From the Department of Biology, University of Padova,
Via G. Colombo 3, 35131 Padova,
Consiglio Nazionale delle
Ricerche, Research Area, Corso Stati Uniti 4, 35100 Padova, and
** Consiglio Nazionale delle Ricerche, Center for Study of the Stability
and Reactivity of Coordination Complexes, Via Marzolo 1, 35131 Padova,
Italy
Received for publication, September 5, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Photosystem II of higher plants and
cyanobacteria is composed of more than 20 polypeptide subunits. The
pronounced hydrophobicity of these proteins hinders their purification
and subsequent analysis by mass spectrometry. This paper reports the
results obtained by application of matrix-assisted laser
desorption/ionization mass spectrometry directly to isolated complexes
and thylakoid membranes prepared from cyanobacteria and spinach.
Changes in protein contents following physiopathological stimuli are
also described. Good correlations between expected and measured
molecular masses allowed the identification of the main, as well as
most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other
supramolecular integral membrane complexes.
Photosystem II is a pigment-protein complex of the thylakoid
membrane of higher plants, eukaryotic algae, and cyanobacteria. It
catalyzes light-induced electron transfer from water to plastoquinone, with associated production of molecular oxygen.
PSII1 consists of a large
complex with a number of polypeptide components, most of which are
integral membrane proteins. A number of extrinsic proteins are also
associated with it at the membrane surface. The entire set of electron
transfer cofactors, including chlorophyll a, pheophytin
a, plastoquinones, and non-heme iron, is associated with the
D1/D2 heterodimer. These two proteins, together with the During the last decade, considerable progress has been made in our
understanding of the organization of polypeptides constituting the
reaction center of PSII, but the topology and functional role of the
small protein subunits are still largely unknown. Some of them are
universal, whereas others are present only in cyanobacteria (PsbU and
PsbV) or only in higher plants (e.g. PsbP, PsbQ, PsbS) (2).
Investigation of the structural and functional roles of the various
PSII subunits requires, as a preliminary step, a suitable method to
detect them in the thylakoid membrane or purified subparticle preparations. The detection and study of stimuli-induced modifications of several PSII components have mainly been based on the use of polyclonal antibodies. However, these are often not available and, when
available, are time-consuming to use.
A different technique, capable of accurately detecting even small
amounts of the various PSII subunits in an integrated system, could be
of great help in studying the function of these proteins. Mass
spectrometry has recently been used for structural biology studies,
also in the field of photosynthesis. A reverse phase high performance
liquid chromatography (HPLC) purification system has been developed for
the separation of PSII reaction center proteins, and the molecular
masses of the resulting purified, intact proteins have been determined
by electrospray ionization mass spectrometry (ESI-MS) (3-6). These
studies demonstrate that ESI-MS is indeed a useful technique for
studying hydrophobic membrane proteins also. Whereas ESI-MS allows
highly accurate measurement of the molecular mass, it suffers from the
important limitation that it cannot be applied in the presence of
detergents, and the proteins, free of salts and detergents prior to MS
analysis, must be purified (7). This paper reports the results
obtained by MALDI mass spectrometry for rapid determination of
non-purified proteins of photosynthetic complexes isolated from spinach
and cyanobacteria.
MALDI Mass Spectrometry--
Measurements were performed on a
REFLEX time-of-flight instrument (Bruker-Franzen Analytik, Bremen,
Germany) equipped with a SCOUT ion source operating in positive linear
mode. Ions, formed by a pulsed UV laser beam (nitrogen laser,
Because of the wide mass range to be examined, two different
instrumental conditions were used. For low molecular masses
(2,000-20,000 Da), pulsed ion extraction was carried out applying a
voltage of 22.3 kV for 300 ns to the second grid. External mass
calibration was carried out using [M + H]+ ions of bovine
insulin and horse myoglobin at m/z 5734 and
16952, respectively, and the corresponding doubly charged species at m/z 2868 and 8476, respectively. For high
molecular masses (20,000-80,000 Da), pulsed ion extraction was used
applying a voltage of 21.9 kV for 400 ns to the second grid. External
mass calibration was carried out using [M + H]+ ions of
horse myoglobin and bovine serum albumin at m/z
16952 and 66431, respectively, and the doubly charged species of bovine serum albumin at m/z 33216. The GuessProt
algorithm was used to determine the predicted molecular masses of the
PSII subunits.
