text:
Structure and function of intact photosystem 1 monomers from the cyanobacterium Thermosynechococcus elongatus.
ABSTRACT Until now, the functional and structural characterization of monomeric photosystem 1 (PS1) complexes from Thermosynechococcus elongatus has been hampered by the lack of a fully intact PS1 preparation; for this reason, the three-dimensional crystal structure at 2.5 A resolution was determined with the trimeric PS1 complex [Jordan, P., et al. (2001) Nature 411 (6840), 909-917]. Here we show the possibility of isolating from this cyanobacterium the intact monomeric PS1 complex which preserves all subunits and the photochemical activity of the isolated trimeric complex. Moreover, the equilibrium between these complexes in the thylakoid membrane can be shifted by a high-salt treatment in favor of monomeric PS1 which can be quantitatively extracted below the phase transition temperature. Both monomers and trimers exhibit identical posttranslational modifications of their subunits and the same reaction centers but differ in the long-wavelength antenna chlorophylls. Their chlorophyll/P700 ratio (108 for the monomer and 112 for the trimer) is slightly higher than in the crystal structure, confirming mild preparation conditions. Interaction of antenna chlorophylls of the monomers within the trimer leads to a larger amount of long-wavelength chlorophylls, resulting in a higher photochemical activity of the trimers under red or far-red illumination. The dynamic equilibrium between monomers and trimers in the thylakoid membrane may indicate a transient monomer population in the course of biogenesis and could also be the basis for short-term adaptation of the cell to changing environmental conditions.
Article: Long-wavelength chlorophylls in photosystem I of cyanobacteria: origin, localization, and functions.
[Show abstract] [Hide abstract]
ABSTRACT: The structural organization of photosystem I (PSI) complexes in cyanobacteria and the origin of the PSI antenna long-wavelength chlorophylls and their role in energy migration, charge separation, and dissipation of excess absorbed energy are discussed. The PSI complex in cyanobacterial membranes is organized preferentially as a trimer with the core antenna enriched with long-wavelength chlorophylls. The contents of long-wavelength chlorophylls and their spectral characteristics in PSI trimers and monomers are species-specific. Chlorophyll aggregates in PSI antenna are potential candidates for the role of the long-wavelength chlorophylls. The red-most chlorophylls in PSI trimers of the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus can be formed as a result of interaction of pigments peripherally localized on different monomeric complexes within the PSI trimers. Long-wavelength chlorophylls affect weakly energy equilibration within the heterogeneous PSI antenna, but they significantly delay energy trapping by P700. When the reaction center is open, energy absorbed by long-wavelength chlorophylls migrates to P700 at physiological temperatures, causing its oxidation. When the PSI reaction center is closed, the P700 cation radical or P700 triplet state (depending on the P700 redox state and the PSI acceptor side cofactors) efficiently quench the fluorescence of the long-wavelength chlorophylls of PSI and thus protect the complex against photodestruction.Biochemistry (Moscow) 03/2014; 79(3):213-20. · 1.15 Impact Factor - SourceAvailable from: Pavlo GordiichukPavlo I. Gordiichuk, Gert‐Jan A. H. Wetzelaer, Dolev Rimmerman, Agnieszka Gruszka, Jan Willem de Vries, Manfred Saller, Daniel A. Gautier, Stefano Catarci, Diego Pesce, Shachar Richter, Paul W. M. Blom, Andreas Herrmann[Show abstract] [Hide abstract]
ABSTRACT: The large multiprotein complex photosystem I, which is at the heart of light-dependent reactions in photosynthesis, is integrated as the active component in a solid-state organic photovoltaic cell. Our experiments demonstrate that photoactive megadalton-protein complexes are compatible with solution processing of organic-semiconductor materials and operate in a dry non-natural environment that is very different from the biological membrane.Advanced Materials 05/2014; · 15.41 Impact Factor - SourceAvailable from: Meng Li[Show abstract] [Hide abstract]
ABSTRACT: Photosystem I (PSI) is a reaction center associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in green and purple bacteria, PSI forms trimeric complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a tetrameric form of PSI in the thermophilic cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821, PSI forms tetrameric and dimeric species. We investigated these species by Blue Native PAGE, Suc density gradient centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis. Transmission electron microscopy analysis of native membranes confirms the presence of the tetrameric PSI structure prior to detergent solubilization. To investigate why TS-821 forms tetramers instead of trimers, we cloned and analyzed its psaL gene. Interestingly, this gene product contains a short insert between the second and third predicted transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which also have a tetrameric form of PSI. These results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to tetrameric oligomer en route to becoming monomeric in plants/algae.The Plant Cell 03/2014; · 9.58 Impact Factor
Page 1
pubs.acs.org/BiochemistryPublished on Web 04/01/2010
r2010 American Chemical Society
4740Biochemistry 2010, 49, 4740–4751
DOI: 10.1021/bi901807p
Structure and Function of Intact Photosystem 1 Monomers from the
Cyanobacterium Thermosynechococcus elongatus†,‡
Eithar El-Mohsnawy,§,rMarta J. Kopczak,§Eberhard Schlodder,)
Bettina Warscheid,@Navassard V. Karapetyan,#and Matthias R€ ogner*,§
Marc Nowaczyk,§Helmut E. Meyer,^
§Plant Biochemistry, Ruhr University Bochum, 44780 Bochum, Germany,
Technical University, 10623 Berlin, Germany,^Medical Proteom-Center, Ruhr University Bochum, 44780 Bochum, Germany,
@Clinical and Cellular Proteomics, Medical Faculty and Center for Medical Biotechnology, Duisburg-Essen University, 45117 Essen,
Germany, and#A. N. Bakh Institute of Biochemistry RAS, 119071 Moscow, RussiarOn leave from Department of Biological and
Geological Sciences, Faculty of Education in Al-Arish, Suez Canal University, Al-Arish, Egypt.
Max-Volmer Laboratory for Biophysical Chemistry,
Received October 23, 2009; Revised Manuscript Received March 15, 2010
ABSTRACT: Until now, the functional and structural characterization of monomeric photosystem 1 (PS1)
complexes from Thermosynechococcus elongatus has been hampered by the lack of a fully intact PS1
preparation; for this reason, the three-dimensional crystal structure at 2.5 A˚resolution was determined with
the trimeric PS1 complex [Jordan, P., et al. (2001) Nature 411 (6840), 909-917]. Here we show the possibility
photochemical activity of the isolated trimeric complex. Moreover, the equilibrium between these complexes
in the thylakoid membrane can be shifted by a high-salt treatment in favor of monomeric PS1 which can be
posttranslational modifications of their subunits and the same reaction centers but differ in the long-
wavelength antenna chlorophylls. Their chlorophyll/P700 ratio (108 for the monomer and 112 for the trimer)
is slightly higher than in the crystal structure, confirming mild preparation conditions.Interaction of antenna
chlorophylls of the monomers within the trimer leads to a larger amount of long-wavelength chlorophylls,
resulting in a higher photochemical activity of the trimers under red or far-red illumination. The dynamic
equilibrium between monomers and trimers in the thylakoid membrane may indicate a transient monomer
population in the course of biogenesis and could also be the basis for short-term adaptation of the cell to
changing environmental conditions.
Photosystem 1 (PS1)1is the largest known multisubunit
membrane protein complex that functions as a solar energy
converter; it catalyzes the transfer of an electron from plasto-
cyanin or cytochrome c6on the lumenal side to ferredoxin (or
flavodoxin) onthecytoplasmicside.AccordingtoX-rayanalysis
of the trimeric PS1 core complex from the cyanobacterium
Thermosynechococcus elongatus, the structureexhibits 12 protein
subunits and 127 cofactors performing light capturing and
electron transfer (1-3). These cofactors include 96 chlorophylls
(Chls), 22 carotenoids (Car), two phylloquinones, three iron-
sulfur (4Fe-4S) clusters, and four lipids for each PS1 monomer.
