Functional studies of the Synechocystis phycobilisomes organizationby high performance liquid chromatography on line with a mass spectrometer Lello Zolla, Maria Bianchetti and Sara Rinal
Trang 1Functional studies of the Synechocystis phycobilisomes organization
by high performance liquid chromatography on line with a mass spectrometer
Lello Zolla, Maria Bianchetti and Sara Rinalducci
Dipartimento di Scienze Ambientali, Universita’ degli Studi della Tuscia, Viterbo, Italy
This study was designed to yield data on the supramolecular
organization of the phycobilisome apparatus from
Synechocystis, and the possible effects of environmental
stress on this arrangement Phycobilisomes were dissociated
in a low ionic strength solution and a quantitative estimation
of the protein components present in each subcomplex was
obtained using liquid chromatography coupled on-line with
a mass spectrometer equipped with an electrospray ion
source (ESI-MS) An advantage of this approach is that
information can be collected on the initial events, which take
place as this organism adapts to environmental changes
Ultracentrifugation of whole phycobilisomes revealed five
subcomplexes; the lightest contained four linker proteins
plus free phycocyanin, the second the core complex, while
the last three bands contained the rod complexes Four
linkers were found in band 1 with higher molecular masses
than those expected from the DNA sequence, indicating that they also contain linked chemical groups UV-B irradiation specifically destroyed the b-phycocyanin and one rod linker, which resulted in the disintegration of the rod complexes The two bilins present in b-phycocyanin give a greater contribution to the UV absorption than the single bilin of the other bilinproteins and probably react with atmospheric oxygen forming toxic radicals The protein backbone is, in fact, protected from damage in anaerobic conditions and in the presence of radical scavengers Cells grown in sulfur- and nitrogen-deficient medium contained significantly reduced levels of b-phycocyanin and one rod linker
Keywords: phycobilisomes; UV-B irradiation; Synechocystis; HPLC; mass spectrometry
1
Phycobilisomes are present in prokaryotic cyanobacteria
and eukaryotic red algae They are highly organized
complexes of various proteins containing bilin as
chromo-phore for light absorption (biliproteins) and linker
poly-peptides [1] The organization of phycobilisomes varies from
organism to organism, and an individual organism has
phycobilisomes that are affected by the environment in
diverse ways [2] In general the macrostructure of
phyco-bilisomes consists of a core (constituted by allophycocyanin)
surrounded by phycocyanin and phycoerythrin (when
present) organized in a periferic structure known as rod [1]
As all these proteins are assembled through specific
interactions with polypeptides called linkers, whose
molecular masses range from 7800 to 100 000 [3,4] The
core is situated in proximity to the photosystem II in
the thylakoid membrane, where chlorophyll a of
photosys-tem II is located The biliproteins have their chromophores
(bilins) arranged to produce rapid and directional energy
migration through the phycobilisomes and to chlorophyll a
in the thylakoid membrane [5] Phycocyanin is the major
constituent of the phycobilisomes, while allophycocyanin
holds the bridging pigments between phycobilisomes and
the photosynthetic lamellae [6] Both phycocyanin and allophycocyanin are composed of two polypeptide chains,
a and b, of approximately 17 000 and 18 000 [7] The a and
b polypeptides contain one or two chromophores, respect-ively In a low ionic strength aqueous medium, phycobili-somes dissociate into various components, and the individual biliproteins, either with or without attached linkers, are obtained [6] The relative stability of biliprotein-linker complexes varies among the biliproteins from differ-ent sources
Information on the supramolecular organization of phycobilisomes has come from electron microscopy research, which showed that cyanobacteria contain various structural types of phycobilisomes but the hemidiscoidal phycobilisomes having a tricylindrical core and six rods are found extensively in most cyanobacteria [1] Allophycocy-anin is organized as a trimer near neutral pH, having three a and three b polypeptides; each of these polypeptides has one chromophore (bilin) Trimers (a3b3) are ringlike assemblies
of three monomers (ab) having threefold symmetry Phy-cocyanin is found in solution as a complex mixture of (a3b3) (a6b6), and other oligomers The hexamers (a6b6) are disk shaped, formed by face-to-face assembly of trimers Rods are formed by face-to-face assembly of these disks From a physiological point of view, arranging the bilinproteins into rods provides the structural basis for efficient energy transfer to respond to variation of environ-mental conditions and to adapt to extreme habitats There are typically two to six disks in a rod depending on the organism and growth conditions It is well documented, for example, that cyanobacterial phycobilisomes adapt to
Correspondence to L Zolla, Dipartimento di Scienze Ambientali
Universita’ degli Studi della Tuscia, Via San Camillo de Lellis 01100
Viterbo, Italy Fax: + 39 0761 357179, E-mail: zolla@unitus.it
Abbreviations: ESI-MS, electrospray ionization mass ionic
spectro-metry; RIC, reconstructed ionic current; ROS, reactive oxygen species.