Preparation of PSII Particles and Isolated RCII from
Spinach--
PSII core complexes from spinach were prepared according
to Ghanotakis et al. (8), and isolation of RCII was
performed as described in Ref. 9.
Preparation of Thylakoid Membranes and PSII from PSI-less
Synechocystis--
Thylakoids were prepared using a modified version
of the procedure of Mayes et al. (10). Briefly, cells were
cultured as previously described (11), harvested by centrifugation
(2000 × g for 5 min at 4 °C), and resuspended in a
breaking buffer containing the following: 20 mM Mes, 5 mM MgCl2, 5 mM CaCl2, 1 mM benzamidine, 1 mM aminocaproic acid, and
25% glycerol (pH 6.35 with NaOH). Cells were broken on ice using a
bead beater (Biospec) by application of 15 30-s cycles at 5-min
intervals. Unbroken cells were eliminated (5-min centrifugation at
2000 × g), and thylakoids were pelleted at
140,000 × g for 30 min. Pellets were then resuspended
in 50 mM Mes and 20 mM sodium pyrophosphate (pH
6.5). Isolated thylakoids were solubilized with 2% (w/v) dodecyl
maltoside for 30 min and loaded on a continuous sucrose gradient
containing 25 mM Mes, 0.5 M sucrose, 10 mM NaCl, 5 mM CaCl2, and 0.03%
dodecyl maltoside (pH 6.5). Three bands, i.e. carotenoids,
cytochrome b6/f, and PSII, separated
out after 17 h of centrifugation at 35,000 rpm. The PSII band was
collected and concentrated in Centricon-100 tubes.
Preparation of RC47 from Synechocystis--
RC47 particles,
containing the PSII reaction center (RCII) and the chlorophyll
a internal antenna CP47 but lacking the other chlorophyll
a internal antenna, CP43, were prepared from PSI-less Synechocystis following the procedure described in
Szabò et
al.2 Briefly,
thylakoid membranes from a PSI-less mutant strain of Synechocystis were solubilized in 10% dodecyl maltoside for
30 min, subjected to anion exchange chromatography, and eluted with a
linear 0-200 mM gradient of MgSO4. The elution
profile comprised a single peak for RC47 at about 110 mM
MgSo4.
SDS-PAGE--
SDS-polyacrylamide gel electrophoresis in
the presence of urea (6 M) was performed as described
previously (12).
Irradiation Conditions--
PSII samples at 90 µg/ml
chlorophyll were irradiated in a cuvette with a 1-cm light path using a
visible light intensity of 700 µmol of photons
m PSII Core and RCII Complexes from Spinach--
The representative
MALDI spectrum of a PSII core complex from spinach in the low molecular
mass range (3000-6000 Da) is shown in Fig.
1A. At least 11 peaks are
detected, and some of them, identifiable as known PSII subunits, are
listed in Table I. It is well known that
PSII core complexes of higher plants contain several low molecular
weight polypeptides, as demonstrated by analysis of the plastidial
genome (13, 14). Morris and co-workers (3, 4, 6) studied polypeptides
purified from preparations of PSII reaction center (RCII) and the RC47
complex of higher plants (pea and spinach, respectively) by HPLC
purification/separation followed by ESI-MS analysis. RC47 is a PSII
subcomplex, resulting from detergent-induced dissociation of internal
antenna CP43 from the PSII core complex. To better identify the
MALDI peaks of Fig. 1A and to compare our results with those
reported by Sharma et al. (3, 4), the RCII from spinach was
prepared and analyzed by MALDI.
MALDI mass spectra for low and high molecular masses of RCII are shown
in Fig. 1, B and C, respectively. SDS-PAGE
analysis and the absorption spectra of the PSII core and RCII
preparations, generally used to identify and characterize the various
PSII subparticles obtained by detergent extraction, are shown in Fig.
2. A small amount of light-harvesting
complex II (LHCII) still present in the preparations is
observable in both the SDS-PAGE and the 20-30-kDa range of the MALDI
spectra (data not shown).
