Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.     

Phycobiliprotein-bilin linkage diversity. II. Structural studies on A- and D-ring-linked phycoerythrobilins

The discovery that the phycocyanobilin group attached to Cys-155 of the beta subunit of C-phycocyanin is D-ring linked (Bishop, J. E., Lagarias, J. C., Nagy, J. O., Schoenleber, R. W., Rapoport, H., Klotz, A. V., and Glazer, A. N. (1986) J. Biol. Chem. 261, 6790-6796) prompted examination of the linkage mode for phycoerythrobilin (PEB) groups attached at the corresponding position in other biliproteins. Appropriate small peptides were obtained by exhaustive enzymatic digestion of Porphyridium cruentum R-phycocyanin (peptide R-PC beta-2TP PEB) and B-phycoerythrin (peptide B-PE beta-2TP PEB). These peptides had the following structures R-PC beta-2TP PEB Gly-Asp-Cys(PEB)-Ser-Ser B-PE beta-2TP PEB Cys(PEB)-Thr-Ser. The spectroscopic and chemical properties of these peptides were compared with those of P. cruentum B-phycoerythrin peptide alpha-1 PEB, Cys(PEB)-Tyr-Arg, in which the bilin is A-ring linked (Schoenleber, R. W., Leung, S.-L., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072-4076). The PEB groups in peptides R-PC beta-2TP PEB and B-PE beta-2TP PEB were shown to be D-ring linked on the basis of the following criteria. Secondary ion mass spectrometry showed the bilins in these peptides and in alpha-1 PEB to have the same mass. The 18'-CH3, 18'-H, and 15-H resonances in the 1H NMR spectra of R-PC beta-2TP PEB and B-PE beta-2TP PEB appear significantly upfield from the corresponding thioether-linked ring A resonances seen in the spectrum of peptide alpha-1 PEB. The CD spectra of the two former peptides showed a strong positive Cotton effect at 300 nm. Such a Cotton effect is absent from the CD spectrum of peptide alpha-1 PEB and those of other A-ring-linked PEB peptides. Refluxing in methanol led to a near-quantitative release of PEB from alpha-1 PEB but no release from R-PC beta-2TP PEB and less than 20% release from B-PE beta-2TP PEB. In conjunction with earlier studies, these results show that distinctive amino acid sequences are found about the attachment sites for A-ring-linked, D-ring-linked, and dilinked (A- and D-ring-linked) bilins on the alpha and beta subunits of cyanobacterial and red algal phycobiliproteins and that the mode of linkage can be correctly predicted from inspection of the amino acid sequence.     

Exclusive A-ring linkage for singly attached phycocyanobilins and phycoerythrobilins in phycobiliproteins. Absence of singly D-ring-linked bilins

Previous spectroscopic studies on the phycocyanobilin-containing peptide beta-2T from Synechococcus sp. 6301 C-phycocyanin and the phycoerythrobilin-containing peptide beta-2TP from Porphyridium cruentum B-phycoerythrin indicated a different single thioether mode of attachment, postulated to be through the D-ring of the tetrapyrrole, in contrast to the A-ring linkage established for the other singly linked bilins in these proteins (Bishop, J.E., Lagarias, J.C., Nagy, J. O., Schoenleber, R.W., Rapoport, H., Klotz, A.V., and Glazer, A.N. (1986) J. Biol. Chem. 261, 6790-6796; Klotz, A.V., Glazer, A.N., Bishop, J.E., Nagy, J.O., and Rapoport, H. (1986) J. Biol. Chem. 261, 6797-6805). The crystal structure of Agmenellum quadruplicatum C-phycocyanin at 2.5-A resolution (Schirmer, T., Bode, W., and Huber, R. (1987) J. Mol. Biol., 196, 677-695) supports an A-ring linkage for all three phycocyanobilins. Consequently we have re-evaluated our proposed structural assignments by further 1H NMR studies. Two-dimensional homonuclear correlated and nuclear Overhauser enhancement spectroscopic data presented here show that all three bilins in Synechococcus 6301 C-phycocyanin are attached solely through the A-ring, complementary to the crystallographic data. The evidence from the NMR data for all bilin peptides examined includes the dipoledipole interactions of the 5-H with the 3-H, 3'-H, and a pyrrole methyl group (7-CH3); the corresponding interactions would not be possible in a D-ring-linked bilin. The 5-H also consistently exhibits allylic J-coupling to the 3-H, supporting A-ring linkage assignment. These data are inconsistent with the alternative D-ring linkage assignment since this would involve J-coupling through five bonds. Examination of the phycoerythrobilin beta-2 position in B-phycoerythrin also reveals an A-ring type of attachment by similar criteria. We conclude that all singly linked bilins are attached through the A-ring.     

Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins

Marine Synechococcus strains WH8103, WH8020, and WH7803 each possess two different phycoerythrins, PE(II) and PE(I), in a weight ratio of 2-4:1. PE(II) and PE(I) differ in amino acid sequence and in bilin composition and content. Studies with strain WH7803 indicated that both PE(II) and PE(I) were present in the same phycobilisome rod substructures and that energy absorbed by PE(II) was transferred to PE(I). Strain WH8103 and WH8020 PE(I)s carried five bilin chromophores thioether-linked to cysteine residues in sequences homologous to those previously characterized in C-, B-, and R-PEs. In contrast, six bilins were attached to strain WH8103 and WH8020 PE(II)s. Five of these were at positions homologous to bilin attachment sites in other phycoerythrins. The additional bilin attachment site was on the alpha subunit. The locations and bilin types in these PE(s) and in the marine Synechocystis strain WH8501 PE(I) (Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9528-9534) are: (table; see text) Since phycourobilin (PUB) (lambda max approximately 495 nm) transfers energy to phycoerythrobilin (PEB) (lambda max approximately 550 nm), inspection of these data shows that the invariant PEB group at beta-82 is the terminal energy acceptor in phycoerythrins. The adaptations to blue-green light, high PUB content and the presence of an additional bilin on the alpha subunit, increase the efficiency of light absorption by PE(II)s at approximately 500 nm.     

Phycobiliprotein-bilin linkage diversity. I. Structural studies on A- and D-ring-linked phycocyanobilins

Phycocyanobilin (PCB) peptides alpha-1 PCB and beta-2T PCB were obtained by proteolytic degradation of Synechococcus 6301 C-phycocyanin. These peptides were found to have the following sequences. alpha-1 PCB Cys(PCB)-Ala-Arg beta-2T PCB Ile-Thr-Gln-Gly-Asp-Cys(PCB)-Ser-Ala. The peptides were examined by 1H NMR, circular dichroism spectroscopy, and secondary ion mass spectrometry. The 1H NMR data confirmed that in each case the bilin was attached through a single linkage, a thioether bond between the cysteinyl residue and the tetrapyrrole moiety. Comparison of the 1H NMR spectra of these peptides with those of appropriate model compounds showed that the thioether linkage in alpha-1 PCB was to the C-3' position and that in beta-2T PCB to the C-18' position on the bilin. Refluxing in neutral methanol under nitrogen led to the release of PCB from alpha-1 PCB but did not release the D-ring-linked tetrapyrrole from beta-2T. The above results together with those of an earlier study (Lagarias, J. C., Glazer, A. N., and Rapoport, H. (1979) J. Am. Chem. Soc. 101, 5030-5037) complete the determination of the mode of linkage of each of the three bilins on C-phycocyanin; two are linked through ring A and one through ring D. This is the first documented report of a singly D-ring-linked bilin.     

In vitro attachment of bilins to apophycocyanin. II. Determination of the structures of tryptic bilin peptides derived from the phycocyanobilin adduct

In vitro reaction of phycocyanobilin (PCB) with apophycocyanin results in the specific addition of the bilin to two of the cysteinyl residues, alpha-Cys-84 and beta-Cys-82, which normally function in PCB attachment (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). These bilin binding sites are designated alpha-1 and beta-1, respectively. Tryptic digestion of the apophycocyanin-PCB adduct releases two major bilin peptides, alpha-1 mesobiliverdin (MBV) and beta-1 MBV, which encompass the two bilin-binding sites. These peptides were examined by 1H NMR and fast atom bombardment mass spectroscopies. The NMR spectra show that the bilin is attached to each peptide through a thioether linkage identical to the linkage observed in the corresponding tryptic peptides, alpha-1 PCB and beta-1 PCB, derived from the natural product, C-phycocyanin. However, the NMR spectra of the adduct peptides lack the resonances corresponding to protons at positions C2 and C3 of ring A seen in the spectra of the alpha-1 PCB and beta-1 PCB peptides. Fast atom bombardment mass spectroscopy shows the masses of the alpha-1 MBV and beta-1 MBV peptides to be 2 atomic mass units lower than those of the alpha-1 PCB and beta-1 PCB peptides, respectively. Comparison of the bilin portion of the NMR spectra of the alpha-1 MBV and beta-1 MBV peptides to the NMR spectra of PCB and mesobiliverdin confirms that the bilin of the two adduct peptides resembles mesobiliverdin in having an extra double bond in the C2-C3 position of ring A. These results show that the major bilin products arising from the reaction of PCB with apophycocyanin differ from the bilins present in C-phycocyanin. The relevance of these results to the biosynthetic pathway for the attachment of tetrapyrroles to phycobiliproteins is discussed.     