(Received 30 July 2001, revised 2 January 2002, accepted 22 January
2002)
Trang 2different light levels through a complex process that involves
changes in the ratio of phycocyanin to phycoerythrin in rods
of certain phycobilisomes to improve light harvesting [8]
Furthermore, modulation of the energy levels of the four
chemically different bilins by a variety of influences
produces more efficient light harvesting and energy
migra-tion It is generally accepted that the linkers govern the
assembly of the biliproteins into phycobilisomes, and,
despite being colorless, in certain cases they have been
shown to improve the energy migration process [7] As some
of the linkers mediate assembly of the biliproteins, they
produce changes in the spectra of biliproteins, and this may
serve to direct energy migration more efficiently through the
phycobilisomes [9,10] Linkers are found in both
cyanobac-terial and red algae phycobilisomes and may constitute 10–
15% of the total mass [11–13] Details of how linkers
perform their tasks are still largely unknown
In this paper we use a recently developed HPLC-ESI-MS
method that allows rapid and efficient separation of
phycobilisomes upon injection of entire subcomplexes [14]
The nondisruptive nature of this method has made it
possible to collect information on the composition of each
subcomplex and on the organization of phycobilisomes
under different environmental conditions Preliminary
studies on how both environmental factors such as UV
radiation and physiological stress such as starvation may
affect this supraorganization are also presented
M A T E R I A L S A N D M E T H O D S
Chemicals
Reagent-grade phosphoric acid, magnesium chloride,
sodium chloride, trifluoroacetic acid, methanol, ethanol,
formamide, as well as HPLC-grade water and acetonitrile,
were obtained from Carlo Erba (Milan, Italy) Sucrose,
Tris, Mes, sodium nitrate, magnesium sulfate, calcium
cloride, citric acid, manganese cloride, cupper sulfate, zinc
sulfate, Hepes, from Sigma, acrylamide and
N,N¢-methy-lene-bis-acrylamide (Bis) from Bio-Rad
Phycobilisome preparation
Synechocystis PCC 6803 was grown at 37°C at 50
lEÆm)2Æs)1in BG11 medium [15] The cells were harvested
by centrifugation at 9800 g
in JA20 Beckman rotor, resuspended in buffer A (20 mM
Mes pH 6.35, 25% glycerol, 5 mM CaCl2Æ2H2O, 5 mM
MgCl2Æ6H2O 1 mMbenzamidine, 1 mMaminocaproic acid)
and disrupted by 15 cycles of 30 s in a Braun Homogenizer
The cell debris was eliminated by centrifugation as before
and the supernatant was spun at 148 000 g
Kontron centrifuge at 4°C [16] The supernatant was
collected and used for HPLC separation without any
further purification
Sucrose gradient ultracentrifugation
The experimental conditions were as reported by Sinha
et al.[17] with the following modification: the dissociated
complexes was loaded onto a 0–60% sucrose gradient in
0.75M phosphate buffer pH 7, and
for 40 h using a Kontron TST 41.14 rotor The blue
pigmented bands were harvested with a syringe and analyzed directly
UV-B and visible light treatment Culture cells in suspension as well as isolated phycobili-somes were exposed for 4 h at 1.8 WÆm)2 at room temperature to artificial UV-B produced by a transillumi-nator (Bio-Rad), with its main output at 312 nm Suspen-sions were gently agitated by a magnetic stirrer during irradiation to ensure uniform distribution
For the visible light treatment, the cells and phycobili-some suspensions were exposed to white fluorescent light, the intensity was fixed at 1000 lEÆm)2Æs)1
Starvation Cells grown in BG11 medium were harvested aseptically, washed once with BG11 lacking of NaNO3and MgSO4, and resuspended in the same medium, and grown for two more days [18]
SDS/PAGE electrophoresis SDS/PAGE analysis was carried out using a Protean II Bio-Rad gel apparatus (180· 160 mm, 1.5 mm thick), using the method described by Schagger [19]: 16.5% T, 5.4% C in the separating gel and 10% T, 3% C in the spacer gel; a constant voltage of 100 kV was applied overnight at room temperature Gels were stained with Coomassie Brilliant Blue R-250 dissolved in acetic acid: methanol: water
10 : 40 : 50 (v/v/v)
High performance liquid chromatography Protein separation by HPLC was performed using a reversed phase Vydac Protein C-4 column (250· 4.6 mm internal diameter
