Prediction of the Structure of an Integral Membrane Protein: The Light-Harvesting Complex II of Rhodospirillum molischianum

  • Xiche Hu
  • Dong Xu
  • Kenneth Hamer
  • Klaus Schulten
  • Juergen Koepke
  • Hartmut Michel


We illustrate in this chapter how one proceeds to predict the structure of integral membrane proteins when a highly homologous structure is unknown. We focus here on the prediction of the structure of the light-harvesting complex II (LH-II) of Rhodospirillum molischianum, an integral membrane protein of 16 polypeptides aggregating and binding to 24 bacteriochlorophyll a’s and 12 lycopenes. Hydropathy analysis was performed to identify the putative transmembrane segments, which were independently verified by multiple sequence alignment propensity analyses and homology modeling. A consensus assignment for secondary structure was derived from a combination of all the prediction methods used. Transmembrane helices were built by comparative modeling. The resulting tertiary structures were then aggregated into a quaternary structure through molecular dynamics simulations and energy minimization under constraints provided by site directed mutagenesis and FT Resonance Raman spectra, as well as conservation of residues. The structure of LH-II, so determined, was an octamer of αβ heterodimers forming a ring with a diameter of 70 Å. We discuss how the resulting structure may be used to solve the phase problem in X-ray crystallography in a procedure called molecular replacement. We will also discuss the exciton structure which results from the circular arrangement of chlorophyls in LH-II.


