Photosynthesis Research 64: 167–177, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
167
Regular paper
Crystallization of dimers of the manganese-stabilizing protein of
Photosystem II
Rina Anati & Noam Adir∗
Department of Chemistry and Institute of Catalysis, Science and Technology, Technion – Israel Institute of
Technology, Technion City, Haifa 32000, Israel; ∗ Author for correspondence (e-mail: [email protected])
Received 10 December 1999; accepted in revised form 24 January 2000
Key words: dynamic light scattering, mass spectrometry, oxygen evolution, photosynthesis, protein crystallization,
protein structure
Abstract
The manganese-stabilizing protein (MSP) of Photosystem II was purified from spinach photosynthetic membranes.
The MSP was crystallized in the presence of calcium. Despite the apparent purity of the isolated protein, the
crystals grew to only about 0.05 mm in their largest dimension. The MSP was analyzed to identify possible
sources of protein heterogeneity that could hinder crystal growth. Tandem reverse-phase HPLC/ electronspray
ionization mass spectrometry analysis of the MSP showed a major peak and four smaller peaks. All five peaks had
molecular masses of 26 535, as expected for mature MSP, indicating the absence of heterogeneities due to covalent
modifications. MALDI mass spectroscopy was utilized to identify heterogeneities in the MSP oligomeric state.
These measurements showed that purified MSP in solution is a mixture of monomers and dimers, while solubilized
MSP crystals contained only dimers. Size-exclusion chromatography and dynamic light scattering were used to
probe the effect of the crystallization conditions on the MSP. Size-exclusion chromatography of concentrated
MSP showed the presence of aggregates and monomers, while dilute MSP contained monomers. Dynamic light
scattering experiments in the absence, or in the presence of 10–50 mM or 100 mM calcium, yielded calculated
molecular mass values of 34 kDa, 48 kDa and 68 kDa, respectively. These changes in the observed molecular
mass of the MSP could have been caused by the formation of dimers and higher oligomers and/or significant
conformational changes. Based on the results reported in this study, a model is presented which details the effect
of oligomeric heterogeneity on the inhibition of MSP crystal growth.
Abbreviations: D – diffusion coefficient; ESI-MS – electronspray ionization mass spectrometry; Hepes – 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; MALDI-MS – matrix-assisted laser desorption ionization/time
of flight mass spectrometry; MSP – manganese-stabilizing protein (also 33 kDa extrinsic protein); OEC –
oxygen-evolving complex; PEG – polyethylene glycol; PS II – Photosystem II; RC II – reaction center II; Rh –
hydrodynamic radius; RP-HPLC – reverse phase high performance liquid chromatography; SDS–PAGE – sodium
dodecyl sulfate-polyacrylamide gel electrophoresis; SEC-HPLC – size exclusion high performance liquid chromatography; tris – tris(hydroxymethyl)aminomethane; Ve – elution volume
Introduction
The primary steps in oxygenic photosynthetic electron transfer occur within the reaction center of Photosystem II (RC II; Ghanotakis and Yocum 1990;
Debus 1992; barber 1998). This large protein–pigment
complex reduces bound quinone while oxidizing water with concomitant release of molecular oxygen
(Hoganson and Babcock 1997). Water oxidation occurs within a unique environment called the oxygenevolving complex (OEC; Ghanotakis and Yocum
1990; Yocum and Pecoraro 1999). The core of the
168
OEC is a tetramanganese complex, which is bound
to the luminal side of RC II by amino acid residues
from intrinsic proteins (Debus 1992). The OEC in intact PS II (in vivo or in isolated thylakoid membranes)
also includes a cluster of three proteins surrounding
the tetramanganese complex (Ghanotakis and Yocum
1990; Seidler 1996). Only one of these proteins, the
psbO gene product, is conserved in all oxygenic photosynthetic organisms (Kuwabara and Murata 1979).
Higher plants and green algae PS II have two additional extrinsic OEC proteins, a 23 kDa protein and a
17 kDa protein (Debus 1992; Campbell et al. 1998).
Cyanobacterial OEC has two alternative proteins, a 12
kDa protein and cytochrome c-550 (Shen et al. 1995,
1998). It has been suggested that the role of the three
OEC proteins is to create a stabilizing environment for
the tetramanganese core.
The psbO gene product has been shown to be
the most important of the OEC subunits for sustained oxygen evolving activity, and is thus known
as the manganese-stabilizing protein (MSP; Debus
1992; Zubrzycki et al. 1998). The MSP is nuclear
encoded, translated in the cytoplasm, transported into
the chloroplast and through the thylakoid membrane
into the luminal space, while being processed to its
mature molecular weight of 26.5 kDa. The number of
MSP copies per RC II has been estimated as either one
(Enami et al. 1991) or two (Betts et al. 1997). The
MSP has been proposed to bind to the luminal face
of RC II by both electron microscopy and electron
diffraction of two-dimensional crystals (Tsiotis et al.