5 , The Separation Group, Hesperia, CA, USA) packed with 5-lm porous butyl silica particles [20,21] This column was operated at a flow rate of 1 mLÆmin)1 for optimum separation efficiency All solutions were filtered through a Millipore (Milan, Italy) type FH 0.5-lm mem-brane filter and degassed by bubbling with helium before use Optimization of chromatographic separations was performed using a Beckman (Fullerton, CA, USA) Gold System with of Model 126 solvent delivery pumps Samples were introduced onto the column by a Model 210 A sample injection valve with either a 20- or a 50-lL sample loop The Vydac C-4 columns were pre-equilibrated with 20% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) trifluoroacetic acid and samples were eluted using a gradient from 20 to 95% (v/v) acetonitrile in 60 min, at a flow rate of
1 mLÆmin)1[14]
Electrospray mass spectrometry The HPLC-ESI-MS experiments were carried out by splitting the outlet of the HPLC and coupling it with a Perkin Elmer API 2000 or API 365 triple quadrupole mass spectrometer equipped with the electrospray ion source [22] For HPLC-MS analysis, with pneumatically assisted elec-trospray, a spray voltage of 5 kV and a sheath gas pressure
of 500 kPa were employed Protein mass spectra were
Trang 3recorded by scanning the first quadrupole; the scan range
was 500–1800 average mass units
ion spectrum of a single protein consists of a series of peaks,
each of which represents a multiply charged ion of the intact
protein having a specific number of protons attached to the basic sites of the amino-acid sequence The m/z values for the ions have the general form [M + zH]/z, where z equals the number of protons attached It follows that the molecular mass can be readily calculated from two meas-ured adjacent m/z values, given the additional information that two adjacent multiply charged ions differ by one charge Once M and z are determined for one pair of peaks, all other m/z signals can be deconvoluted into one peak on a real mass scale, which has a typical peak width at half height
of 10–20 average mass units The mass spectrometer was tuned for chromatographic conditions with a 2 lgÆlL)1 solution of cytochrome c (Sigma) added at a flow-rate of
1 lLÆmin)1 to the column effluent (50 lLÆmin)1, 50% acetonitrile in 0.05% trifluoroacetic acid) by means of a T-piece before entering the ESI source, resulting in a flow rate of 50 lLÆmin)1into the mass spectrometer
R E S U L T S Injection of the total mixture of phycobilisomes from Synechocystis PCC 6803, schematically represented in Fig 1A, into a C4 RP-HPLC system resulted in the separation of the phycobilisome complex in four main peaks and many smaller peaks (Fig 1B), as observed by absorbance detection at 214 nm Simultaneously, the spec-trum of each eluting peak was recorded by a photodiode array detector The chromatogram recorded at 600 nm (see left inset of 1B) shows that only peaks 6, 7, 8 and 9 had an absorption at 600 nm, typically due to the presence of bilin pigments still connected with the polypeptide backbone [14] Coupling the HPLC on line with a mass spectrometer using electrospray as source allowed us to determine the molecu-lar masses of the proteins in each HPLC peak The peaks eluting within 25 min represented linker proteins, while the four main peaks between 28 and 33 min were the phyco-cyanins and allophycophyco-cyanins [15] In Table 1 the experi-mental molecular masses determined by the deconvolution
of the ESI-MS spectra have been correlated to the expected
Table 1 List of the Synechocystis 6803 phycobilisome protein components determined by HPLC-ESI-MS compared with the proteins expected from DNA sequence and those observed by SDS/PAGE The molecular masses were determined by the deconvolution of the ESI-MS spectra recorded during the chromatographic run into a C-4 reverse phase column coupled on-line with by a mass spectrometer equipped with an electrospray ion source (ESI-MS) The values of molecular mass deduced from DNA sequence have been calculated by using the Prot Parameter tools in the EXPASY program The SDS/PAGE molecular masses were deduced by the marker used in the electrophoresis reported in the right inset of Fig 1B.