Integral Membrane Protein Transmembrane Helix Transmembrane Segment Molecular Replacement Resonance Raman Spectrum 
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  1. Allen J, Yeates T, Komiya H, Rees D (1987): Structure of the reaction center from Rhodobacter sphaeroides R-26: The protein subunits. Proc Natl Acad Sci USA 84:6162PubMedCrossRefGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990): Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  3. Argos P, Rao J, Hargrave P (1982): Structural prediction of membrane-bound proteins. European Journal of Biochemistry 128:565PubMedCrossRefGoogle Scholar
  4. Arkin I, Adams P, MacKenzie K, Lemmon M, Brünger A, Engelman D (1994): Structural organization of the pentameric transmembrane α-helices of phospholamban, a cardiac ion channel. Embo Journal 13:4757PubMedGoogle Scholar
  5. Blundell TL, Sibanda BL, Sternberg MJ, Thornton JM (1987): Knowledge-based prediction of protein structures and the design of novel molecules. Nature 326:347PubMedCrossRefGoogle Scholar
  6. Boonstra AF, Visschers RW, Calkoen F, van Grondelle R, van Bruggen EF, Roekema EJ (1993): Structural characterization of the B800–850 and B875 light-harvesting antenna complexes from Rhodobacter-Sphaeroides by electron microscopy. Biochimica et Biophysica Acta 1142:181CrossRefGoogle Scholar
  7. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983): CHARMm: a program for macromolecular energy, minimization, and dynamics calculations.J Comp Chem 4(2): 187–217CrossRefGoogle Scholar
  8. Brünger AT (1990): Extension of molecular replacement: A new search strategy based on Patterson correlation refinement. Acta Cryst A46:46–57Google Scholar
  9. Briinger AT (1992): X-PLOR, Version 3.1 f A System for X-ray Crystallography and NMR. The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale UniversityGoogle Scholar
  10. Brunisholz RA, Wiemken V, Suter F, Bachofen R, Zuber H (1984): The light-harvesting polypeptides of Rhodospirillum rubrum. IL localisation of the amino-terminal regions of the light-harvesting polypeptides B870-« and B870-/? and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+. Hoppe-Seylers Zeitschrift fur Physiologische Chemie 365:689CrossRefGoogle Scholar
  11. Busetta B (1986): Examination of folding patterns for predicting protein topologies. Biochimica et Biophysica Acta 870:327PubMedCrossRefGoogle Scholar
  12. Bylina E, Robles S, Youvan D (1988): Directed mutations affecting the putative bacterio-chlorophyll-binding sites in the light-harvesting I antenna of Rhodobacter capsulatus. Israel Journal of Chemistry 28:73Google Scholar
  13. Chou P, Fasman G (1978): Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology and Related Areas of Molecular Biology 47:45PubMedGoogle Scholar
  14. Cohen FE, Abarbanel RM, Kuntz ID, Fletterick RJ (1986): Turn prediction in proteins using a pattern-matching approach. Biochemistry 25:266PubMedCrossRefGoogle Scholar
  15. Cornette J, Cease K, Margalit H, Spouge J, Berzofsky J, DeLisi C (1987): Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. Journal of Molecular Biology 195:659PubMedCrossRefGoogle Scholar
  16. Cowan SW, Schirmer T, Rummel G, Steiert M, Ghosh R, Pauptit RA, Jansonius, JN, Rosenbusch JP (1992): Crystal structures explain functional properties of 2 E. coli porins. Nature 358(6389):727–733PubMedCrossRefGoogle Scholar
  17. Cramer W, Engelman D, Heijne GV, Rees D (1992): Forces involved in the assembly and stabilization of membrane proteins. Faseb Journal 6:3397PubMedGoogle Scholar
  18. Creighton T, ed. (1992): Protein folding. New York: WH FreemanGoogle Scholar
  19. Crielaard W, Visschers R, Fowler G, van Grondelle R, Hellingwerf K, Hunter C (1994): Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. I. Mutagenesis, effects on binding, function and electrochromic behaviour of its carotenoids. Biochim Biophys Acta 1183:473CrossRefGoogle Scholar
  20. Deisenhofer J, Michel H (1989): The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis. EMBO J 8:2149PubMedGoogle Scholar
  21. Deisenhofer J, Epp O, Mikki K, Huber R, Michel H (1985): Structure of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viridis at 3 Å resolution. Nature 318:618–624PubMedCrossRefGoogle Scholar
  22. Devereux J, Haeberli P, Smithies O (1984): A comprehensive set of sequence analysis programs for the vax. Nucleic Acids Research 12:387PubMedCrossRefGoogle Scholar
