PhotosynthesisResearch 48: 163-170, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Cryptomonad biliproteins: Bilin types and locations G a r y J. W e d e m a y e r 1,2, D a n i e l G. K i d d 1'3 & A l e x a n d e r N. G l a z e r 1,* IDepartment of Molecular and Cell Biology, 229 Stanley Hall #3206, University of California, Berkeley, CA 94720-3206, USA; 2present address: Department of Chemistry, University of California, Berkeley, CA 94720, USA; 3present address: SyStemix, Inc., 3155 Porter Drive, Palo Alto, CA 94304, USA; *Author for correspondence Received 17 November1995;acceptedin revisedform 14 February 1996

Key words: energy transfer, light-harvesting pigments, linear tetrapyrrole, photosynthetic antennae

Abstract

Two cryptophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 569 were purified and characterized with respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the a subunit and three on the 3 subunit. Cr-PC 612 carried phycocyanobilin at a-Cys-18, /~-Cys-82, and fl-Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at 3-DiCys-50,61. Cr-PC 569 carried phycocyanobilin at c~-Cys-18 and/3-Cys-82, a singly-linked Bilin 584 at/3-Cys-158, and a doubly-linked Bilin 584 at/3-DiCys-50,61. This work, in conjunction with earlier studies on Cr-PE 545, Cr-PE 555, Cr-PE 566, and Cr-PC 645, shows that there is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no conserved energy transfer pathway common to all native cryptophycean biliproteins. Only phycocyanobilin or phycoerythrobilin is found at fl-Cys-82; there is greater bilin variability at the other three attachment sites.

Abbreviations: Cr-PC-cryptophycean phycocyanin; Cr-PE-cryptophycean phycoerythrin; DBV-15,16dihydrobiliverdin; M B V - mesobiliverdin; PCB - phycocyanobilin; PEB - phycoerythrobilin; H P L C - high performance liquid chromatography; TFA-trifluoroacetic acid Introduction

Cryptomonads are a small group of unicellular, eukaryotic algae; most are photosynthetic, with biliproteins as major photosynthetic antenna pigments (Allen et al. 1959; 0hEocha and Raftery 1959; Haxo and Fork 1959). These chromoproteins are released in watersoluble form upon cell breakage. Each cryptomonad strain contains only a single spectroscopically distinct type of biliprotein. Currently, seven such classes of cryptophycean biliproteins are recognized, and these have been arbitrarily subdivided into two subclasses: cryptophycean phycoerythrins (Cr-PE), which appear reddish due to the presence of at least one greenabsorbing phycoerythrobilin (PEB) chromophore, and cryptophycean phycocyanins (Cr-PC), which appear

bluish due to the presence of red-absorbing phycocyanobilin (PCB) chromophores. No known cryptophycean biliprotein contains both PEB and PCB. The names of the members of each group incorporate their long wavelength absorption maximum in nanometers. Three different Cr-PEs (Cr-PE 545, Cr-PE 555, and CrPE 566) and four distinct Cr-PCs (Cr-PC 569, Cr-PC 612, Cr-PC 630, and Cr-PC 645) have been described (Hill and Rowan 1989; Wedemayer et al. 1991). These biliproteins are isolated as -,~50,000 kDa c~c~'32 complexes (e.g., MOrschel and Wehrmeyer 1975; Sidler et al. 1985; Guard-Friar and MacColl 1986) which carry one covalently bound bilin on the a and three on the 3 subunit. The a subunits are encoded by a small nuclear multigene family (Jenkins et al. 1990), while the/3 subunit is encoded on the chloroplast genome (Dou-