Type of protein Proteins
Molecular masses
HPLC peak number
Expected from DNA sequence
Measured by HPLC-ESI-MS
Apparent measured by SDS/PAGE
Fig 1 Chromatographic profile of Synechocystis PCC 6803
phyco-bilisome proteins separated by reversed-phase HPLC (A) reports
schematically the phycobilisome organization and their protein
components (B) shows the HPLC chromatogram recorded at 214 nm.
Labeling refers to peak numbers The left inset of (B) shows the spectra
of the four main peaks obtained by the diode array detector The right
inset of (B) reports the SDS/PAGE of phycobilisomes.
Trang 4molecular masses deduced from their DNA sequences of the
phycobilisome protein components and to the apparent
molecular masses observed in SDS/PAGE (right inset of
Fig 1B) The molecular mass values obtained are very close
to those expected; the differences observed (ranging from
580 to 607 Da) were probably due to the presence of
residual bilin pigment still bound to the proteins, whose
molecular mass is 587 Da Proteins with molecular masses
ranging from 32 000 to 37 000 as well as proteins with
molecular masses under 10 000 have been tentatively
attributed to linker proteins However, most of the proteins
found by HPLC-ESI-MS correspond to the bands observed
by SDS/PAGE with the exception of the band over
45 kDa, which was previously interpreted as particle
containing both phycocyanin and allophycocyanin [23] or
ferredoxin [24]
When whole phycobilisomes were suspended in low ionic
strength buffer the interactions between the linkers and
different protein components of phycobilisomes were
destroyed, and various incomplete subcomplexes were
formed [6] With the aim to get information on the
localization and distribution of the phycobilisome
compo-nents, the mixture of subcomplexes was subjected to sucrose
gradient ultracentrifuge separation The results are shown in
Fig 2A: four main blue bands were observed, plus a faint
blue band at the top The first band, corresponding to the
smaller complexes, was faintly colored, the second was the
most colored and the more abundant, whereas the other two
bands, observed at a higher sucrose concentration,
con-tained the higher density subcomplexes Each band was
collected with a syringe and analyzed by RP-HPLC Besides
being more efficient than SDS/PAGE in discriminating
between the four allophycocyanins and phycocyanins,
HPLC separation allowed a quantitative estimation of the
relative amount of each protein component from the area
underlying each peak This was facilitated by the fact that
the biliproteins are strongly conserved and it may
reason-ably be assumed that they have similar optical extinction
coefficients; therefore the area underlying each HPLC peak
allowed a comparison of the relative stoichiometry of each
protein The chromatograms recorded upon injection of
each band into a reversed phase column are reported on the
right part of Fig 2 The first observation to consider is that
band 1, the lightest in the sucrose gradient, showed an
HPLC chromatogram abundant in hydrophilic proteins
such as linkers and a significant amount of a- and
b-phycocyanins, and only traces of the allophycocyanins
On the contrary, HPLC analysis of bands 2–5 revealed that
linker proteins were scarce, while the main components of
the subcomplexes were phycocyanin and allophycocyanin,
although at different stoichiometric ratios In particular,
band 2 contained the highest amount of both, with a
significant reduction
7 of the b-phycocyanin component In
contrast, the other three bands contain essentially a- and
b-phycocyanin in stoichiometric amounts
1 into the HPLC-ESI-MS system revealed the presence of
four proteins with molecular masses ranging from 30 000 to
36 000 (Fig 3) Insets of Fig 3 show the deconvolution
analysis performed on the corresponding ESI-MS spectra of
the HPLC peaks indicated by an arrow Obviously
sucrose-gradient separation has resulted in an enrichment of linker
proteins in band 1 However, the heaviest linker (Mr of
100 000.5) is not revealed, because it is not recovered under
these separation conditions, as confirmed by SDS/PAGE
In fact, it has been reported to remain enclosed in the phycobilisomes unless treated with high salt [25] Further-more, in this sucrose band 1 linker proteins with molecular masses under 10 000 were not found They are detected by ESI-MS into band 5 (data not shown) This result agrees with the hypothesis that these small linkers remain tightly bound to the rod complexes [3]
From this preliminary analysis it may be concluded that the breakdown of phycobilisomes by low ionic strength causes the supramolecular complex to be degra-ded into many subcomplexes, which differ from each other by the percentage and type of bilin proteins they contain
In order to study the influence of certain environmental changes on the supramolecular organization of the
Fig 2 HPLC analysis of phycobilisome complexes (A) Ultracentri-fuge tube containing the phycobilisome apparatus from Synechocystis PCC 6803 once fractionated in its components and loaded onto a 0–60% sucrose gradient in phosphate buffer The individual bands obtained are labeled with number 1–5 starting from the top of tube (B) Panels B1–B5 show the RP-HPLC profile when each sucrose band
is loaded onto a reversed phase C4 column B2 reports the identifica-tion of the mains peaks, previously performed [14].