  23. Donnelly D, Overington JP, Ruffle SV, Nugent JH, Blundell TL (1993): Modeling α-helical transmembrane domains: the calculation and use of substitution tables for lipid-facing residues. Protein Science 2:55PubMedGoogle Scholar
  24. Eccles J, Honig B, Schulten K (1988): Spectroscopic determinants in the reaction center of Rhodo-pseudomonas viridis. Biophys J 53:137–144PubMedCrossRefGoogle Scholar
  25. Eisenberg D (1984): Three-dimensional structure of membrane and surface proteins. Annual Review of Biochemistry 53:595PubMedCrossRefGoogle Scholar
  26. Eisenberg D, Schwarz E, Komaromy M, Wall R (1984): Analysis of membrane and surface protein sequences with the hydrophobic moment plot. Journal of Molecular Biology 179:125PubMedCrossRefGoogle Scholar
  27. Eisenberg D, Weiss R, Terwilliger T, Wilcox W (1982): Hydrophobic moments and protein structure. Faraday Symposia of the Chemical Society 17:109CrossRefGoogle Scholar
  28. Engelman D (1982): An implication of the structure of bacteriorhodopsin: globular membrane proteins are stabilized by polar interactions. Biophys J 37:187PubMedCrossRefGoogle Scholar
  29. Engelman DM, Steitz TA, Goldman A (1986): Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Ann Rev Biophys Biophys Chem 15:321–353CrossRefGoogle Scholar
  30. Fasman G (1989a): Protein conformational prediction. Trends in Biochemical Sciences 14:295PubMedCrossRefGoogle Scholar
  31. Fasman G, ed. (1989b): Prediction of protein structure and the principles of protein conformation. New York: PlenumGoogle Scholar
  32. Fowler G, Sockalingum G, Robert B, Hunter C (1994): Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter Sphaeroides with alterations at α-Tyr-44 and α-Tyr-45. Biochemical Journal 299:695PubMedGoogle Scholar
  33. Gamier J, Osguthorpe D, Robson B (1978): Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journal of Molecular Biology 120:97CrossRefGoogle Scholar
  34. Geourjon C, Deleage G (1994): SOPM: a self optimised prediction method for protein secondary structure prediction. Protein Engineering 7:157PubMedCrossRefGoogle Scholar
  35. Germeroth L, Lottspeich F, Robert B, Michel H (1993): Unexpected similarities of the B800–850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria. Biochemistry 32:5615–5621PubMedCrossRefGoogle Scholar
  36. Hawthornthwaite AM, Cogdell RJ (1991): Bacteriochlorophyll binding proteins. In: Chlorophylls, (Scheer H, ed) pp. 493–528, Boca Raton: CRC PressGoogle Scholar
  37. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH (1990): Model for the structure of Bacteriorhodopsin based on high-resolution electron cryo-microscopy.J Mol Biol 213:899–929PubMedCrossRefGoogle Scholar
  38. Holley LH, Karplus M (1991): Neural networks for protein structure prediction. Methods in Enzymology 202:204PubMedCrossRefGoogle Scholar
  39. Holley LH, Karplus M (1989): Protein secondary structure prediction with a neural network. Proc Natl Acad Sci USA 86:152–156PubMedCrossRefGoogle Scholar
  40. Hu X, Xu D, Hamer K, Schulten K, Koepke J, Michel H (1995): Predicting the structure of the light-harvesting complex II of Rhodospirillum molischianum. Protein Science 4:1670–1682CrossRefGoogle Scholar
  41. Jähnig F (1989): Structure prediction for membrane proteins. In: Prediction of protein structure and the principles of protein conformation, (Fasman G, ed) p. 707, New York: PlenumCrossRefGoogle Scholar
  42. Johnson M, Srinivasan N, Sowdhamini R, Blundell T (1994): Knowledge-based protein modeling. Critical Reviews in Biochemistry and Molecular Biology 29:1PubMedCrossRefGoogle Scholar
  43. Kabsch W, Sander C (1983): Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Bioploymers 22:2577CrossRefGoogle Scholar
  44. Karrasch S, Bullough P, Ghosh R (1995): 8.5Å projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14:631PubMedGoogle Scholar
  45. Kleinekofortg W, Germeroth L, van der Broek J, Schubert D, Michel H (1992): The light-harvesting complex II (B800/850) from Rhodospirillum molischianum is an octamer. Biochimica et Biophysica Acta 1140:102–104CrossRefGoogle Scholar
  46. Koepke J, Hu X, Muenke C, Schulten K, Michel H (1996): The crystal structure of the light harvesting complex II (B800–850) from Rhodospirillum molischianum. Structure (submitted)Google Scholar