The two large subunits, PsaA and PsaB, each consisting of 11
transmembrane helices, coordinate most of the Chls and Cars,
and the redox cofactors of electron transport: primary electron
donor P700, primary electron acceptor A0(Chl a monomer),
secondary electron acceptor A1(phylloquinone), and FX
(iron-sulfur cluster). Terminal electron acceptors FAand FB
(also iron-sulfur clusters) are both coordinated by the PsaC
subunit, which is, with PsaD and PsaE, one of the three extrinsic
subunits on the cytoplasmic side. In contrast to eukaryotes, the
intrinsic PsaF subunit is not involved in docking of either
the electron donor plastocyanin or cytochrome c6in cyano-
bacteria (4). Subunits PsaI and PsaL are responsible for forma-
tion and stabilization of the PS1 trimer in cyanobacteria (5).
Subunits PsaK, -M, and -X seem to play a mere structural role.
The PS1 trimer of T. elongatus is characterized by a molecular
mass of 1068 kDa according to protein and cofactor composi-
molecular structure has been determined. Especially the large
content of cofactors provides more than 30% of its total mass.
high degree of structural similarity (1, 6, 7), but there are some
differences in subunit composition. Subunits PsaX and -M are
found only in cyanobacteria and subunits PsaG and -H only in
†Financial support from the German Research Council, DFG (SFB
480, Project C1, M.R. and M.N.; SFB 498, TP A6, E.S.), the European
Union (SOLAR-H project, M.R. and M.J.K.), the Egypt Government
MCB, and RFBR (Grant 08-04-00143a, to N.V.K.) is gratefully
‡Dedicated to Prof. Dr. Achim Trebst on the occasion of his 80th
*To whom correspondence should be addressed: Ruhr-Universit€ at
Bochum,Universit€ atsstr.150,44780Bochum,Germany.Telephone:þ49
(234) 322-3634. Fax: þ49 (234) 321-4322. E-mail: Matthias.Roegner@
phenolindophenol; Lhca1-Lhca4, light-harvesting complexes of plant
PS1; LWC, long-wavelength chlorophylls; MV, methyl viologen; PS1
and PS2, photosystem 1 and photosystem 2, respectively; P700 and
P700þ, primaryelectrondonorofPS1inthereducedandoxidizedstate,
respectively; β-DM, n-dodecyl β-D-maltoside; PsaA, PsaB, etc., poly-
peptide subunits of the PS1 complex; RT, room temperature; þST and
-ST, salt-treated and non-salt-treated membranes, respectively.
Page 2
ArticleBiochemistry, Vol. 49, No. 23, 20104741
which may be the reason for a different supramolecular organi-
a trimeric complex (3, 8-11), whereas plant PS1 apparently is
exclusively monomeric, most probably due to binding of peri-
trimerization (3, 13, 15, 16).
A high content of the so-called long-wavelength Chls (LWC;
of P700 is a unique property of many cyanobacterial PS1 core
antennae(9,17-21).Thecontentand the spectralcharacteristics
of LWC are highly species-dependent (20, 22), with PS1 trimers
containing usually more red Chls than PS1 monomers (23). Five
Chl a antenna states absorbing at 708 and 719 nm (24-27),
whereas PS1 trimers of Synechocystis sp. PCC 6803 (hereafter
called Synechocystis) contain LWC peaking at shorter wave-
lengths, 708 and 714 nm (28, 29). The most red-shifted Chl
antenna state absorbing at 708 and 740 nm and emitting at
760 nm (77 K) was observed in PS1 trimers of Arthrospira
(Spirulina) platensis (9, 30, 31). The fluorescence of LWC in
these PS1 trimers of A. platensis is quenched by oxidized P700 or
P700 in the triplet state (9, 30, 32). The dependence of the
fluorescence yield of the red-most Chl in PS1 trimers of
A. platensis and T. elongatus on the redox state of P700 allowed
to follow the processes of energy utilization and dissipation in
PS1 (26, 30, 33). Overall, the presence of LWC in many types of
PS1 complexes of cyanobacteria indicates their functional im-
portance. Energy absorbed by these LWCs migrates uphill to
P700 and causes its oxidation (34, 35). As the same 77 K
fluorescence band at 760 nm was found in isolated PS1 trimers,
in membranes, and in whole cells of A. platensis, this was the
direct evidence for preexisting PS1 trimers in membranes of
cyanobacteria (9, 31). Moreover, differences in the fluorescence
emission spectra of PS1 trimers and monomers of A. platensis at
77 K helped to indicate a dynamic equilibrium of the monomer/
trimer ratio within the detergent-free membranes and an energy
exchange between PS1 monomers within a trimer (30, 36).
stable PS1 from which also the most resolved three-dimensional
(3D) structure has been determined (1). To understand the
functional interaction of the three monomers within this well-
a conclusion about its physiological significance, a quantitative
preparation procedure for "nativelike" PS1 monomers is re-
quired. This is hampered by the fact that a routine extraction
from the thylakoid membrane of T. elongatus yields nearly
exclusively trimeric complexes. In the past, attempts to isolate
monomeric PS1 from T. elongatus involved special detergent
treatment using Triton X-100 (37), β-octyl glucopyranoside at
elevated temperatures (38), or sodium dodecyl sulfate and
sodium cholate (8, 39), pre-extraction of carotenoids using
n-heptane (40), or deletion of the central PsaL subunit (3, 11,
41-43), which apparently is required for trimerization (44). All
these attempts resulted in structural and/or functional defects.
Using the detergent n-dodecyl β-D-maltoside (β-DM) in combi-
nation with an osmotic treatment during the passage through a
gel filtration column, Jekow et al. succeeded in partially dis-
sociating PS1 trimers into monomers (45). However, depending
on the detergent (β-DM or sulfobetaine), these monomers
showed a distinctly reduced Chl antenna size and the loss of
PsaL and -K subunits in comparison with PS1 trimers. In any
case, the activity of the obtained monomeric complexes was
considerably reduced, leading to the assumption that only
trimeric PS1 represents the fully active PS1 form. This was
supported by the impaired growth under low-light conditions
reveal 96 Chls (1), and the antenna size of PS1 within cells of the
cyanobacterium A. platensis was even calculated to be 120
Chls (47), indicating that all published methods resulting in
of PS1 in the cell and are not suited for comparison with their
trimeric counterparts.
To combine high-resolution structural data with detailed
functional characterization of PS1 and also to learn about the
smallest functional PS1 unit, we made an attempt to isolate
monomeric PS1 from T. elongatus. Preservation of subunit
composition was achieved by dissociation of trimers into mono-
mers within the membrane phase using high salt prior to
extraction asreported previouslyfor a mesophilic organism(48).
The intactness of this preparation, which was also confirmed by
comparison with a salt-free isolated preparation using an ex-
tremely low detergent concentration for extraction, enabled for
the first time the in depth characterization of a nativelike
monomeric PS1 in comparison with its trimeric counterpart
using both structural and functional state-of-the-art analytical
methods. In addition, this comparison yields information about
functional interactions of the monomers within the trimer,
including the special role of the long-wavelength Chls.
Cell Culture and Preparation of the PS1 Extract.
T. elongatus cells were grown in BG11 medium (49), harvested,
protocols (38, 50, 51). Fresh thylakoid membranes {in buffer A
[0.5 M mannitol, 20 mM HEPES (pH 7.5), 10 mM MgCl2, and
10 mM CaCl2]} or frozen thylakoid membranes [in buffer D
(buffer A with 20% glycerol)] were adjusted to a Chl concentra-
tion of approximately 0.75 mg/mL (in ∼100 mL) with buffer A,
homogenized five times, and centrifuged for 20 min at 8100g
(JLA-16.250 rotor, Beckman). In the case of isolation of PS1
monomers and trimers [with salt treatment (Chart 1)], the
pelleted thylakoid membranes have been washed three times
with buffer A containing 0.05% β-DM to remove the phycobili-
somes. The pellet of the phycobilisome-depleted thylakoid mem-
branes was resuspended and incubated for 20 min in high-salt
extraction buffer (temperature as indicated) at 1 mg/mL Chl,
followed by extraction with β-DM (0.6%) and ultracentrifuga-
tion (1 h at 180000g). For the isolation of PS1 monomers from
β-DM and centrifuged at 8100g for 20 min. After the salt
A containing 2 M (NH4)2SO4], most of the phycobilisomes were
pelleted by ultracentrifugation (1 h at 180000g).
