Functional 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, a nd the possible effects of environmental stress on this arrangement. Phycobilisomes were dissociated in a l ow 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 ch anges. Ultracentrifugation of whole phycobilisomes revealed five subcomplexes; the lightest c ontained four linker proteins plus free phycocyanin, the second the core complex, while the last three bands c ontained the rod complexes. Four linkers were found in band 1 with higher molecular masses than those expected from the DNA s equence, indicating that they also con tain linked c hemical groups. U V-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 s ingle bilin of the other b ilinproteins 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 c ontained significantly reduced levels of b-phycocyanin and one rod linker. Keywords: phycobilisomes; UV-B i rradiation; 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 f rom 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 m embrane, where c hlorophyll a o f photosys- tem I I is located. The biliproteins have their chromophores (bilins) arranged to produce rapid and directional e nergy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane [5]. Phycocyanin is the major constituent of the phycobilisomes, while allophycoc yanin 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 f rom differ- ent sources. Information on the supramolecular organization of phycobilisomes has come from electro n microscopy research, which showed that cyanobacteria contain various structural types of phycobilisomes but the hemidiscoidal phycobilisomes having a tricylindrical c ore 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 ( a 3 b 3 ) are ringlike a ssemblies of three monomers (ab) having threefold symmetry. Phy- cocyanin is found in solution as a complex mixture of (a 3 b 3 ) (a 6 b 6 ), and other oligomers. The hexamers (a 6 b 6 ) are disk shaped, formed by face-to-face assembly of trimers. Rods areformedbyface-to-faceassemblyofthesedisks. From a physiological point of view, arranging the bilinproteins into rods provides the structural basis for efficient energy transfer to res pond t o v ariation of environ- mental conditions and to adapt to extreme h abitats. There are typically two to six disks in a rod depending on the organism and growth c onditions. 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: z olla@unitus.it Abbreviations: ESI-MS, electrospray ionization mass ionic spectro- metry; RIC, reconstructed ionic current; ROS, reactive oxygen species. (Received 30 J uly 2001, revised 2 January 2002, ac cepted 22 January 2002) Eur. J. Biochem. 269, 1534–1542 (2002) Ó FEBS 2002 different light levels through a complex process that i nvolves 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 effi cient 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 t his may serve to direct energy migration more efficiently through the phycobilisomes [9,10]. L inkers are found in both c yanobac- terial and red algae phyc obilisomes and m ay 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 su ch as UV radiation and physiological stress such as starvation may affect this supraorganization are also presented. MATERIALS AND METHODS 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 °Cat50 lEÆm )2 Æs )1 in BG11 medium [15]. The cells were harvested by centrifugation at 9800 g 2 for 10 min at room temperature in JA20 Beckman rotor, resuspended in buffer A (20 m M Mes pH 6.35, 25% glycerol, 5 m M CaCl 2 Æ2H 2 O, 5 m M MgCl 2 Æ6H 2 O1m M benzamidine, 1 m M aminocaproic 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 3 in a TFT 50.38 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 0.75 M phosphate buffer pH 7, and 4 spun at 272 000 g 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- someswereexposedfor4hat1.8WÆm )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 ligh t treatment, the c ells and phycobili- some suspensions were exposed to white fluorescent light, the intensity was fixed at 1000 lEÆm )2 Æs )1 . Starvation Cells grown in BG11 medium were harvested aseptically, washed once with BG11 lacking of NaNO 3 and MgSO 4 , 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 C oomassie Brilliant Blue R-250 dissolved in acetic acid: methanol: w ater 10 : 40 : 50 (v/v/v). High performance liquid chromatography Protein s eparation 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 po rous 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-lmmem- 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 l oop. The V ydac 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 1mLÆmin )1 [14]. Electrospray mass spectrometry The HPLC-ESI-MS experiments were carried out by splitting the outlet of the HPLC and co upling 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 o f 5 kV and a sheath gas pressure of 500 kPa were employed. Protein mass spectra were Ó FEBS 2002 Study of phycobilisomes organization by HPLC-ESI-MS (Eur. J. Biochem. 269) 1535 recorded by scanning the first quadrupole; the scan range was 500–1800 average mass units 6 in 2 s. A typical positive 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 s pecific number of protons attached to the basic sites of the a mino-acid sequen ce. The m/z values for the ions have the general form [M + zH]/z,wherez 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. T he 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 o f 5 0 lLÆmin )1 into the mass spectrometer. RESULTS Injection of the total mixture of phycobilisomes from Synechocyst is PCC 6803, schematically represented in Fig.1A,intoaC4RP-HPLCsystemresultedinthe separation of the phycobilisome complex in four main peaks and many smaller p eaks (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 c hromatogram 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 t o determine the molecu- lar masses of the proteins in each HPLC peak. The peaks eluting within 25 