164 glas 1992). The c~ subunits are not obviously related to any known protein (however, see Sidler et al. 1990), whereas the /3 subunits, regardless of spectroscopic type, have highly conserved amino acid sequences which show >80% identity with those of rhodophytan phycoerythrin/3 subunits (Glazer and Apell 1977; Sidler et al. 1990). For a given biliprotein, the nature, number and locations of its bilins serve as the primary determinants of its visible absorption spectrum and of the pathways of energy transfer within it. We have determined the bilin types and locations in four biliproteins, Cr-PE 545, Cr-PE 555, Cr-PE 566 and Cr-PC 645 (Wedemayer et al. 1991, 1992; Wemmer et al. 1993; Wilbanks et al. 1989). These studies led to the determination of the structures of four hitherto undescribed bilin prosthetic groups. In this paper, we describe the bilin types and locations in representatives of two additional types of biliproteins: Falcomonas daucoides Cr-PC 569 and Hemiselmis HP9001 Cr-PC 612. The new data in conjunction with data obtained earlier pose provocative questions on the role of bilin diversity and on the energy transfer pathways in cryptomonad biliproteins.

Phycobiliprotein

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Trypsin d gestion 1

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Materials and methods

The cryptomonad Falcomonas daucoides, a gift from Drs. David Hill and Richard Weatherbee (University of Melbourne, Australia), was cultured in ' K ' seawater medium as described by Wedemayer et al. (1992). Hemiselmis HP9001 was a gift of Dr T.M. Kana (University of Maryland, Horn Point Environmental Laboratories, Cambridge, MD). This cryptomonad was cultured in 'K' seawater medium (Keller et al. 1987) modified by the aseptic addition of sterile-filtered 10 #g thiamine, 50 pg biotin, and 50 #g cyanocobalamin per liter. Hemiselmis HP9001 cells (--~10 g wet weight) in 50 mM Na-phosphate, 1 mM EDTA, 1 mM NAN3, pH 7.0 (referred to henceforth as 'pH 7.0 buffer') were broken by passage through a French press, the cell debris removed by high speed centrifugation, and the supernatant brought to 85% of saturation by the addition of solid (NH4)2504. The biliprotein-containing slurry was collected by centrifugation, suspended in 20 ml pH 7.0 buffer and stirred for 10 min. Insoluble material was removed by centrifugation and aliquots of the supernatant (7 ml) applied to a Sephacryl S-300 HR column (5 x 30 cm) equilibrated in pH 7.0 buffer at a flow rate of 6 ml min - l . Fractions from a single

Figure 1. Flow chartof proceduresfor isolation and characterization of cryptophyceanbiliprotein bilin peptides. Bilins are depicted as geometric symbols.

blue peak, containing Cr-PC 612 (A614 2> 0.2), were pooled, acidified with 6N HC1 to pH 2.5, and applied to a BioRex 70 column (2×9.5 cm; BioRad, Richmond, CA). The column was washed with 5 column volumes of 0.1M Na-phosphate, pH 2.1, and developed by step elution with urea in the same buffer. The a subunit was eluted with 5 M urea and the/3 subunit with 9 M urea. The c~ and/3 subunit containing fractions were pooled, exhaustively dialyzed against water and then against 1 mM HC1 prior to lyophilization. Falcomonas daucoides Cr-PC 569 and its subunits were obtained in a similar manner. All of the instrumentation and peptide isolation and characterization methods used in this study, as well as peptide nomenclature, are described in detail in Wedemayer et al. 1992. A flow chart of the procedure employed for the isolation and characterization of bilin peptides is shown in Figure 1.