1996; Hankamer et al. 1999; Kuhl et al. 1999). Biophysical measurements performed on a number of site
directed mutants have tried to locate and map the MSP
binding site on the luminal side of RC II (Eaton-Rye
and Murata 1989; Debus 1992; Chu et al. 1994; Qian
et al. 1997; Qian et al. 1999). Spectroscopic investigations of the interaction between the tetramanganese
core and RC II components support an asymmetric
position, closer to the D1 protein side of the reaction center (Evelo et al. 1989; Gilchrist et al. 1995).
The secondary structure of the MSP, as predicted by a
variety of computer algorithms, is about 40% β-sheet,
almost 60% random coil and less than 5% α-helical regions (Xu et al. 1994). While the sequence homologies
of all RC II intrinsic proteins is high, the sequence homology between MSP from spinach and other higher
plants is only 80–90%, and less than 50% compared to
cyanobacterial MSP.
A mutant of the cyanobacteria Synechocystis sp.
6803 lacking the MSP protein was produced which
grew photoautotrophically. However, it had significantly reduced levels of oxygen evolution and an unstable PS II (Burnap and Sherman 1991). However, a
similar deletion mutant in the green alga C. reinhardtii
was non-photosynthetic (Mayfield et al. 1987). Thus,
while the MSP appears not to participate directly in
manganese binding (Ghanotakis and Yocum 1990), its
presence enhances both the activity and stability of
oxygen evolution. These results indicate that the MSP
has a subtle role in both the activity and structure of
the OEC and RC II. A more complete understanding
of the function of the OEC requires structural information, including the three-dimensional structures of the
OEC proteins. Three-dimensional crystals of oxygen
evolving RC II, including the MSP, have been grown
(Adir 1999; Zouni et al. 1999), however, the structure
of the RC II complex has not yet been elucidated.
In higher plants or green algae, the 23 kDa and
17 kDa proteins can be removed from RC II by 1M
NaCl (Miyao and Murata 1984a), which is indicative
of electrostatic interactions with the luminal side of
RC II. The MSP is bound considerably more tightly
to the RC II (Ghanotakis and Yocum 1990) and can
be removed by treatment with either 0.8 M tris buffer
(Ono and Inoue 1986), 1 M CaCl2 (Ono and Inoue
1983) or 2.6 M urea and NaCl (Miyao and Murata
1984b). This suggests that binding of the MSP to PS II
may involve additional forces, which may include hydrogen bonds (Enami et al. 1998) and/or hydrophobic
interactions (Hashimoto et al. 1996). The involvement
of multiple forces may be an indication that conformational changes occur during MSP binding/unbinding.
It has been suggested recently that the MSP belongs
to a family of proteins distinguished by a large degree
of unfolding and refolding (natively unfolded proteins
(Weinreb et al. 1996)) when it binds to or disassociates
from RC II (Hutchison et al. 1998). These authors used
vibrational spectroscopy of bound and unbound MSP,
and determined that between 30 and 40% of the peptide backbone undergoes significant conformational
change. In a different study, the carboxyl-terminal of
MSP was shortened by 3 residues, which resulted in a
significant increase in its apparent molecular mass (as
determined by size exclusion chromatography (SEC)),
and affected its ability to reconstitute active PS II
(Betts et al. 1998). These results were interpreted as
being the result of additional unfolding of the unbound
mutated MSP, due to the truncation of the carboxyl
terminal.
Determination of the three-dimensional structure
of the MSP in its bound state requires the determ-
169
ination of the entire RC II structure. However, the
structure of the free protein could be helpful in understanding how the protein might stabilize the tetramanganese center, whether there are potential metal
binding sites, and might shed light on the structure
of a putative natively unfolded protein (Weinreb et al.
1996). In this paper, we describe the crystallization
of the MSP and the characterization of the oligomeric
state of the MSP in crystal growth conditions.
Materials and methods
Purification and crystallization of the MSP
Thylakoid membranes were obtained from freshly
picked spinach leaves (Berthold et al. 1981) and stored
frozen in the presence of 20% glycerol. Thylakoids
were thawed and washed to remove glycerol, and
PS II membrane fragments were isolated (Berthold
et al. 1981). The MSP was extracted in 0.8 M tris
buffer from the PS II membrane fragments as previously described (Ono and Inoue 1986). The MSP was
precipitated in 80% saturated ammonium sulfate, dialyzed against 20 mM tris (pH = 8.0) and purified on
a DEAE anion exchange column (Toyopearl, Tosohaas). The MSP was eluted using a 0–200 mM NaCl
gradient. The eluted protein was dialyzed against 20
mM tris (pH = 8.0) and concentrated by ultracentrifugation (Centricon-30, Amicon) to 10–20 mg/ml.
