobiol 42:669–678CrossRefGoogle Scholar
  9. Croce R, Morosinotto T, Ihalainen JA, Chojnicka A, Breton J, Dekker JP, van Grondelle R, Bassi R (2004) Origin of the 701-nm fluorescence emission of the Lhca2 subunit of higher plant photosystem I. J Biol Chem 279:48543–48549PubMedCrossRefGoogle Scholar
  10. Davidsson A, Nordén B, Seth S (1980) Measurement of oriented circular dichroism. Chem Phys Lett 70:313–316CrossRefGoogle Scholar
  11. Disch RL, Sverdlik DI (1969) Apparent circular dichroism of oriented systems. Anal Chem 41:82–86CrossRefGoogle Scholar
  12. Frank HA, Violette CA, Taremi SS, Budil DE (1989) Linear dichroism of single crystals of the reaction center from Rhodobacter sphaeroides wild type strain 2.4.1. Photosynth Res 21:107–116Google Scholar
  13. Garab G (1996) Linear and circular dichroism. In: Amesz J, Hoff AJ (eds) Biophysical techniques in photosynthesis. Kluwer, Dordrecht, pp 11–40Google Scholar
  14. Garab G, van Amerongen H (2009) Linear dichroism and circular dichroism in photosynthesis research. Photosynth Res 101:135–146PubMedCrossRefGoogle Scholar
  15. Garab G, Faludi-Daniel A, Sutherland JC, Hind G (1988a) Macroorganization of chlorophyll a/b light-harvesting complex in thylakoids and aggregates: information from circular differential scattering. Biochemistry 27:2425–2430CrossRefGoogle Scholar
  16. Garab G, Leegood RC, Walker DA, Sutherland JC, Hind G (1988b) Reversible changes in macroorganization of the light-harvesting chlorophyll a/b pigment protein complex detected by circular-dichroism. Biochemistry 27:2430–2434CrossRefGoogle Scholar
  17. Garab G, Wells S, Finzi L, Bustamante C (1988c) Helically organized macroaggregates of pigment protein complexes in chloroplasts—evidence from circular intensity differential scattering. Biochemistry 27:5839–5843PubMedCrossRefGoogle Scholar
  18. Garab G, Kieleczawa J, Sutherland JC, Bustamante C, Hind G (1991) Organization of pigment-protein complexes into macrodomains in the thylakoid membranes of wild-type and chlorophyll b-less mutant of barley as revealed by circular dichroism. Photochem Photobiol 54:273–281CrossRefGoogle Scholar
  19. Garab G, Galajda P, Pomozi I, Finzi L, Praznovszky T, Ormos P, van Amerongen H (2005) Alignment of biological microparticles by a polarized laser beam. Eur Biophys J Biophys Lett 34:335–343CrossRefGoogle Scholar
  20. Georgakopoulou S, van der Zwan G, Bassi R, van Grondelle R, van Amerongen H, Croce R (2007) Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization. Biochemistry 46:4745–4754PubMedCrossRefGoogle Scholar
  21. Goss R, Wilhelm C, Garab G (2000) Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy. Biochim Biophys Acta 1457:190–199PubMedCrossRefGoogle Scholar
  22. Hobe S, Prytulla S, Kühlbrandt W, Paulsen H (1994) Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex. Embo J 13:3423–3429PubMedGoogle Scholar
  23. Hofrichter J, Eaton WA (1976) Linear dichroism of biological chromophores. Ann Rev Biophys Bioeng 5:511–560CrossRefGoogle Scholar
  24. Istokovics A, Simidjiev I, Lajko F, Garab G (1997) Characterization of the light induced reversible changes in the chiral macroorganization of the chromophores in chloroplast thylakoid membranes. Temperature dependence and effect of inhibitors. Photosynth Res 54:45–53CrossRefGoogle Scholar