Trang 5Synechocystisphycobilisomes, we subjected whole cells and
isolated phycobilisomes to a number of treatments,
inclu-ding high visible light intensity, UV-B light exposure and
nutrient deficiency conditions
During the visible light treatment, cell cultures were
exposed to 1000 lEÆm)2Æs)1 white light for 3 h under
continuous stirring No significant change was observed
in the blue color of the solution during this time Then
the phycobilisomes were extracted from these cells and
loaded onto a sucrose gradient for ultracentrifuge
separation Five bands were observed as in the control
The HPLC analysis of each band showed
chromato-grams similar to those reported for the control Similarly,
when separated phycobilisomes were subjected to the
same experimental conditions as whole cells, the same
trend was seen Thus it is reasonable to conclude that
visible light does not induce significant and irreversible
rearrangements of the components inside the
subcom-plexes in this organism
In contrast, when either cells or phycobilisomes were
exposed to UV-B radiation, a visible change in the color of
the solution was clearly observed, although different periods
of exposure were required for cells and for isolated
phycobilisomes The treated cell culture assumed an
olive-green pigmentation with time, clearly distinguishable from
the native blue-green of the control strain; the isolated
phycobilisomes gave a much fainter blue pigmentation than
the control HPLC analysis of entire phycobilisomes before
and after illumination revealed the disappearance of the
b-phycocyanin already after 1 h of illumination (Fig 4) In
contrast, SDS/PAGE analysis of the total mixture of
phycobilisomes exposed to the same UV-B irradiation did
not reveal any significant decrease in the total intensity of
the stained bands, confirming the high sensitivity of the
HPLC method (inset A of Fig 4) Nevertheless, Fig 4
(inset B) shows the time course of spectroscopic absorption
recorded on intact phycobilisome undergone to UV-B
irradiation It may be observed that the absorption at
578 nm is more effected, suggesting involvement of bilin chromophores in UV-B damage
Sucrose-gradient ultracentrifugation of UV-B treated phycobilisomes (Fig 5B) showed only two main bands plus a faint one at the top instead of the five observed in the control (Fig 5A), the two heaviest bands having completely disappeared HPLC analysis of the second sucrose bands of UV-B treated phycobilisome revealed that the b-phycocy-anin peak at 214 nm (indicated with an arrow) was missing from both of them, while all the other components were present Thus it may be inferred that b-phycocyanin is the main target of UV-B radiation, and that this component is essential for the formation of all subcomplexes However, the disappearance of a peak at 214 nm in the HPLC chromatogram does not reveal the extent of the damage to b-phycocyanin In fact, it may be that a single chromophore
is destroyed, with consequent decrease of the optical absorption, or the entire protein backbone could be degraded Thus, we analyzed both ultracentrifuge sucrose bands by HPLC-ESI-MS to obtain more details Figure 6 compares the reconstructed ionic current (RIC) recorded by
a mass spectrometer upon injection of band 1 from
Fig 4 Effect of UV-B irradiation on the whole phycobilisome appar-atus Comparison of the RP-HPLC chromatograms obtained for UV-B irradiated phycobilisomes (dashed line) and control sample (dotted line) The arrow indicates the PCb peak Inset A compares the SDS/PAGE of the control (lane C) and UV-B treated (lane UV) samples Inset (B) reports the spectra absorption recorded on phyco-bilisomes undergone to UV-B light at different times Details on chromatographic and electrophoretic separation conditions as repor-ted in the Materials and methods section PC, phycocyanin.
Fig 5 Effect of UV-B irradiation on phycobilisome organization Left part: Ultracentrifuge tubes comparison of untreated (A) and UV-B irradiated (B) phycobilisome apparatus loaded onto 0–60% sucrose gradient The bands are labeled starting from the top of tubes Right part: RP-HPLC chromatogram of the second sucrose band loaded onto a C4 column The arrow indicates the peak affected.
Fig 3 Characterization of linker proteins RP-HPLC of sucrose band
1 analyzed on line with an ESI mass spectrometer The separation
conditions are indicated in the Materials and methods section Insets
report the deconvolution of ESI-MS spectra recorded for each HPLC
peaks The arrows assign each measured molecular mass to the
cor-responding HPLC peaks.