Table I compares our data with the expected mass values of unprocessed
precursors of PSII components and with the results obtained by ESI-MS
(3-6). As may be seen, MALDI data on subunit composition and MW values
in intact spinach PSII cores and RCII complexes closely match those
obtained by Morris and co-workers (3-6) on polypeptides purified from
RCII and RC47. However, in the case of RCII in the low molecular mass
region, we clearly detected an additional peak at
m/z 5928, assigned to the PsbW protein.
Accordingly, this protein has recently been suggested (15) to be the
sixth component of the RCII complex prepared from higher plants. PsbW
has been detected by ESI-MS only in spinach RC47 (6) but not in pea
RCII (3). A peak at m/z 5928 assigned to PsbW is
also visible in the spectrum of the PSII core complex (Fig.
1A). In the high molecular mass region, as expected for RCII, two main peaks are clear at m/z 37,998 and
39,379, attributed to D1 and D2 proteins, respectively (Fig.
1C).
Altogether, these results show that, in particular in the low molecular
mass range, direct measurements on PSII and RCII preparations are quite
accurate and that MW values are comparable with those of isolated
proteins obtained by ESI-MS.
PSII Core Complexes from Cyanobacteria--
Study of the
structural-functional role of the subunit components of supramolecular
complexes has been greatly advanced by the development of molecular
genetic techniques such as the generation of mutants carrying
site-specific modifications or gene deletions. In this respect, the
identification of an easily transformable photosynthetic organism, with
high frequency of homologous recombination, able to survive and
propagate without functional photosystems, has been of great help. The
cyanobacterium Synechocystis sp PCC 6803 fulfils these
requirements, and, moreover, its whole genome has been sequenced.
However, preparation of purified PSII subparticles and their isolated
polypeptide components is even more difficult from cyanobacteria than
from higher plants. A method to reveal and identify PSII components in
cyanobacteria quickly, with no need for extended, time-consuming
purification, would be useful, and it was for this reason that we
focused our attention on Synechocystis.
PSII cores were isolated from a PSI-less mutant cyanobacterium. Part of
the psaAB operon coding for the core complex of PSI was
deleted from the genome of Synechocystis wild-type strain (16). PSI-less cells have approximately six times more PSII than do
wild-type cells, as estimated on a chlorophyll basis. The MALDI spectra
of Fig. 3 were obtained from two
different preparations. In the 20,000-70,000 m/z
range, seven main peaks were detected at m/z
55276 ± 424, 51470 ± 9, 39268 ± 37, 38049 ± 17,
34399 ± 9, 31030 ± 7, and 25750 ± 9 (n = 16 spectra from 8 different preparations). Each
MALDI measurement was highly reproducible for the same sample, and good
reproducibility was observed in the m/z values of
the peaks for samples deriving from different preparations (compare A and B of Fig. 3). However, the relative
intensity of the peaks depended on sample preparation, due to the
presence of varying amounts of interfering materials, such as salts and
lipids (17).
Molecular weight values obtained by MALDI were compared with protein
masses calculated from the nucleotide sequences of PSII components.
Good correlations between calculated and measured values revealed the
following correspondence between measured m/z
values and known PSII components: 55,276 m/z,
CP47 (molecular mass of unprocessed precursor, 55,902 Da);
51,470 m/z, CP43 (molecular mass of unprocessed
precursor, 51,760 Da); 39,268 m/z, D2; 38,049 m/z, D1 (Fig. 3B). It is worth noting
that the two latter values are very close to those ascribed to the
predominant form of D2 and D1 purified from pea by reverse phase HPLC
and detected by ESI-MS (see above). Concerning the other peaks
detectable in the spectra of Fig. 3, that at m/z
25,750 may correspond to the doubly charged species of the ion at
m/z 51,470. The peaks at
m/z 31,030 and 34,399 may represent subunits of
ATP synthase (predicted masses of 30,698 and 34,605 Da, respectively).
Alternatively, the peak at m/z 31,030 may
correspond to the phycobilisome 32-kDa linker peptide (predicted mass
of 30,797 Da). Clear-cut identification of these peaks would require
the use of mutants lacking these proteins.