  25. Johansson LBA, Lindblom G (1980) Orientation and mobility of molecules in membranes studied by polarized light spectroscopy. Quart Rev Biophys 13:63–118CrossRefGoogle Scholar
  26. Keller D, Bustamante C (1986) Theory of the interaction of light with large inhomogeneous molecular aggregates. II. Psi-type circular dichroism. J Chem Phys 84:2972–2980CrossRefGoogle Scholar
  27. Kovacs L, Damkjaer J, Kereïche S, Ilioaia C, Ruban AV, Boekema EJ, Jansson S, Horton P (2006) Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts. Plant Cell 18:3106–3120PubMedCrossRefGoogle Scholar
  28. Kuball HG (2002) CD and ACD spectroscopy on anisotropic samples: Chirality of oriented molecules and anisotropic phases—a critical analysis. Enantiomer 7:197–205PubMedCrossRefGoogle Scholar
  29. Kuball HG, Höfer T (2000a) Chirality and circular dichroism of oriented molecules and anisotropic phases. Chirality 12:278–286PubMedCrossRefGoogle Scholar
  30. Kuball HG, Höfer T (2000b) Circular dichroism of oriented molecules. In: Berova N, Nakanishi K, Woody RW (eds) Circular dichroism: principles and applications. Wiley-VCH, New York, pp 133–157Google Scholar
  31. Kuball HG, Sieber G, Neubrech S, Schultheis B, Schonhofer A (1993) Circular-dichroism of oriented molecules—magnetic dipole and electric quadrupole contribution to the ACD of chirally substituted diaminoanthraquinones. Chem Phys 169:335–350CrossRefGoogle Scholar
  32. Kuball HG, Dorr E, Höfer T, Turk O (2005) Exciton chirality method. Oriented molecules—anisotropic phases. Monatshefte fur Chemie 136:289–324CrossRefGoogle Scholar
  33. Kühlbrandt W, Thaler T, Wehrli E (1983) The structure of membrane crystals of the light-harvesting chlorophyll a/b protein complex. J Cell Biol 96:1414–1424PubMedCrossRefGoogle Scholar
  34. Lambrev P, Várkonyi Z, Krumova S, Kovács L, Miloslavina Y, Holzwarth AR, Garab G (2007) Importance of trimer–trimer interactions for the native state of the plant light-harvesting complex II. Biochim Biophys Acta 1767:847–853PubMedCrossRefGoogle Scholar
  35. Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W (2004) Crystal structure of spinach major light-harvesting complex at 2.72 angstrom resolution. Nature 428:287–292PubMedCrossRefGoogle Scholar
  36. Morosinotto T, Breton J, Bassi R, Croce R (2003) The nature of a chlorophyll ligand in Lhca proteins determines the far red fluorescence emission typical of photosystem I. J Biol Chem 278:49223–49229PubMedCrossRefGoogle Scholar
  37. Morosinotto T, Segalla A, Giacometti GM, Bassi R (2010) Purification of structurally intact grana from plants thylakoids membranes. J Bioenerg Biomembr 42:37–45PubMedCrossRefGoogle Scholar
  38. Moya I, Silvestri M, Vallon O, Cinque G, Bassi R (2001) Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes. Biochem 40:12552–12561CrossRefGoogle Scholar
  39. Norden B, Kubista M, Kurucsev T (1992) Linear dichroism spectroscopy of nucleic-acids. Quart Rev Biophys 25:51–170CrossRefGoogle Scholar
  40. Novoderezhkin VI, Palacios MA, van Amerongen H, van Grondelle R (2005) Excitation dynamics in the LHCII complex of higher plants: modeling based on the 2.72 angstrom crystal structure. J Phys Chem B 109:10493–10504PubMedCrossRefGoogle Scholar
  41. Pearlstein RM (1991) Theoretical interpretation of antenna spectra. In: Scheer H (ed) Chlorophylls. CRC Press, New York, pp 1047–1078Google Scholar