Trang 6control (A) or UV treated (B) separated phycobilisomes We
examined sucrose band 1 because it contained both linkers
and biliproteins It was observed that the b-phycocyanin
peak and the peak corresponding to linker 1 (both indicated
by an arrow) were reduced in treated samples (Fig 6B)
Because in mass spectroscopy the ionic current is strongly
dependent on the protein and not on prosthetic groups, it
could seem reasonable to conclude that both peak
reduc-tions were related to the destruction of the polypeptide
backbone of the native protein However, SDS/PAGE of
each HPLC fraction (inset B, line 2) showed that
b-phycocyanin was partially reduced, but not completely destroyed, contradicting the RIC and UV results Such discrepancies are commonly observed when active oxygen is involved in photodamage of proteins They arise from the fact that in hydrophobic proteins radical attacks occur preferentially in the external hydrophilic region of the protein, a region rich in amino acids with a higher than average contribution to the UV optical absorption and protonation of amino groups by electrospray ionization, leading to falsely high estimates of protein loss by these methods (L Zolla, & S Rinalducci, unpublished results) This consideration suggested that oxygen radicals might be involved in the phenomena described Thus, in order to collect more information on the possible molecular mech-anism by which UV-B affected the phycobilisomes appar-atus, we subjected our sample to UV-B exposure in the presence or in absence of oxygen as well as in the presence of ascorbate, a well-known scavenger of oxygen radical species In anaerobiosis or the presence of ascorbate the b-phycocyanin was not affected by UV-B irradiation (data not shown), confirming the possible involvement of oxygen radicals in the UV-B destruction of the phycobilisome apparatus Interestingly, preliminary electron paramagnetic resonance experiments performed in the presence of the spin trap 5,5-dimethyl-pyrroline N-oxide confirmed the involve-ment of reactive oxygen species (ROS) during UV irradi-ation (I Vass, Institute of Plant Biology, Szeged, Hungary; personal communication)
As a final study, we investigated whether the HPLC method was able to monitor the first steps of phycobilisome changes, in terms of reorganization and protein composi-tion, following altered growing conditions It was previously reported that cyanobacteria grown under sulfur and nitro-gen deficiency, showed an altered phycobilisome organiza-tion [8] With this in mind, Synechocystis cells were taken from exponentially growing cultures and placed in nitrogen-and sulfur-depleted medium for 2 days Figure 7 compares the HPLC chromatograms recorded upon injection of whole phycobilisomes extracted from a control culture with that from cyanobacteria grown in the absence of sulfur and nitrogen It was observed that b-phycocyanin decreased under these experimental conditions together with a linker protein eluting at 18 min (labeled by arrows) and showing a molecular mass of 34 316 Moreover, sucrose gradient analysis of the treated cyanobacteria showed the disappear-ance of the heaviest sucrose band 5 (inset of Fig 7B)
D I S C U S S I O N
In a recent paper the complete resolution of the protein components of phycobilisomes from the cyanobacterium Synechocystis6803, together with the determination of their molecular mass, has successfully been achieved [14] by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS) In the present paper, we have employed this method to make a quantitative estimation of components present in individual subcomplexes obtained by dissociation
in a low ionic strength solution Information was collected
on the possible supramolecolar organization and how some types of environmental stress may interfere with it In low ionic strength conditions phycobilisomes dissociate into water-soluble subcomplexes, which can be separated into
Fig 6 RP-HPLC-ESI-MS chromatograms comparison from control
and UV-B irradiated phycobilisomes (A) Reconstructed ionic current
(RIC) obtained loading the sucrose gradient band 1 onto a C4 column
coupled on line with a mass spectrometer interfaced with an
electro-spray Arrows indicate the peaks affected by UV-B irradiation (B)
RIC of phycobilisomes irradiated with UV-B at 1.8 WÆm)2for 4 h.
Inset of (A) and (B) show the SDS/PAGE of the main HPLC peak
once collected, dried and loaded onto 16.5% T 5.4% C Tris/tricine gel.
Lines 1–4 refer to the PCa, PCb, APCa and APCb, respectively PC,
phycocyanin; APC, allophycocyanin.