Chromatographic Purification and Analysis of PS1.
With the exception of analytical SEC (see below), all chromato-
HIC column (Applied Biosystems) was used as the first purifica-
tion [buffer consisting of 20 mM HEPES (pH 7.5), 1.5 M
ammonium sulfate, 10 mM MgCl2, 10 mM CaCl2, 0.5 M
mannitol, and 0.03% β-DM)], extracted PS1 complexes were
Page 3
4742Biochemistry, Vol. 49, No. 23, 2010El-Mohsnawy et al.
mixed withammonium sulfatefor a final concentration of 1.5M
gradient (1.5 to 0 M) or by two gradient steps (from 1.5 to1.1 M
and from 1.1 to 0 M, in the case of PS1 from non-salt-treated
membranes) of ammonium sulfate at a flow rate of 5 mL/min.
Purified PS1 was dialyzed against buffer [20 mM HEPES (pH
7.5), 10 mM MgCl2, 10 mM CaCl2, 0.5 M mannitol, and 0.03%
β-DM] overnight at 10 ?C before IEC was started as a second
purificationstep. The POROS HQ/M(Applied Biosystems) IEC
column was used as a second purification step. After column
equilibration [buffer consisting of 20 mM HEPES (pH 7.5),
10 mM MgCl2, 10 mM CaCl2, 0.5 M mannitol, and 0.03%
linear gradient (0 to 200 mM) of MgSO4.
MALDI-TOF Analysis of Intact Protein Complexes.
Intact PS1 subunits were analyzed by MALDI-TOF mass
spectrometry according to the method described in ref 52. PS1
and 0.03% β-DM] at a Chl concentration of 0.2-0.3 μg/μL was
or ferulic acid in 1% trifluoroacetic acid and 60% (v/v) aceto-
nitrile], spread on the target plate (0.6 μL), and air-dried before
being used. Analysis was performed on a MALDI-TOF mass
spectrometer (Voyager DE, Applied Biosystems) or alterna-
tively on a qTOF mass spectrometer (QSTAR XL, Applied
Biosystems). Calibration was conducted with standard proteins
(Mix 3, Sequazyme Kit, Applied Biosystems).
Determination of Chl a, Car, and P700 Concentrations.
Chlorophyll concentrations were measured in an 80% (v/v)
acetone/water mixture and calculated according to the method
calculated from the maximal flash-induced absorption change
in the QYregion due to the photooxidation of P700, using
a differential molar extinction coefficient (Δε) of 61000 (
2000 M-1cm-1(54).
The Car content was determined from the absorption spec-
trum of the pigment extract in an 80% acetone/water mixture
vis spectrophotometer. The absorption spectrum of Car was
obtainedbysubtractionofthe spectrumofpureChl a inan80%
acetone/water mixture that has been normalized to the absorp-
tion of the extract in the QYregion. The Chl a/Car ratio was
calculated according to the following equation:
Chl a
at 664 nm for Chl a (53, 56) and 134000 M-1cm-1at 450 nm for
β-carotene (57).
Absorption Spectra. Low-temperature (5 K) absorption
spectra were recorded at a resolution of 0.5 or 1 nm in a Cary-
1E-UV/vis spectrophotometer equipped with an Oxford liquid
was diluted to an OD680of ∼1 in buffer [20 mM MES, 20 mM
CaCl2, 10 mM MgCl2, 5 mM sodium ascorbate, 0.02% β-DM,
and 60-65% glycerol (pH 6.5)].
To analyze the number of Chls contributing to the red
absorption, we decomposed the red region (685-750 nm) of
the 5 K absorbance spectra into Gaussian bands (32). We
obtained the number of Chls contributing to the red absorption
from the ratio of the integrated intensity of the long-wavelength
absorption bands at 710 and 720 nm to the total integrated
intensity of the QYband and the number of Chls per PS1.
Fluorescence Spectra. Fluorescence emission spectra (600-
Chl concentration of the PS1 samples was adjusted with buffer
[20 mM HEPES (pH 7.5), 10 mM MgCl2, 10 mM CaCl2, 0.02%
and to 3 μg/mL for 77 K.
PS1-Mediated Electron Transport Rates. The photo-
uptake according to the Mehler reaction induced by actinic light
in a fiber optic oxygenmeter (FIBOX 2, PreSens). For activity
measurements, the concentrated purified PS1 complexes were
diluted with PS1 activity buffer [30 mM HEPES (pH 7.5), 3 mM
MgCl2, 50 mM KCl, 330 mM mannitol, and 0.03% β-DM] to a
final Chl concentration of 3-5 μg/mL. 2,6-Dichlorophenolindo-
phenol (800 μM) and sodium ascorbate (5 mM) were used as
electron donors, and methyl viologen (0.5 mM) was used as an
electron acceptor. The sample was stirred in darkness for 2 min.
red light (λ > 665 nm, 4000 μE m-2s-1), and far-red light
(λ>715 nm, 700 μE m-2s-1). As a source of actinic light, a
KL2500 LCD (Schott) was used. Calibration of the sensor and all
measurements was conducted at 30 ?C.
Transient Absorption Spectroscopy. Flash-induced absor-
bance changes were measured with the setups described pre-
viously (54). Flash-induced absorbance difference spectra of
P700þminus P700 at RT were measured with PS1 complexes
diluted to 10-15 μg/mL Chl in 20 mM Tricine (pH 7.5), 25 mM
MgCl2, 100 mM KCl, 0.02% β-DM, 5 mM sodium ascorbate,
and 10 μM PMS. P700þFA/B
-minus P700FA/Bspectra recorded
Chart 1: PreparationStepsfortheIsolationandPurificationof
Monomeric and Trimeric PS1 Involving a Salt Treatment Step
Page 4
ArticleBiochemistry, Vol. 49, No. 23, 20104743
after illumination. The concentration of the PS1 complexes and
the buffer composition were identical to those at RT, but the
buffer contained 60-65% glycerol.
Development of a Preparation Procedure for Nativelike
Monomeric PS1. Chart 1 summarizes the developed strategy
prior to extraction, isolation, and purification. This method is
mainly based on the dissociation of trimeric PS1 complexes
within the membrane by a special salt treatment which has been
shown previously for PS1 from the mesophilic cyanobacteria
Synechocystis (48) and A. platensis (30). Prerequisite for an
efficient salt treatment is the minimization of PBS content by a
low-concentration β-DM wash (0.05%). For the subsequent
process of salt dissociation, several salts have been tested: NaCl,
MgCl2exhibited a verylow efficiency indissociating trimers into
monomers as judged from elution profiles of HIC columns or
sucrose density gradients (for details, see the Supporting In-
formation), MgSO4 was partially effective and ammonium
effect was observed at 0.6 M ammonium sulfate at 50 ?C.
Distinctly lower ratios obtained with salt incubation at 20 or
37 ?C indicate that the liquid-crystalline state [> 37 ?C (M. Hato,
PS1 monomers in substantial amounts.
After extraction, PS1 monomers and trimers can be gained at
high purity and homogeneity by two-dimensional chromato-
graphy, consisting of an HIC-HPLC column step, followed by
IEC-HPLC. These fractions were then used for a detailed
biochemical and biophysical characterization.