It is worth noting that the m/z peaks assigned to
the apoproteins of the two internal antennae, CP43
(m/z 51,460) and CP47 (m/z
55,276), are reproducibly very different in their amplitude, despite
the fact that both components have a 1:1 stoichiometric ratio to the
reaction center. This may be related to the different ionization
probability of the two subunits when embedded in the PSII core complex.
It might be speculated that CP43, which is less closely associated with
the core and is located in a more external position with respect to
CP47 (18), offers a higher cross-section for ionization to incoming
laser photons. To confirm unequivocally the assignment of the
m/z peaks to the internal antennae, we prepared
an RC47 particle from the Synechocystis PSII core. This PSII
subparticle, obtained by dissociating the CP43 subunit by partial
detergent solubilization, is the cyanobacterial analogue of the RC47
obtained from higher plants by other groups (6). An immunoblot of its
polypeptide components is shown in Fig.
4C. Whereas the D1, D2, and
CP47 subunits of the PSII core are left unchanged in the RC47 particle
(Fig. 4C, lanes 2, 4, and
8), only a tiny amount of CP43 is contained in the latter (lane 6). If the proposed assignments are correct, we expect
the m/z peak at 51,470 to disappear from the
MALDI spectrum of the RC47 particle. Fig. 4 compares the spectrum of
RC47 (B) with that of the core complex (A). The
expectation is fully matched, because the m/z
peak at 51,470 is highly reduced with respect to the intensity of the
D1 and D2 peaks. It may be noted that the peak at 55,276, attributed to
CP47, maintains approximately the same relative intensity. Therefore,
an eventual shadowing action on CP47 must be exerted by core proteins
other than CP43, in accordance with the recent finding that the two
internal antennae are on opposite sites of the D1-D2 heterodimer,
rather than sequentially located within the PSII core (20). It may also
be noted that detergent treatment on sample A to produce RC47 (Fig.
4B) also eliminates the impurities giving rise to
m/z peaks in the range of 25-35 kDa.
MALDI Spectrum of High MW Subunits of PSII Core Complex in Light
Stress Conditions--
It is known that the D2 and, to an even greater
extent, D1 proteins of higher plants and cyanobacteria are
characterized by high turnover and that their degradation rate is
increased by UV-B radiation (21-25) or an excess of photosynthetic
active radiation (26-29). The usual way to reveal these phenomena is
to measure the D1 and D2 contents of the thylakoid membrane after
exposure to radiation by SDS-PAGE and immunoblotting and then to search for the generated fragments by the same methods. The following experiment aimed at checking whether the MALDI technique could be used
as an alternative, direct method to study the phenomenon. Fig.
5 shows an example of this
application. PSII isolated from PSI-less organisms was left
untreated (Fig. 5A) or was irradiated with visible light
(B) or UV-B light (C) for 30 min. The intensity of each peak was measured in the three samples deriving from the same
preparation and expressed as a percentage of the intensity of the CP43
peak (m/z 51,470), which is known to be
unaffected by the light intensities used here (30). Whereas the
relative intensities of most of the peaks did not change upon exposure to radiation, those corresponding to D1 and D2
(m/z 38,048 and 39,268, respectively) showed
marked variations. In particular, the intensities of the D1 peak
decreased by 63% in visible light-illuminated PSII and by 13% in the
UV-B-treated sample. The intensities of the D2 peak decreased by 45 and
35%, respectively. It is worth stressing the point that the three
spectra (Fig. 5, A-C) were obtained from aliquots of
the same sample, thus ensuring that evaluation of the peak areas was
accurate enough to make quantitative comparisons significant. An
unidentified peak at m/z 31,071 was also found to
be slightly affected by both kinds of irradiation, suggesting that this
protein is involved in the stress response.
Low MW Components of PSII from Cyanobacteria--
Because the low
MW subunits of cyanobacteria and spinach PSII differ in their predicted
masses, the low molecular mass region of the MALDI spectra of PSII core
complex prepared from the PSI-less strain of Synechocystis
was examined (Fig. 6). Detected peaks were tentatively identified with the main known subunits, as listed in
Table II.