Trang 7five different bands upon extended ultracentrifugation The
relative stability of biliprotein-linker complexes is known to
differ among the biliproteins and also depending on the
species However, our data show that in Synechocystis the
linkers are preferentially removed by low ionic strength
solution, particularly the rod linker, and they appear in
band 1, the lightest band in the sucrose gradient In this
band free phycocyanins are also found, confirming a
previous observation by Reuter et al [25] that some
phycocyanins do not participate in supramolecular
organ-ization Interestingly ESI-MS analysis of linkers present in
band 1 reveals the presence of four proteins with Mrvalues
of 33 932, 34 316, 36 608 and 37 118 These molecular
masses are higher than those expected from the DNA
sequence reported in the SwissProt database: 30797.3
(accession number P73204) and 32389.4 (accession number
P73203) This discrepancy suggests that these linker proteins
are subject to post-translational modifications On the other
hand, analysis of intact phycobilisomes by SDS/PAGE
showed two main bands with apparent molecular masses
over 36 000, in agreement with many other reports [1,24],
allowing us to assume that the protein showing molecular
masses in the range 34 000–37 118 may really represent the
linker proteins However, the presence of contaminating
proteins cannot be excluded entirely at the time being
Regarding the content of the other four sucrose gradient
bands, HPLC analysis revealed that a- and b-phycocyanins
are the most abundant components Their presence is particularly prevalent in the heavier bands (bands 3–5), and
it is likely that these heaviest complexes correspond to the rods, the peripheral components of the phycobilisomes apparatus [1] HPLC peaks corresponding to a- and b-phycocyanin show similar intensities, suggesting that they are present in equivalent amounts This is in agreement with the current model where the functional units of all phycobilisomes are disk shaped trimers of closely associated (ab) phycobiliproteins [1–4] An unexpected exception is observed for the subcomplex contained in the sucrose band
2 where the amount of the b-phycocyanin component is significantly reduced The allophycocyanins are mainly found in the second band, which probably corresponds to the core, where the allophycocyanins are prevalently located [1] However, the second band probably contains only remains of dissociated cores, because the intact core complex has an expected molecular mass close to that of a phycobilisome rod, and so should migrate down the gradients The lack of intact cores is probably due to absence in our preparation of the LCMprotein required for the core assembly [25] Data reported previously confirm that different rod subcomplexes are present around the core, which are held together by linker proteins to form the Synechocystisphycobilisome supramolecular organization
In agreement, X-ray crystallography has shown that the hexamers (a6b6) are disk shaped, formed by face-to-face assembly of trimers Rods are formed by face-to-face assembly of these disks [26]
Treatment of phycobilisomes with UV-B destroys this supramolecular organization Data presented here clearly show that exposure of intact Synechocystis cells or isolated phycobilisomes to moderate UV-B intensity (1.8 WÆm)2) induces specific loss of b-phycocyanin and the 37 118-rod linker It is not clear how the latter protein preferentially absorbs more UV-B light than other linkers It is generally accepted that aromatic amino acids can absorb UV, but the DNA sequence of this particular linker does not indicate a high percentage of aromatic amino acids It is more likely that the higher molecular mass measured with respect to that expected is due the presence of a particular functional group that may distinguish this linker from the others Alternatively this linker may be located close to the source
of reactive species generated by photodamage Similarly, Rhodella vs subjected
decreased amount of the linker having an SDS apparent molecular mass of 32 000 [27] Regarding the specific b-phycocyanin damage, three explanations can be sugges-ted: (a) this phycocyanin contains two bilin pigments whereas the other phycobilisomes proteins have only one; (b) it is the more abundant protein; and (c) it is located at the periphery of phycobilisomes In a recent paper the effect
of UV-B irradiation on the isolated phycobilisomes of Synechococcussp PCC 7942 showed photodestruction of both a- and b-phycocyanins, but not of allophycocyanins which also contain bilins [28] Because it is difficult to attribute the specific damage to variations in aromatic amino-acid content, because the amino-acid composition of allophycocyanins and phycocyanins is significantly con-served, it seems reasonable to attribute their greater sensitivity to the external allocation in the supramolecular organization In a recent paper isolated a- and b-phyco-cyanins were irradiated for various lengths of time and
Fig 7 Effect of nutrient deprivation on phycobilisome protein
compo-sition (A) RP-HPLC chromatogram of entire phycobilisomes used as
control (B) The RP-HPLC pattern of phycobilisome isolated from
cells grown in nutrient starvation The arrows indicate the main peaks
affected The growing conditions are reported in Materials and
methods section Insets show the ultracentrifugation tubes obtained
upon loading phycobilisomes from control and starved cyanobacteria
onto a 0–60% sucrose gradient.