MALDI-TOF Analysis of Monomeric versus Trimeric
PS1: What Is the Smallest Fully Functional Unit? Potential
structural differences between monomers and trimers have been
investigated by a detailed MALDI-TOF analysis (Figure 1). To
exclude the secondary effects of the salt treatment on the
monomers, for comparison monomers that have not been
exposed to any salt have also been used (-ST). Additionally,
the detergent concentration used for extraction (0.1% instead of
0.6% β-DM) and the exposure time (5 min instead of 15 min at
RT) was minimized to help keep the monomer in its nativelike
FIGURE 1: MALDI-TOFMSanalysisofpurifiedPS1monomersandtrimersfromT.elongatus.PurifiedPS1complexesweremixedina1/1ratio
TOF). Covered mass range: 2000-10000 (A) and 14000-17000 (B).
Page 5
4744Biochemistry, Vol. 49, No. 23, 2010El-Mohsnawy et al.
amount of extracted monomer is around 1% of the standard
3D crystal structure analysis (1); however, subunits PsaA and
PsaB could be identified only by SDS-PAGE because of their
high mass (not shown). Most remarkably, subunit PsaL which is
easily lost upon dissociation of trimeric PS1 (44, 45) is present in
both monomeric PS1 fractions, which was also confirmed by
immunoblotting (not shown). This indicates the mild conditions
of both purification procedures, with the untreated PS1 mono-
mer showing a slightly higher PsaL content in the immunoblot
than the salt-treated complex (þST).
below 20000 Da with the exception of PsaM are post-transla-
tionally modified (see Table 1). The N-terminal methionine was
removed from six subunits (PsaC, PsaD, PsaE, PsaK, PsaL, and
PsaJ). PsaK was shown to be additionally acetylated, and
N-terminal processing was confirmed for the PsaF subunit (58).
For the evaluation of the quantitative PS1 monomer prepara-
tion involving salt treatment, it is important that no mass
deviation due to high temperature or high salt concentration
was observed.
Spectroscopic Analysis of Monomeric versus Trimeric
PS1. Spectral differences that most likely result from subtle
structural differences between PS1 monomersandtrimerscan be
revealed by comparing absorption spectra and absorption dif-
absorption spectra of PS1 monomers resemble closely those of
trimers except that trimers exhibit higher absorption in the far-
red region (710-725 nm). The fact that both types of monomers
exhibit identical spectra indicates that the combination of high
temperature with high salt treatment during the isolation of the
monomeric complexes did not affect their spectral properties.
The small shoulder at 683.5 nm observed in both PS1 monomers
might indicate a rearrangement of some Chl molecules during
trimerization, resulting in more far-red Chls in the trimer at
720-725 nm at the expense of the Chl absorption between 680
and 85 nm. To obtain more quantitative information about the
red-most chlorophylls, we decomposed the 5 K absorption
spectra of PS1 monomers and trimers in the red region by seven
Gaussian bands. The number of chlorophylls contributing tothe
red absorption can be estimated from the integrated intensity of
the long-wavelength absorption band to the total intensity of the
QYband and the number of chlorophylls per PS1. Both PS1
monomers are characterized by two LWCs absorbing at 720 nm
and fiveor six LWCsabsorbing at710 nm (Table 2). Incontrast,
PS1 trimers exhibit approximately four LWCs absorbing at
720 nm, while there are also five LWCs at 710 nm, in agreement
Figure 3A shows that flash-induced absorption difference
of P700þFA/B
PS1 in the dark-adapted state, with P700 reduced from those
measured after illumination. These difference spectra exhibit a
broad bleaching around 703 nm, a strong absorbance increase at
691 nm, a further bleaching at 685 nm, and several additional
ref 25 for a discussion). Figure 3B reveals also that the high-
resolution spectra detected at 5 K with PS1 monomers and
the arrangement of the reaction center pigments are the same in
the isolated complexes. In addition, P700 of PS1 monomers and
-are virtually identical at RT. Figure 3B presents
Table 1: PS1 Subunit Modifications of Monomeric and Trimeric PS1 According to MALDI-TOF MS Analysis (see Figure 1)
subunitdetermined massa(Da)calculated massa(Da)difference (Da) modification mass spectrometer (matrix)
PsaF (mature)
N-methinone deleted
N-methinone deleted
N-methinone deleted
cleaved N-terminus
formyl group added
formyl group added
N-methinone deleted and
acetyl group added
N-methinone deleted
no modification
N-methinone deleted
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
Voyager DE (Ferulic)
qTOF QSTAR XL (Sinapic)
qTOF QSTAR XL (Sinapic)
aMassspectrometers:qTOF QSTAR XLandVoyagerDE(fordetails,seeExperimentalProcedures).TheoreticalcalculationsofPS1subunits arebasedon
data from CyanoBase.
FIGURE 2: Absorption spectra (5 K) of PS1 monomers and trimers
(buffer consisting of 20 mM HEPES, 10 mM CaCl2, 10 mM MgCl2,
60% glycerol, and 0.03% β-DM).
Page 6
Article Biochemistry, Vol. 49, No. 23, 20104745
Different approaches have been used to analyze the pigment
composition of PS1 monomers and trimers. To determine the
number of Chls per PS1, we measured the concentration of Chl
and the content of P700 for the different samples. The P700
concentration was calculated from the maximal absorption
decrease in the QYregion at ∼702 nm using an extinction
difference coefficient Δε of 61000(2000 M-1cm-1. The iden-
tical absorption difference spectra of monomeric and trimeric
PS1 complexes (see Figure 3) show that the same Δε value might
be used. Using these Δε values, we obtained the following ratios:
112 for PS1 trimers, 108 for native PS1 monomers, and 109 for
for the trimer is slightly higher than for the monomers, but
distinctly higher than the level of 96 Chls per monomer as
determined from the X-ray structure (1). We can, however, not
extinction coefficient taken from the literature (54). Using
redissolved crystals of PS1 trimers from T. elongatus yielded
∼102 ( 6 Chls per P700 with a Δε of 61000 M-1cm-1(55),
whereas only 96 Chls have been identified in the 2.5 A˚struc-
ture (1). This could indicate that the Δε value might be slightly
The Car content wasdetermined byanalyzing the spectrum of
the pigment extract in an 80% acetone/water mixture (see
Experimental Procedures). Both PS1 monomers and trimers
yielded the same ratio of Chl a to Car of 4.55 ( 0.05
(not shown). On the basis of this value and assuming ∼100 Chls
per PS1, 22 ( 1 Car have been calculated per PS1 monomer and
from the 3D structure, i.e., 22 Cars.
The most remarkable difference between purified PS1 mono-
mers and trimers of T. elongatus was revealed in 77 K fluores-
cence emission spectra (Figure 4A). PS1 monomers peaked at
727 nm, while trimers peaked at 732 nm. This characteristic red
shift of 5 nm confirms the higher LWC content of the trimers.
Furthermore, the symmetric form of the observed spectra in
Figure 4A is a good indication of the purity of the isolated PS1
is significantly smaller in trimers (Figure 4B). It is also observed
that the shoulder at 695 nm in untreated monomers (-ST) shifts
to 688 nm with salt treatment (þST). Most probably, this is an
effect of the bound detergent concentration, which is higher for
the detergent-exposed monomers than for trimers, and also
higher for salt-treated monomers than for "native" monomers.
Remarkably, the presented data show only very small (or no)
fluorescence at 680 nm, which is typical for uncoupled Chls; this
confirms the high quality of the PS1 preparations.
To determine the functional activity of the purified PS1
monomers and trimers, we measured the rate of transfer of an
electron from DPIP and ascorbate to methyl viologen as oxygen
trimers showed (within the range of error) very similar oxygen
of Chl)-1h-1. Illumination with red light (λ > 665 nm) resulted
in a decrease in activity that was larger in the case of PS1
monomers than of PS1 trimers [850 and 1100 μmol of O2(mg of
Chl)-1h-1, respectively]. An even greater decrease was found
for the illumination with far-red light (λ>715 nm): 520 and
720 μmol of O2(mg of Chl)-1h-1, respectively.