The identity of a few peaks is still unresolved. For example, the
nature of the peaks at m/z 3570 and 8170 is
unknown, whereas the peak at m/z 8008 may
correspond either to a phycobilisome 7.8-kDa linker peptide or
to a subunit of ATP synthase (calculated masses are 7805 and 7968 Da, respectively).
In an intermediate molecular mass region (data not shown), one peak at
m/z 12,474 may correspond to the psbW precursor
(unprocessed protein mass of 12,590 Da), and another peak at
m/z 14,468 may correspond to cytochrome
c550, also called PsbV (predicted mass of 15,119 Da).
In view of our study of the structural-functional role of the PsbH
subunit of PSII, we were particularly interested in its identification
by MALDI, because this protein cannot be revealed by SDS-PAGE or
immunoblotting in cyanobacteria, in which it is not phosphorylated. The
predicted molecular mass of PsbH for cyanobacteria is 6985 Da. As may
be observed in Fig. 6 and Table II, a peak corresponding to PsbH was
found in the PSII core complex preparation from
Synechocystis.
MALDI Measurements on Whole Thylakoid Membrane--
We checked for
the presence of PsbH protein in the whole thylakoid membrane of
Synechocystis. A peak at m/z 7094 was
observed in the PSI-less preparation (Fig.
7A). For comparison,
thylakoids from a PsbH deletion mutant, IC7 (31, 32), were also
examined (Fig. 7B), but no significant differences could be
observed between the two MALDI spectra except for the lack of the peak
at m/z 7094 in the deletion mutant. It may thus
be concluded that this peak actually corresponds to PsbH. The
m/z value of PsbH measured in whole thylakoids is
~100 Da higher than expected and measured in the PSII core complex.
Although we have no simple explanation for this difference, it may be
due to the lower precision of m/z values when
measured in a rich lipid environment such as that of whole thylakoids.
Although the possibility that PsbH in thylakoids is phosphorylated
cannot be ruled out (33), it is not probable, because the known
phosphorylation site in cyanobacteria is lacking (34).
Analysis of protein components in the membrane multisubunit
complex of photosystem II has traditionally been performed by various
versions of SDS-PAGE and immunoblotting. A great amount of information
has been obtained by these techniques, but their application has been
limited by poor gel resolution and the difficulty of producing suitable
antibodies, especially for the low molecular weight components. The
development of HPLC procedures for the purification of low MW
components and the D1 and D2 proteins of PSII allowed Morris and
co-workers (3-6) to apply mass spectrometry to analysis of PSII
components, using electron spray as the ionization method. This
technique is accurate in determining protein molecular masses and
allowed these authors to characterize post-translational modifications
of some PSII polypeptides. However, it does depend to a great extent on
the proper purification of each subunit, which must be free of salts
and detergents; so it is a rather cumbersome procedure for the
extremely hydrophobic proteins of PSII. We present here the
results of the application of an alternative mass spectrometric
technique, which, although somewhat less accurate in determining exact
molecular masses, does allow quick and simple analysis of the protein
components of the PSII complex, in a single step, on integrated samples
such as the entire PSII core complex or, in some cases, even untreated
photosynthetic membranes.
The first part of the work aimed at comparing our MALDI analysis on
different detergent-extracted subparticles of PSII from higher plants
with results obtained by ESI-MS on isolated subunits. To assign the
observed peaks in the PSII core complex from spinach to the
corresponding proteins, we took into account the masses of unprocessed
precursors and/or comparisons with the MALDI spectra of purified
preparations of RCII. The results shown in Table I compare favorably
with those obtained by ESI-MS, and the molecular mass differences found
are within measurement uncertainty. One interesting result of these
experiments was that, in our isolated RCII preparation, also known as
the D1-D2-cytochrome b559 complex, we also found
the PsbW protein (molecular mass, 5928 Da) as well as the PsbI protein,
which has long been known to belong to this complex, thus confirming
the recent report of Irrgang et al. (15). PsbW usually
escapes detection by SDS-PAGE but was easily clearly visible in the
MALDI spectrum (Fig. 2B). The lack of detection of this
protein in ESI-MS suggests that PsbW is loosely bound to the RCII
complex and may easily be lost during purification.
