The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 1031–1041 | Cite as

Amphipols and Photosynthetic Light-Harvesting Pigment-Protein Complexes

  • Milena OpačićEmail author
  • Grégory Durand
  • Michael Bosco
  • Ange Polidori
  • Jean-Luc Popot


The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the “light-harvesting conformation”, whereas the “quenched conformation” is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30 % shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.


Neoxanthin Fluorescence Light-harvesting complexes Membrane protein A8-35 Non-ionic amphipols 



A specific type of polyacrylate-based amphipol










Circular dichroism




LHCII trimers






Molecular dynamics


Membrane protein


A specific batch of non-ionic APol


Non-ionic APol


Non-photochemical quenching


Size exclusion chromatography


Thiol-based transfer agent



Particular thanks are due to Roberta Croce (VU University Amsterdam) for her support and help throughout this work, as well as for contributing to its writing. We also thank Laura M. Roy and Bart Sasbrink (VU University Amsterdam) for critical reading of the manuscript, and Pengqi Xu for his help with generating Fig. 7. This project was supported by the Chemical Science division of the Netherlands organization for scientific research (NWO-CW) via an ECHO grant to R. Croce, by the CNRS, by University Paris-7, and by the “Initiative d’Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABX-0011-01).

Supplementary material

232_2014_9712_MOESM1_ESM.docx (2.3 mb)
Supplementary material 1 (DOCX 2361 kb)