  47. Kramer HJM, van Grondelle R, Hunter CN, Westerhuis WHJ, Amesz J (1984): Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides. Biochim Biophys Acta 765:156–165CrossRefGoogle Scholar
  48. Kraulis P (1991): MOLSCRIPT—a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24:946–950CrossRefGoogle Scholar
  49. Kühlbrandt W, Wang D-N, Fujiyoshi Y (1994): Atomic model of plant light-harvesting complex by electron crystallography. Nature 367:614PubMedCrossRefGoogle Scholar
  50. Kuhn L, Leigh J (1985): A statistical technique for predicting membrane protein structure. Biochimica et Biophysica Acta 828:351PubMedCrossRefGoogle Scholar
  51. Kyte J, Doolittle RF (1982): A simple method for displaying the hydropathic character of a protein.J Mol Biol 157:105PubMedCrossRefGoogle Scholar
  52. Lattman E (1985): Diffraction methods for biological macromolecules. Use of the rotation and translation functions. Methods in Enzymology 115:55PubMedCrossRefGoogle Scholar
  53. Levitt M (1978): Conformational preference of amino acids in globular proteins. Biochemistry 17:4277PubMedCrossRefGoogle Scholar
  54. Lipman D, Pearson W (1985): Rapid and sensitive protein similarity searches. Science 227:1435PubMedCrossRefGoogle Scholar
  55. Lohmann R, Schneider G, Behrens D, Wrede P (1994): A neural network model for the prediction of membrane-spanning amino acid sequences. Protein Science 3:1597PubMedCrossRefGoogle Scholar
  56. Mackerell A (1995): unpublished researchGoogle Scholar
  57. Mcdermott G, Prince S, Freer A, Hawthornthwalte-Lawless A, Paplz M, Cogdell R, Isaacs N (1995): Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374:517CrossRefGoogle Scholar
  58. McRee D (1993): Practical Protein Crystallography. San Diego: Academic PressGoogle Scholar
  59. Michel H (1991): General and practical aspects of membrane protein crystallization. In: Crystallization of membrane proteins, (Michel H, ed) p. 74, Boca Raton, Florida: CRC PressGoogle Scholar
  60. Michel H, Weyer KA, Gruenberg H, Dunger I, Oesterhelt D, Lottspeich F (1986): The ‘light’ and ‘medium’ subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis: Isolation of genes, nucleotide and amino acid sequence. EMBO J. 5:1149PubMedGoogle Scholar
  61. Olsen JD, Hunter CN (1994): Protein structure modelling of the bacterial light-harvesting complex. Photochem Photobiol 60:521PubMedCrossRefGoogle Scholar
  62. Pearson W (1990): Rapid and sensitive sequence comparison with FASTP and FASTA. Methods in Enzymology 183:63PubMedCrossRefGoogle Scholar
  63. Persson B, Argos P (1994): Prediction of transmembrane segments in proteins utilising multiple sequence alignments. Journal of Molecular Biology 237:182PubMedCrossRefGoogle Scholar
  64. Popot J (1993): Integral membrane protein structure-transmembrane α-helices as autonomous folding domains. Current Opinion In Structural Biology 3:532CrossRefGoogle Scholar
  65. Popot J, de Vitry C (1990): On the microassembly of integral membrane proteins. Annual Review of Biophysics and Biophysical Chemistry 19:369PubMedCrossRefGoogle Scholar
  66. Popot J, Engelman D (1990): Membrane protein folding and oligomerization: the two-stage model. Biochemistry 29:4031PubMedCrossRefGoogle Scholar
  67. Popot J, de Vitry C, Atteia A (1994): Folding and assembly of integral membrane proteins: An introduction. In: Membrane protein structure: experimental approaches, (White, S., ed) p. 41, New York: Oxford University pressGoogle Scholar
  68. Presnell SR, Cohen BI, Cohen FE (1992): A segment-based approach to protein secondary structure prediction. Biochemistry 31:983PubMedCrossRefGoogle Scholar
  69. Rao JM, Argos P (1986): A conformational preference parameter to predict helices in integral membrane proteins. Biochimica et Biophysica Acta 869:197CrossRefGoogle Scholar
  70. Rees D, DeAntonio L, Eisenberg D (1989): Hydrophobic organization of membrane proteins. Science 245:510PubMedCrossRefGoogle Scholar
  71. Ring C, Cohen F (1993): Modeling protein structures: construction and their applications. Faseb Journal 7:783PubMedGoogle Scholar
  72. Rooman M, Wodak S (1988): Identification of predictive sequence motifs limited by protein structure data base size. Nature 335:45PubMedCrossRefGoogle Scholar
  73. Rossmann M, ed (1972): The Molecular Replacement Method. New York: Gordon and BreachGoogle Scholar