Reasoning behind the Preparation of Nativelike PS1
Monomers. While the structure of the monomeric complex
can be deduced from the 3D crystal structure of the trimer
Table2: Chla/P700Ratio,CarotenoidContent,Long-WavelengthChloro-
phylls (LWCs), Photochemical Activity, and P700 Midpoint Redox Poten-
tial of Purified Monomeric and Trimeric PS1 Complexes
Chl a/P700
carotenoid content per monomer
LWC contenta(710 nm/720 nm)
oxygen uptake (WLc)
oxygen uptakeb(RLd)
oxygen uptakeb(FRe)
midpoint potential of P700a(mV)
112 ( 7
22 ( 1
1330 ( 50
108 ( 7
22 ( 1
1330 ( 50
aThe LWC (with absorption peaks at 710 and 720 nm) contents were
determined from 5 K absorption spectra of the corresponding PS1 com-
plexes by Gaussian deconvolution; the photochemical activity is deter-
mined as light-induced oxygen uptake (see Experimental Procedures), and
the P700 midpoint potential is determined by titration of light-induced
absorption changes at 700 nm.
O2per milligram of Chl per hour.
far-red light.
bPhotochemical activity in micromoles of
cWL, white light.
dRL, red light.
FIGURE 3: Flash-induced P700þminus P700 absorbance difference
spectra of trimeric and monomeric PS1 complexes at RT (A) and
were recorded by subtraction of the absorption spectra of PS1 in the
dark-adapted state, with P700 reduced from those measured after
illumination at 5 K (B).
Page 7
4746 Biochemistry, Vol. 49, No. 23, 2010El-Mohsnawy et al.
monomers being merged into one trimer in comparison with the
function of the individual PS1 monomer can only be determined
if a close-to-native preparation of the monomeric complex is
available. Until now, various attempts have been made toisolate
nativelike PS1 monomers from T. elongatus. Among them was
the deletion of the trimer-stabilizing PsaL subunit (3, 11, 41, 42),
an osmotic pressure treatment (45), the extraction by β-octyl
glucopyranoside at elevated temperatures (38), and the use of
mutants with impaired phosphatidylglycerol synthesis which
apparently is required for trimerization (44). All these attempts
resulted in partly damaged monomeric PS1 complexes that
lacked one or more subunits, especially PsaL and/or pigment
molecules (Chl and Car) to various degrees (45). In any case, the
activity of these complexes was considerably reduced, leading to
the assumption that only trimeric PS1 represents the fully active
PS1 form. This was supported by the fact that T. elongatus cells
with deleted psaL, which contained only monomeric PS1,
exhibited impaired growth under low-light conditions (11, 46).
In the special case of T. elongatus, major reasons for the
previous failing to isolate nativelike (i.e., with structural and
functional integrity) PS1monomers mightbe theirsmall amount
in the cells under the preparation conditions (46), their (partial)
lossduring washing with 0.1% β-DM (44), and/or the overlay of
PS1 monomers with the PS2 band in the sucrose density
gradient (50). Results of this study suggest that PS1 complexes
preexist in both monomeric and trimeric form within the
thylakoid membrane of T. elongatus, and that it is possible, if
care is taken, to isolate PS1 monomers in their native form for
further characterization.
Prerequisite for a detailed characterization of monomeric PS1
complexes is the possibility to isolate them in larger quantities
from the cells. As several reports have already shown, it is
trimers, which are dissociated, for instance, by detergent treat-
ment (50). The only promising strategy is to dissociate trimers
them with detergent. In principle, this is possible by a high-salt
treatment of the membranes as shown previously for mesophilic
cyanobacterial cells (59) and confirmed in this report for
T. elongatus. However, in contrast to the mesophile, the optimal
salt concentration was considerably higher for the thermo-
phile (500-600 mM vs 150-200 mM), and the type of salt
(i.e., divalent or monovalent ions) had a severe impact on its
effect. Apparently, divalent sulfate anions are most effective in
screening electrostatic surface charges, suggesting cationic coun-
terions on the PS1 surface are affected by it.
This organism provides an additional unique property. Be-
cause of its thermophilicity, the phase transition temperature of
its membranes is considerably higher than in mesophilic organ-
isms such as Synechocystis (60) which can be used to immobilize
or "freeze" transient compositions of the membranes. At tem-
peratures below the phase transition point, complexes can be
extracted from the membrane in their existing oligomeric status
this is due to the fact that the detergent replaces the lipids that
surround these complexes in their native state.
In the case of PS1, this situation is illustrated by the model
shown in Figure 5. Upon high-salt incubation above the phase
transition temperature, many PS1 trimers dissociate into mono-
mers (Figure 5A,B). Apparently, the added ammonium sulfate
ions neutralize charges in the monomer-monomer interface, in
which calcium ions may also be involved because of their impact
on trimerization (46). This may help to weaken electrostatic
forces(61) andenablelipids todiffuseintothe interface(which is
also promoted by the elevated temperature). These lipids help to
weaken the hydrophobic interaction between the monomers,
since their hydrophobic tails can act as a kind of shield for the
membrane-embedded parts of the proteins (48). The status of
salt-induced monomerization is conserved by rapidly lowering
the temperature to 20 ?C, i.e., distinctly below the phase transi-
tion temperature (Figure 5C). At 20 ?C, both monomeric and
trimeric complexes can be extracted by a mild detergent treat-
of isolated trimers into monomers nor an association of isolated
monomers into trimers is possible under our experimental
conditions (61), the ratio of extracted monomers to trimers (as
evidenced after chromatographic separation) most probably
reflects the ratio present in the membrane in response to the
ammonium sulfate concentration and the chosen temperature
(Figure 5D). If these conditions are chosen appropriately, PS1
monomers can be extracted in quite large amounts which is the
prerequisite for their in depth characterization in comparison
with the trimers.
FIGURE 4: Fluorescence emission spectra of different PS1 prepara-
tions. (A) The 77 K fluorescence spectra of PS1 monomers and
trimers; excitation at 440 nm [buffer consisting of 20 mM HEPES
(pH 7.5), 10 mM CaCl2, 10 mM MgCl2, 0.5 M mannitol, 0.03%
β-DM, and 60% glycerol]. (B) Fluorescence emission spectra of PS1
monomersand trimers atRT; conditions asfor panel A,but without
glycerol. The spectra were normalized by maximum intensity.
Page 8
Article Biochemistry, Vol. 49, No. 23, 20104747
Structural Implications of PS1 Monomers versus Tri-
mers: Protein Part. Our results indicate that under physio-
logical "low-salt" conditions the equilibrium is very much in
favor of the PS1 trimer with a quite small amount of monomer
being extracted. Omitting the salt step indicated that even the
(high) salt treatment caused no detectable loss of subunits or
pigments. Mass spectrometry analysis in combination with
SDS-PAGE and immunoblot analysis showed the complete
set of subunits both for the two monomer preparations and for
the trimer preparation, in agreement with the data from the 3D
X-ray structure of the trimer (1). The identical mass spectra of
monomers (þST and -ST) confirm the efficiency of the pre-
sented method and indicate that the complete assembly of PS1
should occur before the oligomerization process, in agreement
with results obtained for Synechocystis (62). It was reported that
the PsaL subunit is essential (together with PsaM and PsaI) for
the trimerization process (1, 46, 63-65), and it was also shown
that PsaL is easily lost during the isolation procedure (3, 45, 50).
In contrast, both -ST and þST monomers of our preparation
retained their PsaL subunit, confirming the extremely mild
isolation conditions used in our approach.
Additionally, this report could show for the first time the
complete analysis of modifications for all subunits, yielding
essentially no difference between monomers and trimers. Inter-
estingly, the same types of modifications have been shown for
PS2 subunits from T. elongatus, and these have been correlated
with the orientation of the subunits within the complex (66, 67).
According to this analysis, N-termini of PS2 subunits facing the
lumen seem to stay formylated, whereas N-termini facing the
probably because they are accessible for processing enzymes.
Similar results have been gained for the Cyt b6f complex (66).