  1. Althoff T, Mills DJ, Popot J-L, Kühlbrandt W (2011) Assembly of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J 30:4652–4664CrossRefGoogle Scholar
  2. Ballottari M, Girardon J, Dall’Osto L, Bassi R (2012) Evolution and functional properties of Photosystem II light harvesting complexes in eukaryotes. Biochim Biophys Acta 1817:143–157CrossRefGoogle Scholar
  3. Barros T, Royant A, Standfuss J, Dreuw A, Kühlbrandt W (2009) Crystal structure of plant light-harvesting complex shows the active, energy-transmitting state. EMBO J 28:298–306CrossRefGoogle Scholar
  4. Bassi R, Simpson D (1987) Chlorophyll-protein complexes of barley photosystem-I. Eur J Biochem 163:221–230CrossRefGoogle Scholar
  5. Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Banères J-L, Durand G, Zito F, Pucci B, Popot J-L (2012) Non-ionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution NMR. Biochemistry 51:1416–1430CrossRefGoogle Scholar
  6. Belgio E, Johnson MP, Jurić S, Ruban AV (2012) Higher plant photosystem II light-harvesting antenna, not the reaction center, determines the excited-state lifetime-both the maximum and the nonphotochemically quenched. Biophys J 102:2761–2771CrossRefGoogle Scholar
  7. Bowie JU (2001) Stabilizing membrane proteins. Curr Opin Struct Biol 11:397–402CrossRefGoogle Scholar
  8. Breyton C, Tribet C, Olive J, Dubacq J-P, Popot J-L (1997) Dimer to monomer conversion of the cytochrome b6 f complex: causes and consequences. J Biol Chem 272:21892–21900CrossRefGoogle Scholar
  9. Caffarri S, Croce R, Cattivelli L, Bassi R (2004) A look within LHCII: differential analysis of the Lhcbl-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis. Biochemistry 43:9467–9476CrossRefGoogle Scholar
  10. Caffarri S, Passarini F, Bassi R, Croce R (2007) A specific binding site for neoxanthin in the monomeric antenna proteins CP26 and CP29 of Photosystem II. FEBS Lett 581:4704–4710CrossRefGoogle Scholar
  11. Croce R, Zucchelli G, Garlaschi FM, Bassi R, Jennings RC (1996) Excited state equilibration in the photosystem I light-harvesting I complex: P700 is almost isoenergetic with its antenna. Biochemistry 35:8572–8579CrossRefGoogle Scholar
  12. Croce R, Remelli R, Varotto C, Breton J, Bassi R (1999a) The neoxanthin binding site of the major light harvesting complex (LHCII) from higher plants. FEBS Lett 456:1–6CrossRefGoogle Scholar
  13. Croce R, Weiss S, Bassi R (1999b) Carotenoid-binding sites of the major light-harvesting complex II of higher plants. J Biol Chem 274:29613–29623CrossRefGoogle Scholar
  14. Croce R, Canino G, Ros F, Bassi R (2002) Chromophore organization in the higher-plant photosystem II antenna protein CP26. Biochemistry 41:7334–7343CrossRefGoogle Scholar
  15. Croce R, van Amerongen H (2011) Light-harvesting and structural organization of photosystem II: from individual complexes to thylakoid membrane. J Photochem Photobiol, B 104:142–153CrossRefGoogle Scholar
  16. Dahmane T, Giusti F, Catoire LJ, Popot J-L (2011) Sulfonated amphipols: synthesis, properties and applications. Biopolymers 95:811–823CrossRefGoogle Scholar
  17. Dahmane T, Rappaport F, Popot J-L (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences. Eur Biophys J 42:85–101CrossRefGoogle Scholar
  18. Diab C, Tribet C, Gohon Y, Popot J-L, Winnik FM (2007) Complexation of integral membrane proteins by phosphorylcholine-based amphipols. Biochim Biophys Acta 1768:2737–2747CrossRefGoogle Scholar
  19. Dobrikova AG, Várkonyi Z, Krumova SB, Kovács L, Kostov GK, Todinova SJ, Busheva MC, Taneva SG, Garab G (2003) Structural rearrangements in chloroplast thylakoid membranes revealed by differential scanning calorimetry and circular dichroism spectroscopy. Thermo-optic effect. Biochemistry 42:11272–11280CrossRefGoogle Scholar
  20. Etzkorn M, Zoonens M, Catoire LJ, Popot J-L, Hiller S (2014) How amphipols embed membrane proteins: global solvent accessibility and interaction with a flexible protein terminus. J Membr Biol. doi: 10.1007/s00232-014-9657-9 CrossRefGoogle Scholar
  21. Feinstein HE, Tifrea D, Sun G, Popot J-L, de la Maza LM, Cocco MJ (2014) Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis. J Membr Biol. doi: 10.1007/s00232-014-9693-5 CrossRefGoogle Scholar
  22. Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276:32403–32406CrossRefGoogle Scholar
  23. 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 46:4745–4754CrossRefGoogle Scholar