Protein concentration was measured by UV absorption
measurements (Stoscheck 1990). MSP was crystallized using the hanging drop vapor diffusion method.
Equal volumes of MSP (10–15 mg/ml) and the reservoir solution (50–100 mM CaCl2 , 5–10% PEG400 in
50 mM HEPES pH = 7.5).
Analytical techniques
SDS–PAGE (14% acrylamide resolving gels) was performed using the Laemmli buffer system (Laemmli
1970).
SEC-HPLC analysis of MSP was performed on a
PL-GFC 1000 Å HPLC column (Polymer Laboratories) equilibrated in 50 mM tris (pH = 8.0) with 100
mM NaCl, at a flow rate of 0.5 ml/min and monitored
at 276 nm. The MSP was analyzed at different concentrations with sample volumes of 5 µl. The elution
profile was used to calculate the molecular mass based
on standard proteins (Sigma Chemicals).
RP-HPLC, ESI-MS and MALDI-MS were performed by the Protein Analysis Center in the De-
partment of Biology, Technion (Haifa, Israel). RPHPLC was performed on a 2.1×30 mm C-8 column
(AQUAPORE RP-300, Applied Biosystems), and
eluted with a linear 15–65% acetonitrile gradient
(ACN) in 0.05 TFA, at 1.25%/ min and a flow rate of
50 µl/min. ESI-MS (LCQ, Finnigan), was performed
in the positive ion mode. The mass estimation of the
proteins was done using the deconvolution algorithm,
which transforms an ESI mass spectral plot of relative
abundance versus mass–charge ratios, into a plot of relative abundance versus mass. Each sequence of multiply charged ion peaks in the acquired mass spectrum
(corresponding to one sample component) is converted
into a single peak positioned at the molecular mass
in the deconvoluted spectrum. For MALDI-MS (2E,
Micromass UK), the MSP (1 mg/ml) was deposited
on the metal target as sinapic acid (Fluka) derivatives (Beavis and Chait 1989), and the mass spectrum
was determined in the positive ion mode. Two mass
standards, trypsinogen (23.980 kDa) and bovine serum
albumin (66.430 kDa), were used for mass calibration.
MALDI-MS of MSP microcrystals was performed by
removal of the crystals from the growth drop, centrifugation, resuspension in protein free crystallization
mother liquor (repeated twice) to wash the crystals
from possible contamination from uncrystallized protein. Following the third centrifugation, the crystals
were solubilized in 1–2 µl of tris buffer.
Dynamic light scattering was performed on a
DynaPro MSTC (ProteinSolutions Inc.). The temperature for all measurements was 20 ◦ C. All protein
samples (12 µl) were filtered, and the optimal protein
concentration was found to be 1 mg/ml. The results were analyzed by the ProteinSolutions Dynamics
software package. Molecular masses were calculated
using two calculation models (volume shape hydration model or standard curve model) included in the
software package.
Results and discussion
Crystallization of spinach MSP
Figure 1 shows a SDS–PAGE analysis of the purified
MSP from spinach. The MSP can be easily extracted
from salt washed thylakoid membranes using either
tris buffer (Ono and Inoue 1986), 1 M CaCl2 (Ono
and Inoue 1983) or 2.6 M urea (Betts et al. 1996).
Isolation of proteins for the screening of crystallization conditions requires the protein to be highly pure,
170
Figure 1. SDS–PAGE of MSP purification steps. (1) PS II membranes, (2) supernatant of NaCl wash, (3) NaCl washed PS II membranes, (4)
MSP from tris extracted, NaCl washed PS II membranes, (5) tris washed PS II membranes, (6) MSP from 80% ammonium sulfate precipitation
step, (7) purified MSP after anion-exchange chromatography, (8) molecular weight standards (Sigma Chemicals, wide range). Mr , relative
molecular masses (kDa).
Figure 2. MSP microcrystals. The crystals were removed from their growth drop, washed in reservoir solution and treated with a protein crystal
specific blue dye (IzitTM , Hampton Research). Bar indicates 0.05 mm.
171
homogeneous and devoid of small molecule additives due to the purification process. We chose the
tris wash method (Ono and Inoue 1986) as the least
problematic of the three possible methods. Following
precipitation with 80% saturated ammonium sulfate
purification and anion-exchange chromatography, the
MSP is essentially pure (Figure 1, lane 7). The purified
MSP could be concentrated in buffer to up to 25 mg/ml
without the formation of precipitate. However screening for crystallization conditions was hampered by
the very low solubility of the MSP in most typical
precipitating reagents (i.e. ammonium sulfate, PEG
etc.), indicating that the properties of the MSP in
solution were modified. We thus began screening for
conditions including low concentrations of salts, in
the addition of low molecular weight stabilizing molecules (sucrose, glycerol, PEG 400). Microcrystals
of the MSP (Figure 2) grew in the presence of 25–
100 mM CaCl2 , and the addition of low concentrations
of PEG400 was able to somewhat increase their size.