PsaK and PsaM are processed, although their N-termini are
exposed to the lumen (1), and PsaJ still keeps the initial formyl
modification while its N-terminus faces the cytoplasm. This
suggests a different mechanism for the assembly of PS1, espe-
cially concerning its subunits PsaK, PsaM, and PsaJ, in compar-
ison with PS2 or the Cyt b6f complex.
Finally, the combination of salt treatment and "routine"
detergent extraction allowstheisolationof atleast10times more
monomeric PS1 than the "extreme mild treatment" and for this
reason was established as a routine preparation procedure for
monomers with upscaling capacity.
Structural and Functional Implications of PS1 Mono-
mers versus Trimers: Reaction Center and Antenna Chls.
Spectroscopic analysis such as the 5 K absorption spectra
(Figure 2) or the determination of the pigment content
(Table 2) suggests that PS1 monomers closely resemble PS1
trimers. The Chl/P700 ratio, reflecting the Chl antenna size per
T. elongatus and for the 3D crystal structure of the trimer from
the same organism. While the first HPLC-purified monomeric
PS1 displayed, like the trimer, 85 ( 5 Chls/P700 [determination
of P700 by light-induced absorption change (38, 50)], this value
could not be improved by a different preparation procedure,
yielding 85 or 65 Chls/P700 upon extraction by β-DM or
treatment during the preparation process. In contrast, our
monomeric complexes showed ∼108 ( 7 Chls/P700 (trimer,
112 ( 8 Chls/P700), indicating that there is no loss of antenna
pigments during the preparation, although these numbers might
be slightly too large due to the used extinction difference
coefficient (see Results). The Chl/Car ratio is within the error
margins the same for PS1 monomers and trimers, resulting in
∼22 Cars per PS1 which is in agreement with the X-ray
structure (1). These results indicate that the antenna size and
organization do not change during the oligomerization process
and confirm the conclusion that the improved isolation method
gave nativelike PS1 monomers.
There is one interesting exception. At 5 K, the absorption
FIGURE 5: Model illustrating the experimental steps taken to moni-
tor the equilibrium between PS1 monomers and trimers in the
thylakoid membrane of T. elongatus. (A) At <30 ?C (solid lipid
phase), PS1 trimers are surrounded by lipids. Hydrophobic inter-
action (i.e., the central PsaL subunit in combination with Chl and
Car) and electrostatic interaction (involving Ca2þions) may help to
stabilize the trimer. (B) Heating to 50 ?C (mobile lipid phase) in the
presence of a high salt concentration. Weakened electrostatic inter-
actions between PS1 monomers due to increased ion concentration.
Diffusion of lipids into the PS1 trimer contact area weakens hydro-
phobic interactions between the monomers and establishes an equi-
librium between monomeric and trimeric PS1. (C) Cooling to 20 ?C
(solid lipid phase). PS1 monomers and trimers are surrounded by
lipid molecules ("trapped equilibrium"). (D) Lipids of extracted PS1
tion of monomeric PS1 in the thylakoid membrane, the isolation of
intact PS1 monomers is possible; additionally, preexisting PS1
trimers are isolated.
Page 9
4748 Biochemistry, Vol. 49, No. 23, 2010El-Mohsnawy et al.
less pronounced in the monomers. According to analysis by
Gaussian deconvolution, the PS1 monomers contain approxi-
mately two fewer LWCs than the trimers (see Table 2), which is
also confirmed by the 5 nm blue shift in the 77 K fluorescence
trimers (see Figure 4A). This effect can be used as a "quality
control" for isolated PS1 monomers. There is also agreement
istic 77 K main fluorescence peaks of monomeric and trimeric
PS1 (730 and 760 nm, respectively) confirmed the successful
in vitro oligomerization of isolated monomeric PS1 from
A. platensis into trimer (31).
to the absence of the L-subunit leading to the loss of LWCs
monomers of this report, too, exhibit a decreased red-most
absorption band, two explanations seem feasible. (a) It is quite
possible that trimerization causes conformational changes of
assembled monomers which lead to a stronger coupling of two
(or more) Chl molecules; this may induce formation of a Chl
aggregate within each monomer, resulting in an increased red-
shifted absorption. (b) Alternatively, strong interaction between
peripheral Chls of different monomers within the trimer might
lead to a red-shifted low-energy exciton band. Two such Chls
can be identified, for instance, from the X-ray structure of
T. elongatus trimers: Chl 11601 coordinated by subunit M of
one monomer and Chl 11801 coordinated by a phospholipid of
the other (Chl numbering according to Protein Data Bank entry
1JB0) approach by the trimerization to an edge-to-edge dis-
tance of ∼5 A˚. Especially the M subunit-coordinated Chl is not
well connected to the energy transfer network within the mono-
mer, but apparently better connected to Chls of the adjacent
in the transfer of energy among the monomers integrated within
the trimer (for a more detailed discussion, see refs 15 and 68). If
we attribute the additional LWCs in the PS1 trimer to these two
P700. It should be noted that Car 7 is closest to the proposed
M1 of the adjacent monomer.
different "red" Chl pools in monomeric and trimeric PS1
complexes from T. elongatus (69) isolated according to the
method outlined here. Because of the mild preparation condi-
tions, the origin of the red compartments in the monomer could
be compared with that of the trimer. These results indicated that
either the monomeric or oligomeric state. It is not clear why the
two additional LWCs observed in trimers could not be revealed
in these data.
The number of Chls that are disconnected from the Chl
antenna network is another indicator for the quality of a
preparation. Such Chls lose their ability to transfer energy to
other Chls which results in a fluorescence emission at ∼670-
680 nm. The fact that both PS1 monomers and trimers at 77 K
showed no emission at this wavelength confirms the absence of
such Chls in our preparations (see Figure 4A). At RT, fluores-
cence emission spectra of the various PS1 preparations show a
smaller in trimers (Figure 4B). This might be partly explained by
of the bound detergent concentration. The surface area which is
exposed to the detergent β-DM most probably is larger for
monomers than for trimers; this results in a tension force that
a disconnection from the antenna network. A further increase in
the detergent concentration or the use of more aggressive
detergents would result in a loss of these Chls due to incorpora-
tion into detergent micelles. This would explain the lower
Chl/PS1 ratio observed in earlier PS1 preparations.
Spectral properties of the reaction center can be monitored by
time-resolved absorption difference spectroscopy. Absorbance
difference spectra reflect the changes in absorption upon light-
induced formation of transient functional states (charge-
separated states or3P700) within the reaction center. Due to
the difference, only pigments with altered electronic state and
we measured the flash-induced absorption difference spectra
of PS1 monomers and trimers attributed to the formation of
high-resolution spectra at 5 K were almost identical for mono-
mers and trimers, this indicates identical spectral properties and
an identical arrangement of the reaction center pigments. In
addition, the identical midpoint potential for P700 in monomers
and trimers strongly suggests that the surroundings of P700 and
the redox properties of the components involved in electron
transfer do not change upon oligomerization (Table 2).
The light-induced electron transport activity (DPIP f MV) is
also the same for PS1 trimers and monomers using high-white
light illumination. The rate of ∼1350 μmol of O2(mg of Chl)-1
h-1corresponds to the transport of 55 electrons per PS1 per
second,with the rate-limiting step being most likely the donation
of an electron to P700þby DPIP. Using red and far-red
illumination, the electron transport activity is higher for trimers
in the far-red region can be related to their higher LWC content
which might contribute by uphill-energy transfer to P700,
especially under low-light conditions (19, 35).
Physiological Implications of PS1 Dynamics. The com-
parison of nativelike PS1 monomers, produced by various
procedures, with PS1 trimers revealed that monomers most
probably preexist in thylakoid membranes in equilibirium with
trimers. As indicated by our extraction under extremely mild
conditions, this equilibrium under "normal", i.e., stress-free,
conditions is very much in favor of trimeric PS1, leaving less
than 10% of all PS1 for the monomer. A similar ratio has also
been found for membranes of the mesophilic cyanobacterium
Synechococcus PCC 7002 (65).
