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Biophysics Masters Course 2002

This course explores the biophysics of photosynthetic membranes, focusing on the tubular membranes in Rb. Sphaeroides and grana membranes in green plants. Topics include membrane proteins, membrane organization, and biophysical techniques like linear dichroism spectroscopy.

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Biophysics Masters Course 2002

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  1. Biophysics Masters Course 2002 Photosynthetic Membranes Jan P. Dekker

  2. Contents 1a. Tubular membranes in Rb. Sphaeroides 1b. Grana membranes in green plants

  3. Biophysics Masters Course 2002 1a. Tubular membranes in Rb. Sphaeroides

  4. Membrane proteins in Rb. sphaeroides chromatophores

  5. Membranes of Rb. Sphaeroides RC-LH1 Freeze-etching pictures of intracytoplasmic RC-LH1 membranes present in Rb. sphaeroides WT (A) and a mutant deleted in pufX (B). Scale bar = 50 nm. From [4]. + pufX - pufX

  6. Membrane proteins in Rb. sphaeroides chromatophores Jamieson et al., EMBO J. 21, 2002, 3927

  7. Membrane proteins in Rb. sphaeroides chromatophores Jamieson et al., EMBO J. 21, 2002, 3927 Without pufX: RC surrounded by a ring of 16 LH1 complexes

  8. Proposed organization of RC-LH1 rings and of dimeric bc1 complex without pufX. Fig. showing model structures for protein complexes of the photosynthetic chain, taken from crystallographic data. LH1 and LH2 rings from Schulten's lab; bc1-complex dimer, from Ed Berry's coordinates for the chicken heart mitochondrial complex (but including only the three catalytic subunits). (Crofts et al.)

  9. Membranes of Rb. Sphaeroides RC-LH1 Freeze-etching pictures of intracytoplasmic RC-LH1 membranes present in Rb. sphaeroides WT (A) and a mutant deleted in pufX (B). Scale bar = 50 nm. From [4]. + pufX - pufX

  10. + pufX (A) Low-dose electron micrographs of native tubular membranes from Rb. sphaeroides. Samples were negatively stained with 1% uranyl acetate. Bar = 100 nm for all the tubes. (B) Fourier transform of one tube from digitized scan. The arrow indicates a resolution at 25 Å. (C) Filtered image from masked Fourier transform of electron micrographs. Bar = 200 Å. From [1].

  11. Tubular Membranes Projection map at 20 Å resolution after processing and averaging of negatively stained native tubular flat membrane from Rb. sphaeroides. The unit cell is outlined in black. Positive density representing the protein is shown as solid lines and negative density as dotted lines. From [1].

  12. Tubular Membranes Proposed model of the photosynthetic unit of Rb. sphaeroides viewed from above the membrane. The projection structure of the RC of Rb. sphaeroides and ¾ of LH1 from Rs. rubrum are shown in red and green, respectively. From [1]. • Conclusions: • RC-LH1 dimer • No closed LH1 ring • Questions: • What is in the middle? • Is Cyt. bc1 complex present?

  13. A Amino acid sequence of the PufX proteins of Rb.capsulatus and Rbsphaeroides according to the nucleotide sequence of the corresponding pufX genes (14, 16). Dotted lines indicate possible identities in amino acid sequence if the polypeptides were correspondingly aligned.

  14. Schematic representation of the supramolecular organization of the photosynthetic apparatus of R. sphaeroides view from above the membrane. The LH1 complexes are in green, the RCs in yellow and the cyt bc1 complex in blue. This has been deduced from the projection map at 20 Å resolution of negatively stained tubular membranes. From [4].

  15. Biophysical Technique: Linear Dichroism

  16. Analysis of membranes by LD spectroscopy Chlorophyll a Qx (630 nm) Qy (670 nm) Chlorophylls have two oppositely oriented electronic transitions

  17. Analysis of membranes by LD spectroscopy Transition dipole LD = Apar - Aperp a LD / 3A = ½(3cos2a – 1)F Normal No LD if: 3cos2a = 1 a = 54.7o (magic angle) Aperp Apar

  18. Analysis of membranes by LD spectroscopy (a) The main Qy absorption of the LH1-only membranes (solid line) and the RC-only membranes (dashed line) at 77 K; the latter were frozen in the dark. P = special pair, B = accessory BChls, H = BPheos. (b) LD spectra of these membranes at 77 K in the same spectral region. From [2]. P: a = 90 degrees (assumed) B: a = 66 degrees H: a = 35 degrees

  19. Analysis of membranes by LD spectroscopy • LD spectra of RC-LH1 membranes at 77 K with (solid line) and without (dashed line) the pufX protein present. Inset: Magnification of the spectra in the accessory BChl and Bpheo region. From [2]. • LD - pufX: as expected • LD + pufX: • LH1: a > magic angle (exp.) • P: a < magic angle (?, not) • B: a < magic angle (not) • H: a > magic angle (exp)

  20. Analysis of membranes by LD spectroscopy 77 K dark-minus-light absorption (a) and LD (b) spectra of RC-LH1 membranes at 77 K with (solid line) and without (dashed line) the pufX protein present. From [2]. These spectra confirm the (very) small a for P.

  21. Model of the organization of RC-LH1 core complexes in tubular membranes. (c) The RC pigments P (red), B (green), H (blue) and QB (orange), with orientation consistent with the LD measurements on the + pufX membranes. (b) Model of the RC with LH1 ring (red) and inserted pufX protein (green) together with a cytochrome bc1 complex (yellow). (a) Longe-range organization of RC-LH1 complexes in + pufX (left) and – pufX (right) membranes. From [2].

  22. Various models for the organization of RC (color), LH1 (pink), pufX (black) and the cytochrome bc1 complex (grey). From [3].

  23. Analysis of membranes by LD spectroscopy LD of cytochromes in tubular membranes (Kinga Sznee, unpublished results). One of the cytochromes shows anunusual orientation in the + pufX membranes (arrows), which points to a non-random orientation of the cytochrome bc1 complex in these membranes.

  24. LD of cytochrome b6f complex Qx Qy

  25. LD of CP43 complex Qy Qx

  26. 1a. Tubular Membranes in Rb. Sphaeroides • Literature: • C. Jungas, J.-L. Ranck, J.-L. Rigaud, P. Joliot and A. Vermeglio (1999) Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides. EMBO J. 18, 534-542 • R.N. Frese, J.D. Olsen, R. Branvall, W.H.J. Westerhuis, C.N. Hunter and R. Van Grondelle (2000) The longe-range supraorganization of the bacterial photosynthetic unit: A key role for PufX. Proc. Natl. Acad. Sci. USA 97, 5197-5202 • P.A. Loach (2000) Supramolecular complexes in photosynthetic bacteria. Proc. Natl. Acad. Sci. USA 97, 5016-5018 • A. Vermeglio and P. Joliot (2002) Supramolecular organisation of the photosynthetic chain in anoxygenic bacteria. Biochim. Biophys. Acta 1555, 60-64

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