Transcript Rb sphaeroides

Biophysics Masters Course 2002
1. Photosynthetic Membranes
Jan P. Dekker
Contents
1a. Tubular membranes in Rb. Sphaeroides
1b. Grana membranes in green plants
Biophysics Masters Course 2002
1a. Tubular membranes in
Rb. Sphaeroides
Membrane proteins in Rb. sphaeroides
chromatophores
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
Membrane proteins in Rb. sphaeroides
chromatophores
Jamieson et al., EMBO J. 21, 2002, 3927
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
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; bc1complex dimer, from Ed Berry's
coordinates for the chicken heart
mitochondrial
complex
(but
including only the three catalytic
subunits). (Crofts et al.)
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
+ 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].
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].
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?
A Amino acid sequence of the PufX proteins of Rb. capsulatus and Rb sphaeroides
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.
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].
Biophysical Technique:
Linear Dichroism
Analysis of membranes by LD spectroscopy
Chlorophyll a
Qx (630 nm)
Qy (670 nm)
Chlorophylls have two
oppositely oriented
electronic transitions
Analysis of membranes by LD spectroscopy
Transition dipole
LD = Apar - Aperp
a
Normal
Aperp
Apar
LD / 3A = ½(3cos2a – 1)F
No LD if:
3cos2a = 1
a = 54.7o (magic angle)
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
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)
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.
Model of the organization of RCLH1 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 RCLH1 complexes in + pufX (left) and
– pufX (right) membranes. From [2].
Various models for the
organization of RC
(color), LH1 (pink),
pufX (black) and the
cytochrome bc1
complex (grey). From
[3].
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.
LD of cytochrome b6f complex
Qx
Qy
LD of CP43 complex
Qy
Qx
1a. Tubular Membranes in Rb. Sphaeroides
Literature:
1.
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
2.
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
3.
P.A. Loach (2000) Supramolecular complexes in photosynthetic bacteria. Proc.
Natl. Acad. Sci. USA 97, 5016-5018
4.
A. Vermeglio and P. Joliot (2002) Supramolecular organisation of the
photosynthetic chain in anoxygenic bacteria. Biochim. Biophys. Acta 1555,
60-64