Crystals did not grow in the absence of calcium. In
some cases, MSP crystals grew out of drops containing
protein precipitate. Attempts to increase crystal size
by microseeding or macroseeding have so far been
unsuccessful.
Characterization of MSP before and after
crystallization
MSP heterogeneity is not due to covalent
modifications
The cessation of crystal growth could be due to many
factors. One of the possible problems is the presence
of heterogeneities in the protein sample due to posttranslational modifications, oxidation of amino acid
residues or partial proteolysis. In order to identify
possible covalent modifications to the MSP, the purified protein was subjected to reverse-phase HPLC in
tandem with electronspray ionization mass spectrometry (ESI-MS). Figure 3a shows the separation of the
purified MSP by RP-HPLC. A single major peak, corresponding to more than 99% of the total MSP, eluted
with a retention time of 25.35 min. Four additional
peaks eluted at 20.87, 22.54, 30.70 and 32.88 min.
All five protein-containing fractions were analyzed by
ESI-MS. The major peak (Figure 3b), as well as the
other four minor peaks, contained MSP with a molecular weight of 26 535.0. This is very close to the
theoretical molecular weight of the MSP as calculated
from the spinach psbO gene, and similar to a previous
report of MALDI-MS analysis of the MSP (Zubrzycki
et al. 1998). The elution of MSP at more than one
position by RP-HPLC could be due to a differential
affinity to the column matrix, due to either a conformational difference and/or the presence of oligomeric
forms of MSP. The results of this experiment indicated
that the MSP is very pure, as well as homogeneous on
the polypeptide level.
Determination of the oligomerization state of the MSP
Since the MSP in solution did not contain covalent
heterogeneities, we wanted to determine the oligomerization state of the crystallized MSP. MALDI-MS
(Winston and Fitzgerald 1997) is a mass spectrometric
technique that preserves protein–protein contacts and
thus was used to identify the oligomeric state of the
MSP. Figure 4A shows a mass spectrum of purified
MSP. The MSP separated into a major fraction at molecular mass equivalent to a monomeric form and a
substantial amount of dimeric MSP. When solubilized
MSP microcrystals were analyzed by MALDI-MS,
a single major peak at about 52 kDa was resolved
(Figure 4B), indicating that the microcrystals contain
dimeric MSP. The signal–noise ratio is much poorer
in the crystalline sample due to the extremely small
amount of protein. A previously reported account of
MALDI-MS analysis of soluble MSP (Zubrzycki et al.
1998) reported monomeric MSP. However in this report, an unidentified peak at about 53 kDa in the mass
spectrogram is also present, which may be dimeric
MSP.
MSP dimers were also recently obtained by
cross-linking with a zero-length cross-linking reagent
(Enami et al. 1998). In this report, the authors used
very dilute MSP to avoid intermolecular cross-links;
however, a substantial amount of MSP dimers formed.
It should be noted that the MSP used in this report was
isolated using the 1 M CaCl2 method. Thus, there is a
possibility that the dimerization of MSP is due to the
presence of calcium. Preliminary results from crosslinking experiments with glutaraldehyde indicate that
the MSP forms cross-linked dimers, and that the presence of calcium increases the amount of dimeric MSP
(data not shown).
Identification of conformational or quaternary
changes to monomeric MSP
The MALDI-MS results indicate that only MSP dimers crystallize. Thus, it was important to try and
elucidate the parameters that determine the oligomeric
state of the MSP. SEC-HPLC is a method that can
differentiate between molecules of different molecular
172
Figure 3. Analysis of purified MSP for possible covalent heterogeneities. (A) RP-HPLC separation of MSP isoforms prior to crystallization.
(B) ESI-MS of the main peak in (A). See ‘Materials and methods’ section for experimental details.
173
Figure 4. Identification of MSP in different oligomerization states. (A) MALDI mass spectrum of MSP (1 mg/ml) prior to crystallization. (B)
MALDI mass spectrum of solubilized MSP crystals. Standards used for calibration were trypsinogen (molecular mass = 23 980) and bovine
serum albumin (molecular mass = 66 430). See ‘Materials and methods’ section for additional experimental details.
174
Table 1. Effect of protein concentration and calcium on apparent
molecular mass of the MSP by size-exclusion high performance
chromatographya
MSP concentration (mg/ml)
CaCl2
Molecular mass (kDa)b
1
1
11
11
–
50 mM
–
50 mM
41.1 ± 2.9c
38.3 ± 5.8
54.8 ± 4.9c
∼350 kDad
42.2 kDad
a For experimental details, see ‘Materials and methods’.
b Standards used were carbonic anhydrase (29 kDa), bovine serum
albumin (66 kDa), alcohol dehydroginase (150 kDa), β-amylase (200
kDa) and apoferritin (443 kDa).
c Results are the average of 4 experiments.
d Sample eluted as two peaks.
masses or states of unfolding. The calculated molecular masses are based on the relative size of a series of
standard proteins, which are typically globular. A protein with an extended structure (as has been suggested
to be the case for the MSP) will elute at a volume
equivalent to an apparently larger molecular mass.