165 Results

Characterization ofHemiselmis HP9001 Cr-PC 612 and its a and/3 subunits Native Cr-PC 612 exhibits absorption maxima (relative intensities) at 335 (0.163), 372 (0.158), 578 (0.94), and 614 nm (1.00). (The absorption maximum would require the designation of this protein as Cr-PC 614. To conform with older literature, we have named this protein Cr-PC 612.) Chromatography of the a subunit on a reversed-phase C4 HPLC column (Swanson and Glazer 1990) gave a single chromoprotein peak with absorption maxima at 355 and 662 nm diagnostic of polypeptide-bound PCB. The sequences of the/3 subunits of cryptomonad biliproteins are highly conserved (reviewed in Wedemayer and Glazer 1995) and consistently yield three bilin peptides of predictable size on complete tryptic digestion, /3-1 with the bilin attached at Cys-82,/3-2 with the bilin at Cys- 158, and/3-3 with the bilin doublylinked at Cys-50 and Cys-61 (Wedemayer et al. 1991, 1992). The Cr-PC 612/3 subunit tryptic peptides were separated by gel filtration on Sephadex G-50 in 30% (v/v) aqueous acetic acid. The/3-3 peptide eluted first, followed by/3-2, and then/3-1. The peak fractions for each of these peptides were pooled and then individually repurified by chromatography on a Ca reversedphase column. The absorption spectrum of the purified /3-1 peptide in 10 mM aqueous TFA showed absorption maxima at 348 nm and 650 nm and that of/3-2 at 344 and 647 nm. These spectra corresponded closely to those of PCB-peptides of known bilin structure from other proteins. The amino acid compositions of /3-1 and /3-2 (data not shown) were consistent with the assignment of these as the tryptic peptides containing the/3-Cys 82 and/3-Cys 155 bilin attachment sites based on homology with the sequences of other cryptomonad biliprotein/3 subunits (see Glazer and Wedemayer 1995). Upon chromatography on a C4 reversed-phase column, the /3-3 chromopeptide was resolved into two peaks with distinctive absorption spectra, pink and purple, with very similar amino acid compositions that matched the/3-DiCys-50,61 attachment site. The pink /3-3 fractions turned purple on standing in room light and exhibited a spectrum similar to that of the purple fraction. Reapplication to the Ca column again resulted in separation of the forms. The absorbance spectrum of the photostationary state attained in daylight had maxima at 339 and 591 nm with a broad shoulder at 562

nm. A similar photoconvertible peptide, the Cr-PC 645 /3-DiCys-50,61 15,16-dihydrobiliverdin (DBV) chromopeptide described by Wedemayer et al. (1992), has peaks at 331 and 562 nm and a shoulder at 590 nm. The small difference most likely represent slightly different proportions of the pink and purple conformers. The studies on Hemiselmis HP9001 Cr-PC 612 indicate that the a subunit carries PCB and that the /3 subunit carries PCB at Cys-82 and Cys-158 and a DBV doubly-linked at Cys-50 and Cys-61.

Spectroscopic characteristics of Falcomonas daucoides Cr-PC 569 and its a and/3 subunits Native Cr-PC 569 in pH 7.0 buffer shows absorption maxima at 380, 570, and 637 nm with relative intensities of 0.14, 1.0, and 0.71, respectively. The a and/3 subunits were separated by chromatography on BioRex 70 with an acid urea step gradient (Glazer and Fang 1973) with the a subunits eluting in 6.5 M urea and the/3 subunits at 9 M. The absorption maxima (relative intensities) of the a subunit were at 356 (1.00) and 665 nm (0.96) while those of the /3 subunit were at 311 (0.42), 350 (0.41), and 593 nm (1.00).

Characterization of the bilin peptide from Cr-PC 569 a subunit Cr-PE 569 a subunit was digested with Pronase and the peptides fractionated by C l8 reversed-phase HPLC. One major bilin peptide was obtained. This peptide, (FAD) a-Cys- 18-Pr 1, was repurified to apparent homogeneity by HPLC and found to have an amino acid composition Cys, Gly and an absorption spectrum with two intense maxima at 346 and 640 nm in 10 mM aqueous TFA (Figure 2A). This spectrum is diagnostic of a thioether-linked PCB.

Fractionation and characterization of Cr-PC 569 fl subunit tryptic peptides The tryptic peptides of CropC 569/3 subunit were fractionated by gel filtration on Sephadex G-50 in 30% (v/v) acetic acid. The chromopeptides eluted as two well-separated broad peaks designated as Pool I and Pool II. The Pool I peptides were subjected to sequential Pronase and pepsin degradation and fractionated on a C18 reversed-phase HPLC column using a sodium phosphate/acetonitrile solvent system. Only two major chromopeptide peaks were seen in the elution pro-

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Cryptomonad biliproteins: Bilin types and locations.

Two crytophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 69 were purified and characterized with resp...
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