The physiological significance of this monomer-trimer equi-
librium may be many-fold. (1) Especially in the case of thermo-
philic organisms such as T. elongatus, the trimer may be
dominant because of its higher stability based on hydrophobic
FTIR nvestigations (Kopczak, Dzafic, M€ antele, and R€ ogner,
unpublished observations). (2) Besides better thermostability,
the PS1 trimers may also yield a higher photostability due to the
higher content of red-most chlorophylls which help in the
dissipation of excess absorbed energy (36). (3) Stress conditions
such as Fe limitation (70) have been reported to induce mono-
merization. Combined with this, the dynamic adaptation to
changing light conditions by rearrangement of phycobilisome
contact with the photosystems (known as state transitions) may
-atRT(Figure3A) and5K(Figure3B).Aseventhe
Page 10
ArticleBiochemistry, Vol. 49, No. 23, 20104749
be induced by reversible monomerization/trimerization, which
has been suggested (61, 65). (4) Especially the structural and
functional integrity of the monomer strongly indicates a role as
ofthetrimer.Thisisinagreement withthecaseofphotosystem 2
from the same organism, where a fully competent monomeric
PS2 could be shown to precede the dimeric complex (52), but
different from the case of the cytochrome b6f complex, which
requires the formation of a dimer for gaining activity (Gomolla
and R€ ogner, unpublished observations). While the dynamics of
monomeric PS2 complexes as "intermediates" in biogenesis and
repair could be shown due to the high turnover rate of their D1
subunit (52), such processes are considerably delayed in the case
of PS1 with reported lifetimes of several days (71). For this
the medium to gain sufficient material for comparative charac-
terization with the trimer. Such a change in equilibrium may
occur under physiological conditions by light and/or other stress
conditions which still must be shown.
However, prerequisite for all these dynamic adaptations is an
appropriate fluidity of the membrane which has been shown in
this report for T. elongatus. The combination of all reported
processes indicates a considerable potential for the dynamics of
PS1 in the thylakoid membrane and may be the basis for its
longevity and stress tolerance. Moreover, the comparison with
PS2 shows that the monomer-oligomer equilibrium in the
membrane is a universal mechanism for the regulation of the
activity and stability of the photosynthetic apparatus.
Conclusions. (1) Monomeric PS1 is fully competent for all
major reactions of trimeric PS1 and can be isolated as an intact
complex with identical antenna size, subunit composition
(including posttranslational modifications), and photochemical
activity. (2) The dynamicequilibrium betweenthe PS1 monomer
and trimer in the thylakoid membrane is under nonstress condi-
transition temperature of the surrounding lipids. (3) The struc-
tural equilibrium between monomer and trimer is reflected by a
"functional" equilibrium with the trimer showing two additional
long-wavelength chlorophylls.
We thank Claudia K€ onig and Regina Oworah-Nkruma for
excellent technical assistance.
Details of the biochemical isolation procedure, including
quantification of the monomer/trimer ratio by HPLC elution
is available free of charge via the Internet at http://pubs.acs.org.
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Latest Rover Stories
NASA prototype lunar lander Morpheus experienced a malfunction this week during testing, leading the future spacecraft to crash and burn.
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Written in the style of a thriller but solidly based on an array of sources, this study reinterprets the entire sea campaign in the Pacific, using intelligence as the missing key to the Allied success. It examines every aspect of the secret war of intelligence -- from radio dispatches and espionage to vital information from prisoners and document ... Read More Written in the style of a thriller but solidly based on an array of sources, this study reinterprets the entire sea campaign in the Pacific, using intelligence as the missing key to the Allied success. It examines every aspect of the secret war of intelligence -- from radio dispatches and espionage to vital information from prisoners and document translation -- showing how U.S. intelligence outsmarted Japan nearly every step of the way. The resulting assessment is a virtual rewriting of history that challenges previous conceptions about the Pacific conflict. John Prados relates the growing intelligence knowledge on both sides to the progress and outcome of naval actions. Along the way he offers a wealth of revelations that include data on how the United States caught the superbattleship Yamato and the impact of intelligence on the initial campaigns in the Philippines and Netherlands East Indies and the escape of American codebreakers from Corregidor. He also provides colorful vignettes of personalities who shaped the secret intelligence war. This ambitious work is not simply a rundown of code-breaking successes, but an astonishing demonstration of how the day-to-day accumulation of knowledge can produce extraordinary results. Its accounting of Japanese intelligence is unprecedented in detail. Its reassessment of battles and campaigns is presented not just in terms of troops or ships but in how the secret war actually played out. Lauded as a major new study when published in hardcover in 1995, the book remains the most comprehensive study written. For sheer drama and gut-level operational practicality, it ranks with the very best. Read Less
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^ +1That's what I did too on my 500 rebuild.. I sent the guy I bought them from on ebay a message to let him know this was an issue, and he replied that he'd sold "hundreds" of these kits and never had a complaint. Not likely!


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Break the Limits!
You are trapped in a room and the only thing you can do is think.
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Popular Songs
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Economists warn on Tory cuts
More than 50 economists have signed a letter in support of Prime Minister Gordon Brown's economic proposals and warning Tory cutbacks could tip the country back into recession.
In a letter leaked to The Daily Telegraph, the 58 economists warn that plans by Conservative leader David Cameron (pictured) to £6bn-worth of "efficiency" savings in Government could lead to job losses and "destabilise" the recovery.
Economists Lord Layard, Lord Skidelsky, Lord Peston and Sir David Hendry are among the 58 who signed the letter.
It says: "This cut is described as efficiency savings. But in macroeconomic terms it is just a cut by another name. It will lead directly to job losses and indirectly to further falls in spending through the standard multiplier process."
"At a time when recovery is delicate it could even affect confidence to the degree that we are tipped back into recession - with much larger job consequences."
It adds: "This is not the time for such a destabilising action. The recovery is still fragile. Firms and households are saving more to rebuild their balance sheets, so that firms are investing less and households are spending less."
Meanwhile, the Conservatives say that more than 400 firms back its proposals to stop most of Labour's National Insurance rises.
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Readers' comments (1)
Oh yes, economists - the ones who should have prevented us from getting into this mess in the first place - remind me why we should listen to them.
As we all know, £6 billion sounds like a lot, but it is a drop in the ocean as a proportion of GDP and the UK's debt burden. Bear in mind that Labour is already planning £15 billion of cuts. Why should another £6 billion make such a difference? It's not much more than a rounding error when you look at the balance sheet of UK plc.


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Panoramic shot of the Colossuem in Redscale... different
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The BIG List: 37 Greatest Signature Ad Libs
#1. Diddy: "Uh-huh, yeah!"
The king of the ad libs! Whether he's talking reckless (like he did earlier this year on Jay Electronica's "The Ghost of Christopher Wallace"), yelling at haters (like on Nas's hit "Hate Me Now") or just filling up space to let the beat rock (like on his own hit, "All About The Benjamins"), Diddy has perfected the art of ad libbing.


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Standing around doin nothin and tugging on your scrotum sack when there is work to be done.Dragging Balls
Guy 1: What do you do at work all day?
Guy 2: Nothin but pull stones
Guy 2: Nothin but pull stones
by rdbrooks
March 07, 2008
1 more definition
To "Pull Stone" means to "exit" or get out quickly. Derived from the action of car wheels on a road surface.
"I saw them jack the bank... and pull stone something fierce."
by Eric White
October 31, 2005


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A work of almost breathtaking visual beauty that manages to ravish the heart while dazzling the eye simultaneously, neither at the expense of the other.
A work of almost breathtaking visual beauty that manages to ravish the heart while dazzling the eye simultaneously, neither at the expense of the other.
Avery's more than capable behind the camera, he just needs to be met halfway by his screenwriting, which dwells in overly familiar territory.
"The Ballad of Narayama" is a Japanese film of great beauty and elegant artifice, telling a story of startling cruelty. What a space it opens…
Patrice Leconte's "Monsieur Hire" is a tragedy about loneliness and erotomania, told about two solitary people who have nothing else in common. It involves a…
"Life Itself" won the 2015 Best Documentary award from the Producer's Guild of America.