We first wanted to identify possible effects of the
concentration of the MSP on its size (Table 1). The
protein, which is injected into the SEC-HPLC column,
undergoes dilution by a factor of 50–100, thus lowering the effective protein concentration analyzed by a
similar factor. Dilute MSP (1 mg/ml) in the absence
or presence of calcium, elutes with an apparent molecular mass of about 40 kDa. This is similar to results
obtained by other groups (Murata 1979; Betts et al.
1998; Hutchison et al. 1998; Lydakis-Simantiris et al.
1999). When more concentrated MSP (11 mg/ml) was
analyzed in the absence of calcium, the protein eluted
with an apparent molecular mass of about 55 kDa,
which could have been due to either MSP dimerization
or due to extensive unfolding. A similar apparent size
increase was reported when MSP carboxyl terminal
deletion mutants were analyzed by SEC-HPLC (Betts
et al. 1998). In the case of the mutants, the increase
was interpreted as a further unfolding of the MSP
(Betts et al. 1998). However, when the MALDI-MS
results (above) are taken into account, it seems more
likely that a shift in the equilibrium towards dimer
formation occurs when the MSP is concentrated.
When concentrated MSP was analyzed in the presence of calcium (Table 1), the protein eluted as two
fractions, a high molecular mass aggregate (∼350
kDa) and monomers (42 kDa). Thus, calcium promoted, or stabilized, self-aggregation in concentrated
MSP. The presence of monomeric MSP may have been
due to the dilution effect, indicating that aggregation
due to concentration of the MSP may be reversible.
Dynamic light scattering (DLS) is an additional
method that can asses the size distribution of molecules and identify possible situations of polydispersity which are deleterious to crystallization (FerreD’Amare and Burley 1997). In DLS, it is not possible
to analyze high protein concentrations, however no
additional dilution occurs. We performed the DLS
measurements on MSP in buffer without calcium, or
in buffer with calcium in order to mimic the crystallization conditions (Table 2). MSP (at 1 mg/ml)
in buffer was best fit as a single scattering molecule
(monomodal) with a hydrodynamic radius of 2.7 ±
0.1 nm and a diffusion coefficient of 8.1 ± 0.1 ×
10−7 cm2 s−1 . A recently published study of MSP
using analytical centrifugation methods (Zubrzycki et
al. 1998) reported a diffusion coefficient of 7.7 ×
10−7 cm2 s−1 . Using a specific volume of 0.7317 ml/g
(Zubrzycki et al. 1998), and a shape-hydration factor
for a somewhat extended protein (ProteinSolutions
DynaPro manual), the calculated MSP molecular mass
was 34.3 ± 3.2 kDa. In a previously reported analysis of MSP by DLS, at lower protein concentrations
(Zubrzycki et al. 1998), MSP was reported to have a
similar diffusion coefficient of 7.52 × 10−7 cm2 s−1 .
When DSP was performed on MSP in the presence
of either 10–50 mM or 100 mM CaCl2 , the apparent diffusion coefficient decreased to 7.0 ± 0.1 ×
10−7 cm2 s−1 and 6.5 ± 0.2 × 10−7 cm2 s−1 respectively. Using the same parameters indicated above, this
results in calculated molecular masses of 48.0 ± 2.9
kDa and 67.5 ± 9.2 kDa, respectively. While the possibility that the changes in the diffusion coefficient
due to unfolding in the presence of calcium could not
be fully discounted, these results strongly suggested
a process of calcium induced dimerization followed
by further aggregation. Since dimers might actually be
more globular in structure, a different molecular mass
calculation could be used (based on a standard curve
in the commercial software). Using this calculation,
the MSP molecular mass is 56.1 ± 6.2 kDa (Table 2),
which is similar to the results from the SEC-HPLC experiment described above. The results obtained in the
presence of 10–50 mM CaCl2 may represent a mixture
of monomers and dimers, which is not resolvable by
DLS.
175
Table 2. Dynamic light scattering measurements of different MSP samples. All results are based on the
Regularization cumulants analysis
Sample
Da (10−7 cm2 s−1 )
Rbh (nm)
Molecular massc (kDa)
Molecular massd (kDa)
MSP
8.1±0.1
2.7±0.1
34±3.2
32.5±2.5
MSP
+10–50 mM CaCl2
7.0±0.1
3.0±0.1
48±2.9
42.5±2.2
MSP
+100 mM CaCl2
6.5±0.2
3.4±0.2
67.5±9.2
56.1±6.2
a D, diffusion coefficient.
b R , hydrodynamic radius.
h
c Volume shape hydration model (Dynamics analysis software, Protein Solutions Inc.).
d Standard curve model (Dynamics analysis software, Protein Solutions Inc.).