min represented linker p roteins, while the four main peaks between 28 and 33 min were the phyco- cyanins and allophycocyanins [15]. In Table 1 the experi- mental molecular masses determined by the deconvolution of the ESI-MS spe ctra have been correlated t o 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 o f molecular mass deduced from DNA sequence have been c a lculated by u sing the Prot Parameter t oo ls in the EXPASY program. The SDS/PAGE molecular masses were deduced by the marker used in the e lectrophoresis 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 Bilinproteins PHAA 17 280.0 17 860 18 000 6 PHAB 17 215.6 17 820 17 000 7 PHCA 17 586.6 18 180 17 500 8 PHCB 18 126.4 19 320 18 000 9 Linkers PYC1 7800 7542 – 1 having low M r PYS1 9322.3 9242 – 2 Vcf20 12 554.6 Linkers PYR1 32 520.6 33 932 > 36 000 4 having high M r PYR2 30 797.3 36 608 < 39 000 3 CpCG 27 932.0 CPCG 28 522.5 45 000 APCE 100 295.8 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 recor ded at 214 nm. Labeling refers to peak numbe rs. The left inset of (B) s hows the spe ctra of the fou r main peaks obtained by the diode array detector. Th e right inset of (B) reports the SDS/PAGE of phycobilisomes. 1536 L. Zolla et al. (Eur. J. Biochem. 269) Ó FEBS 2002 molecular masses deduced from their DNA sequences of the phycobilisome protein components and to the apparent molecular masses observed in S DS/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 p roteins. 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 p hycobilisomes 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 t op. 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 c onserved 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 s toichiometry of each protein. The chromatograms recorded upon injection of each band into a reversed phase column are reported on the right part of F ig. 2. T he first observation to consider is that band 1, the lightest in the sucrose g radient, showed an HPLC chromatogram abundant in hydrophilic proteins such as linkers and a significant amount of a-and b-phycocyan ins, and only traces of the allophycocyanins. On the contrary, HPLC a nalysis 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-phycocyan in in stoichiometric amounts 8 . Injection of band 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 ( M r of 100 000.5) is not revealed, b ecause it is not recovered under these separation conditions, as confirmed by SDS/PAGE. In fact, it h as been reported t o remain enclosed i n the phycobilisomes unless treated with high salt [25]. Further- more, in this sucrose band 1 linker p roteins with m olecular masses under 1 0 000 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 ro d 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 b ilin proteins they contain. In order to study the influence of certain environmen tal changes on t he supramolecular organization of the Fig. 2. HPLC analysis of phycobilisome complexes. (A) Ultracentri- fuge tube containing the phycobilisome apparatus from Synechocystis PCC6803oncefractionatedinitscomponentsandloadedontoa 0–60% sucrose gradient in phosphate buffer. The ind ividual bands obtained are labeled with number 1–5 st arting from the t op of tube. (B) Panels B 1–B5 s how t he RP- HPLC p rofi le whe n e ach s ucrose b and is loaded onto a reversed phase C4 column. B2 reports the identifica- tion of the mains peaks, previously performed [14]. Ó FEBS 2002 Study of phycobilisomes organization by HPLC-ESI-MS (Eur. J. Biochem. 269) 1537 Synechocystis phycobilisomes, 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 cond itions. During the visible light treatment, cell cultures were exposed to 1000 lEÆm )2 Æs )1 white light for 3 h under continuous stirring. No significant change was observed in the blue c olor of the solution during this time. Then the phycobilisomes were extracted from these cells and loaded onto a sucrose g radient 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 su bjected 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 chan ge 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 thenativeblue-greenofthecontrolstrain;theisolated phycobilisomes gave a much fainter blue pigmentation than the control. HPLC analysis of entire phycobilisomes before and after illumination reve aled the d isappearance 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 h igh sensitivity of the HPLC method (inset A of Fig. 4). Nevertheless, Fig. 4 (inset B) shows the time course of spectroscopic a bsorption recorded on intact phycobilisome undergone to UV-B irradiation. It may be observed t hat the absorption at 578 nm is more effected, suggesting involvement o f 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 t he 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 P Cb 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. Le ft part: Ult racentrifu ge tubes comparison of untreated (A) and UV-B irradiated (B) phycobilisome apparatus loaded onto 0–60% sucrose gradient. The bands are labeled starting f rom the top of tubes. Righ t part: RP-HPLC chro matogram of the second sucrose band loaded onto a C4 column. The arrow indicates the peak a ffected. Fig. 3. Characterization of linker proteins. RP-HPLC of sucrose band 1analyzedonlinewithanESImassspectrometer.Theseparation 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. 1538 L. Zolla et al. (Eur. J. Biochem. 