An excerpt of Eric Kohn's Indiewire article on Oscar snubs.
A note of thanks from Chaz Ebert to the wonderful people behind "Life Itself."
A look at Kimberly Pierce's 2013 version of "Carrie."
A look at Kimberly Pierce's 2013 version of "Carrie."
An FFC comments on Roger Moore's best James Bond film, "The Spy Who Loved Me."
A report from the opening night of the 30th Annual Santa Barbara International Film Festival.
An interview with Anna Boden & Ryan Fleck of "Mississippi Grind".
'The Glass House" brings skilled technique to a plot that's a foregone conclusion. Since it's clear from early in the film what must have happened and why, it's a film about waiting for the characters to catch up to us. The movie's trailer doesn't help, with its comprehensive betrayal of the movie's key secrets. It should even be a secret that this is a thriller--we should walk in thinking it's about kids surviving the loss of their parents. No chance of that.
The film opens with one of those irrelevant shock buttons that have become annoying in recent years--five or 10 minutes that have nothing to do with the rest of the story, but fool us with misleading footage. In this case, there's a horror scene, and then we see it's a film, and then we see the heroine and her friends watching it--and, yes, they're cute as they giggle at their own reactions, but openings like this are empty stylistic exercises. Once was a time when the well-made film used its opening scenes to dig in, not just spin its wheels.
The movie was directed by the TV veteran Daniel Sackheim, who worked on "ER," "The X-Files," "Law & Order," and other series that are smarter than this. It stars Leelee Sobieski, one of the best young actresses, as Ruby Baker, who with her little brother, Rhett (Trevor Morgan), is orphaned when their parents die in a car crash. The family lawyer (Bruce Dern) explains that the parents had arranged for their close friends Erin and Terry Glass to be their guardians in the case of tragedy, and soon the kids are moving into the Glasses' big glass house (uh huh), which is luxurious, although Ruby and Rhett are a little too old to be sharing the same bedroom. It's a detail like that we find annoying. Why would the Glasses, who have acres of living space on their Malibu hilltop, put the kids into one room? Given the Glasses' long-term plans, why not make the kids as happy as possible? There's a kind of thriller in which the events unfold as they might in real life, and we have to decide which way to take them--and another kind of thriller, this kind, where the events unfold as a series of ominous portents, real and false alarms, and music stingers on the soundtrack. The first kind of thriller is a film, the second is a technical exercise.
What makes "The Glass House" sad is that resources have been wasted. Diane Lane and Stellan Skarsgard, as the Glasses, are so good in the dialed-down "realistic scenes" that we cringe when they have to go over the top and make everything so very absolutely clear for the slow learners in the audience. Sobieski is fine, too--as good an upscale Los Angeles high school student as Kirsten Dunst in the recent "Crazy/Beautiful," but in a genre exercise that strands her instead of going someplace interesting and taking her along.
It's good to see Bruce Dern again. He's one of those actors, like Christopher Walken, who you assume on first glance has a secret evil agenda. Here he's the family lawyer who the kids can or can't trust, and is wise enough to play the character absolutely straight, with no tics or twitches, so that he keeps us wondering--or would, if Wesley Strick's screenplay wasn't one of those infuriating constructions where the key outside characters turn up at the wrong times, believe the wrong people and misinterpret everything.
Speaking of turning up, Sobieski's character turns up at too many right times. How fortunate that she drops in on Mr. Glass's office just at the right moment to eavesdrop, unobserved, on crucial dialogue. And how unfortunate that she seems to be proving the Glasses right and herself wrong when a social worker walks in on a crucial moment and, of course, misinterprets it.
If you want to see a great movie about a couple of kids endangered by a sinister guardian, rent "The Night of the Hunter." Watching "The Glass House" has all the elements for a better film, but doesn't trust the audience to keep up with them. Having criticized the Strick screenplay, I should in fairness observe that the way it usually works is, the writer puts in the smart stuff and then it comes out in the story conferences with executives who figure if they don't understand it, nobody will.
This message came to me from a reader named Peter Svensland. He and a fr...
A look at Kimberly Pierce's 2013 version of "Carrie."
A note of thanks from Chaz Ebert to the wonderful people behind "Life Itself."


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Around the Nation
6:14 am
Tue November 26, 2013
Minnesota Woman Embraces Her Full Name
By editor
Originally published on Tue November 26, 2013 12:28 pm
Good morning, I'm David Greene.
A woman in Minneapolis turns 43 years old on Thursday. Dr. Happy Thanksgiving Reynolds says she was born to hippie parents, who hadn't picked out a name ahead of time. When their daughter arrived on Thanksgiving Day, they took it as a sign. Far from being embarrassed about her name, Dr. Reynolds embraces it. She says it even helped her get job interviews. Adding, quote, "I'm someone you are not going to forget based on the name." Surely, indeed.
Well, let me say this: Happy Birthday, Happy Thanksgiving.
It's MORNING EDITION. Transcript provided by NPR, Copyright NPR.


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Q&A: f(x) Reacts to Topping K-Pop Hot 100 & World Albums Chart (Exclusive)
By Jeff Benjamin, New York and Jessica Oak, Billboard Korea
Backstage at KCON 2013, the K-pop group talks their big year and details filming a Funny or Die skit with Anna Kendrick.
Girl groups were in short supply at KCON 2013. Though, given f(x)'s whirlwind 2013, they likely would have more than sufficed even without a last-minute addition of Crayon Pop at the American K-pop convention.
Despite being away from the K-pop scene for over a year, the female quintet saw their domestic and international popularity maintained with new single "Rum Pum Pum Pum" hitting No. 1 on the K-Pop Hot 100. Meanwhile, its accompanying full-length album, "Pink Tape," not only topped Billboard's World Album chart, but also placed on Billboard's Heatseekers Albums chart -- meaning a Billboard 200 entry wasn't too far away.
Falling in line with past singles such as "Pinocchio (Danger)" and "NU ABO," the 12 tracks on "Pink Tape" also mix fizzy electro-pop, icy harmonies and strange, yet addictive, hooks. One could argue the quirky confections on the LP represent the contrasting personalities of the five f(x) members -- on display as the quintet sit down for an interview with Billboard hours before opening KCON's grand finale K-pop concert.
Cool and calm, members Sulli and San Francisco-bred Krystal appear guarded at first. However, undeniable f(x) sweetheart Luna wears a huge smile on her face the entire chat and during the KCON concert -- at one point appearing to stray from her group's choreography to wave to fans on the other side of the stage. 26-year-old leader Victoria is nice, but nervous; likely anxious to present her band in the best light possible. Meanwhile, Los Angeles native Amber enters the room like a sledgehammer of confident energy, perhaps from being most comfortable speaking in English and having sister Jackie backstage.
But minutes into talking with f(x), the girls' personalities blend into one with a collective concern for fans and a genuine excitement seeing their hard work result in success -- even reserved Krystal breaks character for a second, loudly exclaiming "Really?!" after a mention that f(x) topped World Albums. Billboard gets f(x)'s reactions on the year so far.
KCON 2013: 15 Things Seen & Heard
Welcome to Los Angeles, f(x). You're here today to perform at KCON 2013, but you made your K-pop debut in 2009. How did you decide what three songs to perform today?
Krystal: We're performing our new song "Rum Pum Pum Pum," "Electric Shock" and "Hot Summer." The reason why we're performing them is that "Rum Pum Pum Pum" is our new song; it's our first time performing it in the States. "Electric Shock" is really noisy. And we're performing "Hot Summer" because it's so hot outside.
"Rum Pum Pum Pum" has a lot of layers and harmonies. Are you at all nervous about performing it live tonight?
Sulli: The song is difficult to sing live, but our main concern is that we just want our fans to enjoy our performances.
NEXT: "We were like, 'Dude! It's been a year. How are we going to do?!'"


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