Figure 5. Schematic diagram of the proposed mechanism for MSP crystallization. Words or phrases in italics are chemical additions or physical
changes which shift the equilibrium to the state indicated by the nearest arrow.
Mechanism of MSP crystallization
The experimental results described above can be combined to explain the mechanism of MSP crystallization. Figure 5 shows a diagram depicting the equilibrium of the various states of MSP. In the first stage,
MSP is removed from its RC II bound state into solution by high concentrations of calcium, tris or urea.
The protein is probably released as a monomer with
a certain degree of unfolding (Lydakis-Simantiris et
al. 1999) as indicated by both SEC-HPLC and DLS
experiments (Tables 1 and 2). Unbound MSP can form
dimers as seen in Figure 4A and in previously published studies (Enami et al. 1998; Zubrzycki et al.
1998). The presence of calcium can induce further
dimerization (Table 2) and can induce further aggregation at higher protein concentrations (Table 1). The
aggregation is reversible, with the release of monomeric MSP. Thus we propose that when the concentrated MSP is mixed with 25–50 mM calcium (in the
process of crystallization) dimers and aggregates form.
This mixture of states could be the reason for lag time
in crystal formation (∼2 months). Eventually, most of
the MSP is in the aggregated form, however the aggregate is in equilibrium with monomers, which can
dimerize in the presence of calcium, and the MSP dimers can then crystallize. The presence of additional
monomeric MSP (and perhaps higher order oligomers)
that bind to the growing crystal, may be the reason
behind the small crystal size. We are now attempting
to identify conditions that promote dimerization of the
MSP monomers released from RC II and inhibit the
formation of higher aggregates.
A question that arises from this study is the possible relevance of MSP dimerization in vivo. The ratio
of MSP to RC II is controversial (Enami et al. 1991;
Betts et al. 1997). If the native bound state of MSP is a
dimer, with one of the monomers bound strongly to the
RC II and the second copy bound weakly to the first
monomer, then some methods of preparation could
176
cause the loss of one MSP, while others might not. On
the other hand, it is possible that bound MSP binds
additional MSP only in non-physiological protein isolation conditions (i.e. high calcium concentration), and
that there is only a single bound MSP per RC II. There
have also been reports of unbound MSP found in the
luminal space of thylakoid membranes (Hashimoto et
al. 1996), which could be the source of MSP for the
formation of dimers.
It has been established that RC II goes through a
continuous cycle of movement from the grana lamella
to the stroma lamella, due to the need for D1 protein
replacement (Prasil et al. 1992). Since the newly synthesized D1 found in RC II in stroma lamella is in its
precursor form (Adir et al. 1990), and thus cannot bind
MSP (Diner et al. 1988), it seems likely that prior to
its movement out of the grana lamella, the MSP is released into the luminal space. Unbound and unfolded
proteins might be recognized by proteolytic systems in
the photosynthetic membrane (Ostersetzer and Adam
1997; Itzhaki et al. 1998), leading to a loss of MSP.
However, the MSP does not turnover in conditions
where the D1 protein turnsover (Prasil et al. 1992).
Formation of MSP dimers might inhibit proteolysis,
until a recycled RC II with a processed D1 protein
arrives from the stroma lamella, and rebinds the MSP.
Acknowledgements
This work was supported by the Israel Science Foundation founded by the Israeli Academy of Sciences and
Humanities (366/99-1) and the TechnionV.P.R. Fund
– M. and F. Pollard Biochemistry Research Fund. We
would like to thank Dr Michal Harel of the Dept. of
Structural Biology of the Weizmann Institute for her
help with the DLS measurements.
References
Adir N (1999). Crystallization of the oxygen-evolving reaction
centre of Photosystem II in nine different detergent mixtures.