  42. Schellman J, Jensen HP (1987) Optical spectroscopy of oriented molecules. Chem Rev 87:1359–1399CrossRefGoogle Scholar
  43. Simidjiev I, Barzda V, Mustárdy L, Garab G (1997) Isolation of lamellar aggregates of the light-harvesting chlorophyll a/b protein complex of photosystem II with long-range chiral order and structural flexibility. Anal Biochem 250:169–175PubMedCrossRefGoogle Scholar
  44. Standfuss J, van Scheltinga ACT, Lamborghini M, Kühlbrandt W (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5A resolution. EMBO J 24:919–928PubMedCrossRefGoogle Scholar
  45. Steinbach G, Besson F, Pomozi I, Garab G (2005) Differential polarization laser scanning microscopy: biological applications. Proc SPIE 5969:59692CCrossRefGoogle Scholar
  46. Sutherland JC (1978) The magnetic optical activity of porphyrins. The porphyrins. Academic Press, New York, pp 225–248Google Scholar
  47. Sutherland JC, Holmquist B (1980) Magnetic circular dichroism of biological molecules. Ann Rev Biophys Bioeng 9:293–326CrossRefGoogle Scholar
  48. Swenberg CE, Geacintov NE (1976) Selective light scattering from oriented photosynthetic membranes. In: Birks JB (ed) Excited states in biology. Wiley, London, pp 288–300Google Scholar
  49. Szabó M, Lepetit B, Goss R, Wihelm C, Mustárdy L, Garab G (2008) Structurally flexible macro-organization of the pigment-protein complexes of the diatom Phaeodactylum tricornutum. Photosynth Res 95:237–245PubMedCrossRefGoogle Scholar
  50. Tinoco I, Mickols W, Maestre MF, Bustamante C (1987) Absorption, scattering, and imaging of biomolecular structures with polarized-light. Annu Rev Biophys Biophys Chem 16:319–349PubMedCrossRefGoogle Scholar
  51. Tunis-Schneider MJ, Maestre MF (1970) Circular dichroism spectra of oriented and unoriented deoxyribonucleic acid films—a preliminary study. J Mol Biol 52:521–541PubMedCrossRefGoogle Scholar
  52. van Amerongen H, Struve WS (1995) Polarized optical spectroscopy of chromoproteins. In: Sauer K (ed) Methods in enzymology vol. 246 biochemical spectroscopy. Academic Press, San Diego, pp 259–283Google Scholar
  53. van Amerongen H, Valkunas L, van Grondelle R (2000) Photosynthetic excitons. World Scientific, SingaporeCrossRefGoogle Scholar
  54. Völker S, Ono T, Inoue Y, Renger G (1985) Effect of trypsin on PS-II particles—correlation between Hill-activity, Mn-abundance and peptide pattern. Biochim Biophys Acta 806:25–34CrossRefGoogle Scholar
  55. Yang C, Boggasch S, Haase W, Paulsen H (2006) Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids. Biochim Biophys Acta 1757:1642–1648PubMedCrossRefGoogle Scholar
  56. Yang C, Lambrev P, Chen Z, Jávorfi T, Kiss AZ, Paulsen H, Garab G (2008) The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II. Biochim Biophys Acta 1777:1463–1470PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Yuliya Miloslavina
    • 1
  • Petar H. Lambrev
    • 1
  • Tamás Jávorfi
    • 2
  • Zsuzsanna Várkonyi
    • 1
  • Václav Karlický
    • 1
  • Joseph S. Wall
    • 3
  • Geoffrey Hind
    • 3
  • Győző Garab
    • 1
  1. 1.Institute of Plant Biology, Biological Research CenterHungarian Academy of SciencesSzegedHungary
  2. 2.Diamond Light Source Ltd.DidcotUK
  3. 3.Biology DepartmentBrookhaven National LaboratoryUptonUSA

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