269) Ó FEBS 2002 control (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 t he 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. S uch discrepancies are commonly observed when active oxygen is involved in photodamage of proteins. T hey 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 g roups by electrospray ionization, leading to falsely high e stimates of protein loss by these methods (L. Zolla, & S. Rinalducci, unpublished results). This consideration suggested that oxygen radicals might be involved in the phe nomena described. Thus, in order to collect more info rmation on the p ossible molecular mech- anism by which UV -B affecte d the p hycobilisomes appar- atus, we subjected our sample to UV-B exposure in the presence or in absence o f oxygen as w ell as in the presen ce 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 p aramagnetic 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 o f P lant Biology, Szeged, Hungary; personal communication) 9 . As a final study, we investigated whether t he 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-d epleted 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 h eaviest sucrose band 5 (inset of Fig. 7B). DISCUSSION In a recent paper the complete resolution of the protein components of phycobilisomes from the cyanobacterium Synechocystis 6803, together with the determination of their molecular mass, has s uccessfully been achieved [14] by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). I n t he present paper, we have employed this method to make a quantitative estimation of components present in individual subcomplexes obtained by dissociation in a low ion ic strength solution. Information was collected on the possible supramolecolar organization and how some types of environmental stress may interfere with i t. In low ionic strength conditions phycobilisomes dissociate into water-soluble s ubcomplexes, which can be separated into Fig. 6. RP-HPLC-ESI-MS chromatograms comparison from control and UV-B irradiated phycobilisomes. (A) Reconstructed ionic cu rrent (RIC) obtained l oading the sucrose gradient band 1 onto a C4 c olumn coupled on line with a mass spectrometer inter faced 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 )2 for 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. Ó FEBS 2002 Study of phycobilisomes organization by HPLC-ESI-MS (Eur. J. Biochem. 269) 1539 five 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 l ow 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 sup ramolecular organ- ization. Interestingly ESI-MS analysis of linkers present in band 1 reveals the p resence of four proteins with M r values 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 p roteins 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 molecu lar masses in the range 34 000–37 118 may really represent the linker p roteins. 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-andb-ph ycocyanins are the most abundant components. Their presence is particularly prevalent in the heavier bands (bands 3–5), a nd 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 i n the su crose band 2 where the amount of the b-phycocyanin component is significantly reduced. The allophycocyanins are mainly found in the second band, which p robably 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 m ass close to that of a phycobilisome rod, and so should migrate down the gradients. The lack of intact cores is probably due to absence i n our preparation of the L CM protein r equired 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 Synechocyst is phycobilisome supramolecular organization. In agreement, X-ray crystallography has shown that the hexamers (a 6 b 6 ) are disk shaped, f ormed 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 Syn echocystis ce lls 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 o ther linkers. It is generally accepted that aromatic amino acids can absorb UV, but the DNA sequence of th is particular linker does not indicate a high percentage of aromatic amino acids. It is more likely that the h igher 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 s ource of reactive species generated by photodamage. Similarly, Rhodella vs. subjected 10 to medium visible light shows a decreased amount of the linker having an SDS apparent molecular mass of 32 0 00 [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 t he more abundant protein; and (c) it is located at the periphery o f phycobilisomes. In a recent paper the effect of UV-B irradiation on the isolated phycobilisomes of Synechococc us sp. PCC 7942 showed photodestruction of both a-andb-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 p aper isolated a-andb-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 tu bes obtained upon loading phycobilisomes from control and starved cyanobacteria onto a 0–60% sucrose gradient. 1540 L. Zolla et al. (Eur. J. Biochem. 269) Ó FEBS 2002 analyzed by HPLC on a reversed-phase column; both phycobiliproteins s howed similar photo destruction quan- tum yields [29]. On the other hand, irradiation at 280 or 640 nm caused the same extent o f 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 s ignificantly a ffected. This specific damage was observed a fter 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, b ecause protonation of positive a mino acids, which occurs during electrospray ionization, is independent of the presence of an aromatic g roup [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 s pecific 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 be en subjected to chemical dest ruction from a ctive oxygen radical (I. Vass, personal communication). This also agrees with the previous study of He et al. [31] who observe d 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 relatedtodamagetob-phycocyanin, because it i s the most abundant biliprotein in all sub complexes. 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 k nown 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 o f the subcomplexes. T his 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 h igh 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 t he 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 th e 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 d ays 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 phyc obilisomes and m ay increase the understanding of the molecular mechanisms underlying their physiological adaptations to environmental condi- tions. 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