  24. Gilmore AM, Yamamoto HY (1991) Zeaxanthin formation and energy-dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport. Plant Physiol 96:635–643CrossRefGoogle Scholar
  25. Giusti F, Popot J-L, Tribet C (2012) Well-defined critical association concentration and rapid adsorption at the air/water interface of a short amphiphilic polymer, amphipol A8-35: a study by Förster resonance energy transfer and dynamic surface tension measurements. Langmuir 28:10372–10380CrossRefGoogle Scholar
  26. Giusti F, Rieger J, Catoire L, Qian S, Calabrese AN, Watkinson TG, Casiraghi M, Radford SE, Ashcroft AE, Popot J-L (2014) Synthesis, characterization and applications of a perdeuterated amphipol. J Membr Biol. doi: 10.1007/s00232-014-9656-x CrossRefGoogle Scholar
  27. Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot J-L (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22:1281–1290CrossRefGoogle Scholar
  28. Gohon Y, Dahmane T, Ruigrok R, Schuck P, Charvolin D, Rappaport F, Timmins P, Engelman DM, Tribet C, Popot J-L, Ebel C (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J 94:3523–3537CrossRefGoogle Scholar
  29. Jansson S (1999) A guide to the Lhc genes and their relatives in Arabidopsis. Trends Plant Sci 4:236–240CrossRefGoogle Scholar
  30. Johnson MP, Ruban AV (2009) Photoprotective energy dissipation in higher plants involves alteration of the excited state energy of the emitting chlorophyll(s) in the light harvesting antenna II (LHCII). J Biol Chem 284:23592–23601CrossRefGoogle Scholar
  31. Kleinschmidt JH, Popot J-L (2014) Folding and stability of integral membrane proteins in amphipols (submitted).CrossRefGoogle Scholar
  32. Lambrev PH, 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 1767:847–853CrossRefGoogle Scholar
  33. Levi V, Rossi JP, Echarte MM, Castello PR, Gonzalez Flecha FL (2000) Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions. J Membr Biol 173:215–225CrossRefGoogle Scholar
  34. Liao M, Cao E, Julius D, Cheng Y (2014) Single particle electron cryo-microscopy of a mammalian ion channel. Curr Opin Struct Biol 27:1–7CrossRefGoogle Scholar
  35. Liguori N, Roy LM, Opačić M, Durand G, Croce R (2013) Regulation of light-harvesting in the green alga Chlamydomonas reinhardtii: the C-terminus of LHCSR is the knob of a dimmer switch. J Am Chem Soc 135:18339–18342CrossRefGoogle Scholar
  36. Liu ZF, Yan HC, Wang KB, Kuang TY, Zhang JP, Gui LL, An XM, Chang WR (2004) Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428:287–292CrossRefGoogle Scholar
  37. Lund S, Orlowski S, de Foresta B, Champeil P, le Maire M, Møller JV (1989) Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum. J Biol Chem 264:4907–4915PubMedGoogle Scholar
  38. Miloslavina Y, Wehner A, Lambrev PH, Wientjes E, Reus M, Garab G, Croce R, Holzwarth AR (2008) Far-red fluorescence: a direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching. FEBS Lett 582:3625–3631CrossRefGoogle Scholar
  39. Morosinotto T, Mozzo M, Bassi R, Croce R (2005) Pigment-pigment interactions in Lhca4 antenna complex of higher plants photosystem I. J Biol Chem 280:20612–20619CrossRefGoogle Scholar
  40. 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. Biochemistry 40:12552–12561CrossRefGoogle Scholar
  41. Mozzo M, Morosinotto T, Bassi R, Croce R (2006) Probing the structure of Lhca3 by mutation analysis. Biochim Biophys Acta 1757:1607–1613CrossRefGoogle Scholar
  42. Müller P, Li XP, Niyogi KK (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol 125:1558–1566CrossRefGoogle Scholar
  43. Nagy JK, Kuhn Hoffmann A, Keyes MH, Gray DN, Oxenoid K, Sanders CR (2001) Use of amphipathic polymers to deliver a membrane protein to lipid bilayers. FEBS Lett 501:115–120CrossRefGoogle Scholar
  44. Natali A, Roy LM, Croce R (2014) In vitro reconstitution of light-harvesting complexes of plants and green algae. J Vis Exp. doi: 10.3791/51852
  45. Pandit A, Shirzad-Wasei N, Wlodarczyk LM, van Roon H, Boekema EJ, Dekker JP, de Grip WJ (2011) Assembly of the major light-harvesting complex II in lipid nanodiscs. Biophys J 101:2507–2515CrossRefGoogle Scholar
  46. Pascal AA, Liu ZF, Broess K, van Oort B, van Amerongen H, Wang C, Horton P, Robert B, Chang WR, Ruban A (2005) Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436:134–137CrossRefGoogle Scholar
  47. Perlmutter JD, Popot J-L, Sachs JN (2014) Molecular dynamics simulations of a membrane protein/amphipol complex. J Membr Biol. doi: 10.1007/s00232-014-9690-8 CrossRefGoogle Scholar