Acta Crystallogr D Biol Crystallogr 55: 891–4
Adir N, Shochat S and Ohad I (1990). Light-dependent D1 protein
synthesis and translocation is regulated by reaction center II. Reaction center II serves as an acceptor for the D1 precursor. J Biol
Chem 265: 12563–8
Barber J (1998). Photosystem II. Biochim Biophys Acta 1365: 269–
277
Beavis RC and Chait BT (1989) Cinnamic acid derivatives as
matrices for ultraviolet laser desorption mass spectrometry of
proteins. Rapid Commun Mass Spectrom 3: 432–435
Berthold DA, Babcock GT and Yocum CF (1981) A highly resolved, oxygen-evolving Photosystem II preparation from spinach thylakoid membranes. FEBS Lett. 134: 231–234
Betts SD, Ross JR, Pichersky E and Yocum CF (1996) Coldsensitive assembly of a mutant manganese-stabilizing protein
caused by a Val to Ala replacement. Biochemistry 35: 6302–7
Betts SD, Ross JR, Pichersky E and Yocum CF (1997) Mutation
Val235Ala weakens binding of the 33-kDa manganese stabilizing protein of Photosystem II to one of two sites. Biochemistry
36: 4047–53
Betts SD, Lydakis-Simantiris N, Ross JR and Yocum CF (1998)
The carboxyl-terminal tripeptide of the manganese-stabilizing
protein is required for quantitative assembly into Photosystem
II and for high rates of oxygen evolution activity. Biochemistry
37: 14230–6
Burnap RL and Sherman LA (1991) Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein
of Photosystem II is not essential for O2 evolution. Biochemistry
30: 440–6
Campbell KA, Gregor W, Pham DP, Peloquin J M, Debus RJ and
Britt RD (1998) The 23 and 17 kDa extrinsic proteins of Photosystem II modulate the magnetic properties of the S1-state
manganese cluster. Biochemistry 37: 5039–45
Chu HA, Nguyen AP and Debus RJ (1994) Site-directed Photosystem II mutants with perturbed oxygen-evolving properties. 2.
Increased binding or photooxidation of manganese in the absence
of the extrinsic 33-kDa polypeptide in vivo. Biochemistry 33:
6150–7
Debus RJ (1992) The manganese and calcium ions of photosynthetic
oxygen evolution. Biochim Biophys Acta 1102: 269–352
Diner BA, Ries DF, Cohen BN and Metz JG (1988) COOH-terminal
processing of polypeptide D1 of the Photosystem II reaction center of Scenedesmus obliquus is necessary for the assembly of the
oxygen-evolving complex. J Biol Chem 263: 8972–80
Eaton-Rye JJ and Murata N (1989) Evidence that the aminoterminus of the 33 kDa extrinsic protein is required for binding to
the Photosystem II complex. Biochim Biophys Acta 977: 219–26
Enami I, Kaneko M, Koike H, Sonoike K, Inoue Y and Katoh
S (1991) Total immobilization of the extrinsic 33 kDa protein in spinach Photosystem II membrane preparations. Protein
stoichiometry and stabilization of oxygen evolution. Biochim
Biophys Acta 1060: 224–232
Enami I, Kamo M, Ohta H, Takahashi S, Miura T, Kusayanagi M,
Tanabe S, Kamei A, Motoki A, Hirano M, Tomo T and Satoh
K (1998) Intramolecular cross-linking of the extrinsic 33-kDa
protein leads to loss of oxygen evolution but not its ability of
binding to Photosystem II and stabilization of the manganese
cluster. J Biol Chem 273: 4629–34
Evelo RG, Styring S, Rutherford AW and Hoff AJ (1989) EPR
relaxation measurements of Photosystem II reaction centers; influence of s-state oxidation and temperature. Biochim Biophys
Acta 973: 428–442
Ferre-D’Amare AR and Burley SK (1997) Dynamic Light Scattering in Evaluating Crystallizability of Macromolecules. Meth
Enzym 276: 157–166
Ghanotakis DF and Yocum CF (1990) Photosystem II and the Oxygen evolving complex. Ann Rev Plant Physiol Plant Mol Biol 41:
255–276
Gilchrist ML Jr, Ball JA, Randall DW and Britt RD (1995) Proximity of the manganese cluster of Photosystem II to the redoxactive tyrosine YZ. Proc Natl Acad Sci USA 92: 9545–9
Hankamer B, Morris EP and Barber J (1999) Revealing the structure of the oxygen-evolving core dimer of Photosystem II by
cryoelectron crystallography. Nat Struct Biol 6: 560–4
177
Hashimoto A, Yamamoto Y and Theg SM (1996) Unassembled
subunits of the photosynthetic oxygen-evolving complex present
in the thylakoid lumen are long-lived and assembly-competent.
FEBS Lett 391: 29–34
Hoganson CW and Babcock GT (1997) A metalloradical mechanism for the generation of oxygen from water in photosynthesis.
Science 277: 1953–6
Hutchison RS, Betts SD, Yocum CF and Barry BA (1998) Conformational changes in the extrinsic manganese stabilizing protein can
occur upon binding to the Photosystem II reaction center: An
isotope editing and FT-IR study. Biochemistry 37: 5643–5653
Itzhaki H, Naveh L, Lindahl M, Cook M and Adam Z (1998)
Identification and characterization of DegP, a serine protease associated with the luminal side of the thylakoid membrane. J Biol
Chem 273: 7094–8
Kuhl H, Rogner M, Van Breemen JF and Boekema EJ (1999) Localization of cyanobacterial Photosystem II donor-side subunits
by electron microscopy and the supramolecular organization of
Photosystem II in the thylakoid membrane. Eur J Biochem 266:
453–459
Kuwabara T and Murata N (1979) Purification and Characterization
of the 33 kDa protein of spinach chloroplasts. Biochim Biophys
Acta 581: 228–236
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680–5
Lydakis-Simantiris N, Hutchison RS, Betts SD, Barry BA and
Yocum CF (1999) Manganese stabilizing protein of Photosystem
II is a thermostable, natively unfolded polypeptide. Biochemistry
38: 404–14
Mayfield SP, Bennoun P and Rochaix JD (1987) Expression of the
nuclear encoded OEE1 protein is required for oxygen evolution and stability of Photosystem II particles in Chlamydomonas
reinhardtii. Embo J 6: 313–8
Miyao M and Murata N (1984a) Calcium ions can be substuituted
for the 24 kDa polypeptide in photosynthetic oxygen evolution.