Trang 8analyzed by HPLC on a reversed-phase column; both
phycobiliproteins showed similar photodestruction
quan-tum yields [29] On the other hand, irradiation at 280 or
640 nm caused the same extent of damage, indicating that
both tryptophan and bilin absorption are involved in the
phenomenon observed In contrast, our experiments
showed that whether entire phycobilisomes or whole cells
were irradiated with UV-B, only the b-phycocyanin
com-ponent was significantly affected This specific damage was
observed after the first hour of irradiation by both optical
absorption and RIC decrease However, although the
optical decreases observed could be correlated to the
damage of aromatic amino acids, this could not explain
the RIC decrease, because protonation of positive amino
acids, which occurs during electrospray ionization, is
independent of the presence of an aromatic group [30]
Thus, the simplest explanation is that the two bilins present
in b-phycocyanin give a greater contribution to the optical
absorption than the single bilin of the other biliproteins
More experiments are in progress in our laboratory to
localize the reactive species generated by photodamage and
the specific role that oxygen may play in this
photodestruc-tive phenomenon Preliminary EPR experiments have
shown the presence of ROS in the solution of irradiated
phycobilisomes, indicating that the protein has been
subjected to chemical destruction from active oxygen radical
(I Vass, personal communication) This also agrees with
the previous study of He et al [31] who observed reactive
oxygen species generated from phycobiliproteins after
photosensitization and the recent study of Zhang et al [32]
In any case, it is not surprising that the disappearance of
all subcomplexes as a consequence of UV-B irradiation is
related to damage to b-phycocyanin, because it is the most
abundant biliprotein in all subcomplexes
It is interesting that visible light stress seems not to
influence the internal composition of each subcomplex in
contrast to UV irradiation In fact, it is well known that
adaptation of cyanobacterial phycobilisomes to light by
complementary chromatic adaptation is a complex process
that changes the ratio of phycocyanin to phycoerythrin in
rods of certain phycobilisomes to improve light harvesting
in changing habitats [1] Thus in Synechocystis where
phycoerythrin is not present, visible light may only induce
an alternative arrangement of the subcomplexes This agrees
with the previous observation that upon light adaptation
some phycocyanin (ab)3 units were released and
conse-quently the length of phycobilisome rods resulted reduced
[27] In any case, cyanobacteria seem to be better adapted
than higher plants to endure high intensity visible light [1]
Finally, we have here presented an example of a response
to an environmental change that does not interact directly
with the chromophore, but is supposed to involve the
biosynthetic apparatus of the cells Synechocystis grown
under sulfur- and nitrogen-deficient conditions contained
less b-phycocyanin; this only had a marked effect on band 5,
the heaviest one, consistent with the idea that this band
contains mainly b-phycocyanins Probably during nitrogen
starvation b-phycocyanin represents the main source of
nitrogen for cyanobacteria [33] Moreover, the linker with
molecular mass 34 316 is also lost, raising the possibility
that this rod linker, which is different to that destroyed by
UV-B, plays a role in binding the heaviest subcomplex
present in band 5 However, it is worth emphasizing that all
this evidence is obtained by the HPLC method during the first days of starvation, allowing us to monitor the first steps
of phycobilisome reorganization
In conclusion, the data reported here demonstrate that the use of analytical methods with greater resolving powers can reveal the initial events in the process of damage, which are well observed by HPLC after 3 h of UV-B irradiation or
3 days of starvation, but not by SDS/PAGE analysis Moreover, by the HPLC-ESI-MS method, a minimum manipulation of samples for component analysis is neces-sary which may help to eliminate artifacts This knowledge
is expected to shed light on the composition and supramo-lecular organization of phycobilisomes and may increase the understanding of the molecular mechanisms underlying their physiological adaptations to environmental condi-tions The technique also lends itself to the screening of phycobiliproteins for interesting structural features or the characterization of mutant phycobiliproteins lacking one or more bilin peptides
A C K N O W L E D G E M E N T S
The authors are grateful to Dr Sergio Gallo (PE Biosystems, Rome, Italy) for the mass spectrometer measurements, Prof Giorgio Giacom-etti for providing the cyanobacteria strain and Dr Cristina ProiGiacom-etti Zolla for valuable assistance in the preparation of the samples This work was supported by the CE Project CIPA CT93 0202, MURST 40% and COST Contract ERB IC15CT 980126.
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