  48. Picard M, Dahmane T, Garrigos M, Gauron C, Giusti F, le Maire M, Popot J-L, Champeil P (2006) Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study. Biochemistry 45:1861–1869CrossRefGoogle Scholar
  49. Polovinkin V, Gushchin I, Balandin T, Chervakov P, Round E, Schevchenko V, Popov A, Borshchevskiy V, Popot J-L, Gordeliy V (2014) High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase. J Membr Biol. doi: 10.1007/s00232-014-9700-x CrossRefGoogle Scholar
  50. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775CrossRefGoogle Scholar
  51. Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Rappaport F, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408CrossRefGoogle Scholar
  52. Privé GG (2007) Detergents for the stabilization and crystallization of membrane proteins. Methods 41:388–397CrossRefGoogle Scholar
  53. Rosenbusch JP (2001) Stability of membrane proteins: relevance for the selection of appropriate methods for high-resolution structure determinations. J Struct Biol 136:144–157CrossRefGoogle Scholar
  54. Ruban AV, Berera R, Ilioaia C, van Stokkum IHM, Kennis JTM, Pascal AA, van Amerongen H, Robert B, Horton P, van Grondelle R (2007) Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450:575–578CrossRefGoogle Scholar
  55. Ruban AV, Johnson MP, Duffy CDP (2012) The photoprotective molecular switch in the photosystem II antenna. Biochim Biophys Acta 1817:167–181CrossRefGoogle Scholar
  56. Sharma KS, Durand G, Pucci B (2011) Synthesis and determination of polymerization rate constants of glucose-based monomers. Des Monomers Polym 14:499–513CrossRefGoogle Scholar
  57. Sharma KS, Durand G, Gabel F, Bazzacco P, Le Bon C, Billon-Denis E, Catoire LJ, Popot J-L, Ebel C, Pucci B (2012) Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation. Langmuir 28:4625–4639CrossRefGoogle Scholar
  58. Tifrea D, Pal S, Cocco MJ, Popot J-L, de la Maza LM (2014) Increased immuno accessibility of MOMP epitopes in a vaccine formulated with amphipols may account for the very robust protection elicited against a vaginal challenge with C. muridarum. J Immunol 192:5201–5213CrossRefGoogle Scholar
  59. Tribet C, Audebert R, Popot J-L (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93:15047–15050CrossRefGoogle Scholar
  60. Tribet C, Audebert R, Popot J-L (1997) Stabilization of hydrophobic colloidal dispersions in water with amphiphilic polymers: application to integral membrane proteins. Langmuir 13:5570–5576CrossRefGoogle Scholar
  61. Tribet C, Diab C, Dahmane T, Zoonens M, Popot J-L, Winnik FM (2009) Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins. Langmuir 25:12623–12634CrossRefGoogle Scholar
  62. van Oort B, van Hoek A, Ruban AV, van Amerongen H (2007) Aggregation of light-harvesting complex II leads to formation of efficient excitation energy traps in monomeric and trimeric complexes. FEBS Lett 581:3528–3532CrossRefGoogle Scholar
  63. van Oort B, Amunts A, Borst JW, van Hoek A, Nelson N, van Amerongen H, Croce R (2008) Picosecond fluorescence of intact and dissolved PSI-LHCI crystals. Biophys J 95:5851–5861CrossRefGoogle Scholar
  64. Wientjes E, Oostergetel GT, Jansson S, Boekema EJ, Croce R (2009) The role of Lhca complexes in the supramolecular organization of higher plant photosystem I. J Biol Chem 284:7803–7810CrossRefGoogle Scholar
  65. Wientjes E, van Amerongen H, Croce R (2013) LHCII is an antenna of both photosystems after long-term acclimation. Biochim Biophys Acta 1827:420–426CrossRefGoogle Scholar
  66. Zoonens M, Giusti F, Zito F, Popot J-L (2007) Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer. Implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46:10392–10404CrossRefGoogle Scholar
  67. Zoonens M, Popot J-L (2014) Amphipols for each season. J Membr Biol. doi: 10.1007/s00232-014-9666-8 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Milena Opačić
    • 1
    Email author
  • Grégory Durand
    • 2
    • 3
  • Michael Bosco
    • 2
    • 3
  • Ange Polidori
    • 2
    • 3
  • Jean-Luc Popot
    • 4
  1. 1.Department of Physics and Astronomy, Faculty of SciencesVU University AmsterdamAmsterdamThe Netherlands
  2. 2.Equipe Chimie Bioorganique et Systèmes AmphiphilesUniversité d’AvignonAvignonFrance
  3. 3.Institut des Biomolécules Max Mousseron (UMR 5247)Montpellier Cedex 05France
  4. 4.Unité Mixte de Recherche 7099, Centre National de la Recherche Scientifique and Université Paris 7Institut de Biologie Physico-Chimique (FRC 550)ParisFrance

Personalised recommendations