  74. Sali A, Blundell TL (1993): Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology 234:779PubMedCrossRefGoogle Scholar
  75. Schuler G, Altschul S, Lipman D (1991): A workbench for multiple alignment construction and analysis. Proteins: Structure, Function, and Genetics 9:180CrossRefGoogle Scholar
  76. Segrest J, Loof HD, Dohlman J, Brouillette C, Anantharamaiah G (1990): Amphipathic helix motif: Classes and properties. Proteins, Struct Funct Genet 8:103CrossRefGoogle Scholar
  77. Sundstrom V, van Grondelle R (1991): Dynamics of excitation energy transfer in pho-tosynthetic bacteria. In: Chlorophylls, (Scheer H, ed) pp. 627–704, Boca Raton: CRC PressGoogle Scholar
  78. Treutlein H, Schulten K, Deisenhofer J, Michel H, Brünger A, Karplus M (1988): Molecular dynamics simulation of the primary processes in the photosynthetic reaction center of Rhodopseudomonas viridis. In: The Photosynthetic Bacterial Reaction Center: Structure and Dynamics, (Breton J, Verméglio A, eds) volume 149 of NATO ASI Series A: Life Sciences pp. 139–150. Plenum New YorkGoogle Scholar
  79. Tuffery P, Etchebest C, Popot J, Lavery R (1994): Prediction of the positioning of the seven transmembrane α-helices of bacteriorhodopsin. A molecular simulation study. Journal of Molecular Biology 236:1105PubMedCrossRefGoogle Scholar
  80. van Grondelle R, Sundstrom V (1988): Excitation energy transfer in photosynthesis. In: Photosynthetic Light-Harvesting Systems, (Scheer H, ed) pp. 403–438, Berlin, New York: Walter de Gruyter and CoGoogle Scholar
  81. Visschers RW, Crielaard W, Fowler GJ, Hunter CN, van Grondelle R (1994) Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. II. A low temperature spectroscopy study of structural aspects of the pigment-protein conformation. Biochim Biophys Acta 1183:483CrossRefGoogle Scholar
  82. von Heijne G (1994a): Decoding the signals of membrane protein sequence. In: Membrane protein structure: experimental approaches, (White S, ed) p. 27, New York: Oxford University pressGoogle Scholar
  83. von Heijne G (1994b): Membrane proteins: from sequence to structure. Annual Review of Biophysics and Biomolecular Structure 23:167CrossRefGoogle Scholar
  84. von Heijne G (1992): Membrane protein structure prediction—hydrophobicity analysis and the positive-inside rule. Journal of Molecular Biology 225:487CrossRefGoogle Scholar
  85. von Heijne G (1988): Transcending the impenetrable: how proteins come to terms with membranes. Biochimica et Biophysica Acta 947:307Google Scholar
  86. von Heijne G, Manoil C (1990): Membrane proteins: from sequence to structure. Protein Engineering 4:109CrossRefGoogle Scholar
  87. Weiss M, Kreusch A, Nestel U, Weite W, Weckesser J, Schulz G (1991): The structure of porin from Rhodobacter capsulatus at 1.8 Å resolution. FEBS Lett 280:379PubMedCrossRefGoogle Scholar
  88. White SH (1994): Hydropathy plots and the prediction of membrane protein topology. In: Membrane protein structure: experimental approaches, (White SH, ed), New York: Oxford University pressGoogle Scholar
  89. Zuber H (1993): Structural features of photosynthetic light-harvesting systems. In: The Photo synthetic Reaction Center, (Deisenhofer J, Norris JR, eds) p. 43, San Diego: Academic PressGoogle Scholar
  90. Zuber H (1986): Structure of light-harvesting antenna complexes of photosynthetic bacteria, cyanobacteria and red algae. Trends Biochem Sci 11:414CrossRefGoogle Scholar
  91. Zuber H (1985): Structure and function of light-harvesting complexes and their polypeptides. Photochem Photobiol 42:821CrossRefGoogle Scholar
  92. Zuber H, Brunisholz R (1991): Structure and function of antenna polypeptides and chlorophyll-protein complexes: Principles and variability. In: Chlorophylls, (Scheer H, ed) pp. 627–692, Boca Raton: CRC PressGoogle Scholar
  93. Zvelebil MJ, Barton GJ, Taylor WR, Sternberg MJ (1987): Prediction of protein secondary structure and active sites using the alignment of homologous sequences. J Mol Biol 195:957PubMedCrossRefGoogle Scholar

Copyright information

© Birkhäuser Boston 1996

Authors and Affiliations

  • Xiche Hu
  • Dong Xu
  • Kenneth Hamer
  • Klaus Schulten
  • Juergen Koepke
  • Hartmut Michel

There are no affiliations available

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