A number of peaks in the 4500-5500 m/z region of
PSII core complexes (Fig. 1A) were not identified; they may
have been impurities or, more probably, D1 fragments still associated
with the PSII core (35). Some unidentified minor peaks were also found
in the high molecular mass region of the purified RCII spectrum, showing the sensitivity of MALDI analysis to impurities in preparations (Fig. 1C).
The second part of our work was application of the MALDI technique to
the photosynthetic membrane of Synechocystis, to check whether it could identify PSII protein components in this organism, which is a particularly interesting system frequently adopted for
studies applying the molecular genetic approach to structural and
functional studies of PSII. Attribution of the peaks observed in the
MALDI spectra was made easier by knowledge of the molecular masses of
all the precursors of thylakoid proteins in this organism.
When the PSII complex was isolated from the PSI-less strain of
Synechocystis, the MALDI spectrum turned out to be highly
reproducible even in the high MW region, which is generally the most
unfavorable for this technique. Such good resolution and
reproducibility was not obtained, in the same region, for PSII cores
isolated by detergent treatment of the thylakoid membrane of spinach.
This may have been because of the large quantity of detergent needed in
the latter case, which is known to lead to signal suppression in the MALDI ionization process (36). Most of the known components in
both high and low molecular mass regions were identified using the
expected molecular masses, and by comparing subparticles and/or mutant
strains lacking one PSII protein component.
We also attempted the resolution of whole, untreated photosynthetic
membrane by MALDI-MS. One particularly interesting result was
identification of the PsbH protein in the photosynthetic membrane of
Synechocystis. This small subunit of the PSII core was
originally detected as a 9-kDa phosphoprotein in pea chloroplasts (37). The physiological roles proposed for it include regulation of photoinhibition (38), control of the electron transfer from Qa to Qb
(10), and stabilization of the PSII dimer in higher plants (39, 40).
Interestingly, the psbh gene product from cyanobacteria is
truncated at the N terminus site and lacks the phosphorylation site
(19). This renders its detection in cyanobacteria extremely difficult
by immunoblotting using a polyclonal antiserum produced against the
spinach phosphoprotein; however, no specific antibodies against PsbH
from Synechocystis have yet been obtained. Furthermore, the PsbH protein cannot be detected by staining of SDS-PAGE.3 Thus, the
reported clear-cut detection of PsbH in the thylakoid membrane of
Synechocystis will greatly help further work in progress on
this important subunit.
Assignment of the various m/z peaks to PSII
polypeptide subunits, based on the expected molecular masses, was
confirmed in all cases in which an independent check could be applied,
as in the case of D1, D2, CP43, CP47, and H subunit. This
confirmation and the favorable comparison of MALDI results with
those obtained by ESI mass spectrometry on higher plant PSII particles
provide good proof that this technique can be of great help in the
identification and analysis of the polypeptide subunits of large
integral membrane complexes. In addition to structural information
concerning the composition of complexes from a number of different
organisms, MALDI can also be applied to study the effects of various
stimuli or stress conditions. This is demonstrated by the changes
induced by exposure of a PSII complex to UV-B radiation or excessive
visible light in their MALDI spectra. As expected, a significant
decrease in the relative abundance of the peaks due to D1 is observed
after visible light stress and of those of both D1 and D2 proteins upon exposure to UV-B, in agreement with previous results (21-29). The observed significant increase in the number of peaks in the low molecular mass region (data not shown) also indicates the possibility of detecting fragments originating from radiation-induced degradation of PSII proteins.
In conclusion, the present work provides evidence that the MALDI
technique may be applied directly to photosynthetic protein complexes
without the need to purify subunits prior to MS analysis. This novel
approach, in combination with genetic engineering, may greatly
contribute to unraveling the role of the various protein subunits of
photosystems and other supramolecular membrane complexes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
subunits of cytochrome b559 and the PsbI
protein, constitute the so-called reaction center II (RCII), which is
the smallest PSII subparticle still able to perform light-induced charge separation (1). Two large integral membrane proteins, CP43 and
CP47, each coordinating a number (12-15) of chlorophyll a molecules, and several low molecular mass (<10
kDa) polypeptides are constituents of the PSII core complex, which is
very similar in higher plants and prokaryotic cyanobacteria.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= 337 nm), were accelerated to 25 kV. The use of this beam did not
result in degradation of photosynthetic proteins during measurements.