FEBS Lett 170: 350–354
Miyao M and Murata N (1984b).Role of the 33-kDa polypeptide
in preserving Mn in the photosynthetic oxygen evolution system
and its replacement by chloride ions. FEBS Lett 168: 118–120
Ono T and Inoue Y (1983) Mn-preserving extraction of the 33- 24and 16 kDa proteins from oxygen evolving PS II particles by
divalent salt washing. FEBS Lett 164: 255–260
Ono T and Inoue Y (1986) Effects of removal and reconstitution of
the extrinsic 33, 24, and 16 kDa proteins on flash oxygen yield in
Photosystem II particles. Biochim Biophys Acta 850: 380–389
Ostersetzer O and Adam Z (1997) Light-stimulated degradation
of an unassembled Rieske FeS protein by a thylakoid-bound
protease: the possible role of the FtsH protease. Plant Cell 9:
957–965
Prasil O, Adir N and Ohad I (1992) Dynamics of Photosys-
tem II: Mechanism of Photoinhibition and Recovery Processes.
In: Barber J (ed) The Photosystems: Structure, Function and
Molecular Biology, pp 295–348. Elsievier Science Publishers,
Amsterdam
Qian M, Al-Khaldi SF, Putnam-Evans C, Bricker TM and Burnap
RL (1997) Photoassembly of the Photosystem II (Mn)4 cluster in
site-directed mutants impaired in the binding of the manganesestabilizing protein. Biochemistry 36: 15244–52
Qian M, Dao L, Debus RJ and Burnap RL (1999) Impact of mutations within the putative Ca2+-binding lumenal interhelical a-b
loop of the Photosystem II D1 protein on the kinetics of photoactivation and H2O-oxidation in Synechocystis sp. PCC6803.
Biochemistry 38: 6070–81
Seidler A (1996) Intermolecular and intramolecular interactions of
the 33-kDa protein in Photosystem II. Eur J Biochem 242: 485–
90
Shen JR, Burnap RL and Inoue Y (1995) An independent role of
cytochrome c-550 in cyanobacterial Photosystem II as revealed
by double-deletion mutagenesis of the psbO and psbV genes in
Synechocystis sp. PCC 6803. Biochemistry 34: 12661–8
Shen JR, Qian M, Inoue Y and Burnap RL (1998) Functional characterization of Synechocystis sp. PCC 6803 delta psbU and delta
psbV mutants reveals important roles of cytochrome c-550 in
cyanobacterial oxygen evolution. Biochemistry 37: 1551–8
Stoscheck CM (1990) Quantitation of Proteins. Meth Enzymol 182:
50–68
Tsiotis G, Walz T, Spyridaki A, Lustig A, Engel A and Ghanotakis
D (1996) Tubular crystals of a Photosystem II core complex. J
Mol Biol 259: 241–8
Weinreb PH, Zhen W, Poon AW, Conway KA and Lansbury PT Jr
(1996) NACP, a protein implicated in Alzheimer’s disease and
learning, is natively unfolded. Biochemistry 35: 13709–15
Winston RL and Fitzgerald MC (1997) Mass spectrometry as a
readout of protein structure and function. Mass Spectrom Rev
16: 165–79
Xu Q, Nelson J and Bricker TM (1994) Secondary structure of the
33 kDa, extrinsic protein of Photosystem II: A far-UV circular
dichroism study. Biochim Biophys Acta 1188: 427–31
Yocum CF and Pecoraro VL (1999) Recent advances in the understanding of the biological chemistry of manganese. Curr Opin
Chem Biol 3: 182–7
Zouni A, Luneberg C, Fromme P, Schubert WD, Saenger W and
Witt HT (1999) Characterization of single crystals of Photosystem II from the thermophilic cyanobacterium Synechococcus
elongatus. In: Garab G (ed) Photosynthesis; Mechanisms &
Effects, Vol 2. Kluwer Academic Publishers, Dordrecht, The
Netherlands
Zubrzycki IZ, Frankel LK, Russo PS and Bricker TM (1998)
Hydrodynamic studies on the manganese-stabilizing protein of
Photosystem II. Biochemistry 37: 13553–8