Sinapinic acid (saturated solution in acetonitrile:water (50:50 v:v))
was used as a matrix. Samples, at chlorophyll concentrations of 9-250
µg/ml, were diluted 2- to 10-fold with a 0.1% trifluoroacetic
acid aqueous solution. 5 µl of the diluted sample solution was
mixed with the same volume of matrix solution, and 1 µl of the
resulting mixture was deposited on a stainless steel sample holder and
allowed to dry before introduction into the mass spectrometer.
2 s
1 and a UV-B
light intensity of 5 µmol of photons m
2
s
1. A Vilbert-Lourmat 215M lamp was used as a
UV-B source, wrapped in cellulose diacetate foil (0.15 mm thick) to
screen out any UV-C component emitted by the UV-B source.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
MALDI spectra of PSII from spinach: core
complex in the 3-6-kDa range (A) and PSII reaction
center in the 3-20-kDa range (B) and the 30-70-kDa
range (C). m/z values of
main peaks are reported in Table I. In all spectra, the ordinate scale
corresponds to intensity of peaks in arbitrary units. Cyt,
cytochrome.
Proposed identification of measured m/z peaks in PSII or RCII from
spinach
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Fig. 2.
A, SDS-PAGE of PSII core complex
(lane 1) and PSII reaction center (lane 2) from
spinach; 1.5 µg of chlorophyll/lane (silver-staining). B,
absorption spectrum of reaction center. Same preparations as in Fig.
1.
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Fig. 3.
MALDI spectra of two distinct preparations of
PSII core complex from Synechocystis.
m/z values are indicated in A;
assignment of the main peaks is shown in B.
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Fig. 4.
MALDI spectra of PSII core complexes
(A) and RC47 subparticle (B) from
Synechocystis. Immunoblots (C) of PSII
core complexes (lanes 1, 3, 5, and
7) and RC47 (lanes 2, 4, 6,
and 8). Lanes 1 and 2, 3 and 4, 5 and 6, and 7 and
8 were probed with antibodies specific for D1, D2, CP43, and
CP47, respectively. PSII samples were loaded at 0.25 µg of
chlorophyll.
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Fig. 5.
MALDI spectra of PSII core complexes from
Synechocystis: untreated sample (A)
and samples irradiated with visible (B) and UV-B light
(C). See "Materials and Methods"
for irradiation conditions.
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Fig. 6.
MALDI spectrum in the low MW region of PSII
core complex isolated from Synechocystis.
Proposed identification of m/z peaks of low MW subunits of PSII
from Synechocystis
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Fig. 7.
MALDI spectra of whole thylakoid membranes of
wild type (A) and IC7 Synechocystis
mutant (B).
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. E. Bergantino and E. Bergo and Prof. R. Barbato for discussions, Dr. M. Bianchetti for help in preparing some samples, and Profs. J. Barber and W. F. J. Vermaas for the IC7 and PSI-less mutants of Synechocystis, respectively.
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FOOTNOTES |
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* This work was supported by European Union Grant IC15CT98, Italian Ministry of the University and Scientific Research under program PRIN99 and by Consiglio Nazionale delle Ricerche grant "Target Project on Biotechnology."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Recipient of a Young Researcher grant from the University of Padova.
To whom correspondence should be addressed. Tel.:
39-049-8276326; Fax: 39-049-8276344; E-mail:
gcometti@civ.bio.unipd.it.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M008081200
2 I. Szabò, F. Rigoni, M. Bianchetti, D. Carbonera, F. Pierantoni, R. Seraglia, and G. M. Giacometti, Submitted for publication.
3 E. Bergantino, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
PSII, photosystem
II;
RCII, isolated reaction center of PSII comprising D1, D2, and
subunits of cytochrome b559, and PsbI
proteins;
HPLC, high performance liquid chromatography;
ESI, electrospray ionization;
MS, mass spectrometry;
MALDI, matrix-assisted
laser desorption/ionization;
Mes, 4-morpholineethanesulfonic acid;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis;
MW, molecular
weight.
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