Posttranslational modifications of the beta subunit of a cryptomonad phycoerythrin. Sites of bilin attachment and asparagine methylation

Determination of the partial amino acid sequence of the beta subunit of cryptomonad strain CBD phycoerythrin 566 established the nature, locations, and modes of attachment of the three bilin prosthetic groups and revealed a site of posttranslational methylation. Isolation of peptides cross-linked by a phycobiliviolin led to an unambiguous assignment of two thioether linkages, from residues beta-Cys-50 and beta-Cys-61 to this bilin. Two bilins were attached through single thioether linkages, a phycobiliviolin at beta-Cys-158 and a phycoerythrobilin at beta-Cys-82 (the residue numbering is that for B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Morisset, W., Wehrmeyer, W., and Zuber, H. (1985) Biol. Chem. Hoppe-Seyler 366, 233-244). The partial sequences (99 residues) established for phycoerythrin 566 beta subunit showed a 79% identity with that of the red algal Porphyridium cruentum B-phycoerythrin beta subunit. A particularly remarkable finding is that the unique methylasparagine residue at position beta-72, highly conserved in cyanobacterial and red algal phycobiliproteins (Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chem. 262, 17350-17355), is also present at beta-72 in the cryptomonad phycoerythrin. Comparison of the locations of donor and acceptor bilins in cryptomonad phycoerythrin with those found for cyanobacterial and red algal phycobiliproteins showed different favored pathways of energy migration in the cryptomonad protein.     

Characterization of the bilin attachment sites in R-phycoerythrin

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.     

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.     

In vitro attachment of bilins to apophycocyanin. III. Properties of the phycoerythrobilin adduct

Addition of phycoerythrobilin (PEB) to apophycocyanin at pH 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues alpha-Cys-84 and beta-Cys-82 to be the sites of bilin addition. The product of PEB addition at the alpha-Cys-84 site was shown by 1H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the beta-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a lambda max of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins.     

R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins

A new member of the phycocyanin family of phycobiliproteins, R-phycocyanin II (R-PC II) has been discovered in several strains of marine Synechococcus sp. R-PC II has absorption maxima at 533 and 554 nm, a subsidiary maximum at 615 nm, and a fluorescence emission maximum at 646 nm. It is the first phycoerythrobilin (PEB)-containing phycocyanin of cyanobacterial origin. The purified protein is made up of alpha and beta subunits in equal amounts and is in an (alpha beta)2 aggregation state. The alpha and beta subunits of this protein are homologous to the corresponding subunits of previously described C- and R-phycocyanins as assessed by amino-terminal sequence determination and analyses of sequences about sites of bilin attachment. R-PC II carries phycocyanobilin (PCB) at beta-84 and PEB at alpha-84 and beta-155 (residue numbering is that for C-phycocyanin), whereas in C-phycocyanin PCB is present at all three positions. In R-phycocyanin, the bilin distribution is alpha-84 (PCB), beta-84 (PCB), beta-155 (PEB). In both R-phycocyanin and R-phycocyanin II excitation at 550 nm, absorbed primarily by PEB groups, leads to emission at 625 nm from PCB. These comparative data support the conclusion that the invariant beta-84 PCB serves as the terminal energy acceptor in phycocyanins.     

Molecular architecture of a light-harvesting antenna. Quaternary interactions in the Synechococcus 6301 phycobilisome core as revealed by partial tryptic digestion and circular dichroism studies

The core of the phycobilisomes of Synechococcus 6301 (Anacystis nidulans) strain AN112 consists of two cylindrical elements each made up of the same four distinct subcomplexes: A (alpha AP beta AP)3; B (alpha AP beta AP)2 . 18.3K . 75K; C (alpha 1APB alpha 2AP beta 3AP) . 10.5K; and D (alpha AP beta AP)3 . 10.5K, where alpha AP and beta AP are the subunits of allophycocyanin, alpha APB is the subunit of allophycocyanin B, and 18.3K, 75K, and 10.5K are polypeptides of 18,300, 75,000, and 10,500 Da, respectively. An 18 S subassembly containing subcomplexes A and B has previously been characterized (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086; Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901, 902-908). A ternary core subassembly, containing complexes A, B, and C, was isolated from a limited tryptic digest of AN112 phycobilisomes and characterized with respect to composition and spectroscopic properties. Isolation of this ternary subassembly also establishes that subcomplex D must occupy a terminal position in each of the two core cylinders. Spectroscopic studies of the individual complexes, A-D, of the subassemblies AB and ABC, and of intact AN112 phycobilisomes showed core assembly-dependent changes in the circular dichroism spectra indicative of changes in the environment and/or conformation of the bilin chromophores within the individual subcomplexes. Two terminal energy acceptors are present in the phycobilisome core, alpha APB and 75K. No indication of interaction between the chromophores on these polypeptides was detected by circular dichroism spectroscopy. This result indicates that the bilins on alpha APB and 75K act as independent energy acceptors rather than as exciton pairs.     

Chromophore composition of the phycobiliprotein Cr-PC577 from the cryptophyte Hemiselmis pacifica

The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αβ)(α′β) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the β-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position β-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position β-Cys-50 and β-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the β-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.     

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 respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the subunit and three on the subunit. Cr-PC 612 carried phycocyanobilin at -Cys-18, -Cys-82, and -Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at -DiCys-50,61. Cr-PC 569 carried phycocyanobilin at -Cys-18 and -Cys-82, a singly-linked Bilin 584 at -Cys-158, and a doubly-linked Bilin 584 at -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 -Cys-82; there is greater bilin variability at the other three attachment sites.Abbreviations Cr-PC cryptophycean phycocyanin - Cr-PE cryptophycean phycoerythrin - DBV 15,16-dihydrobiliverdin - MBV mesobiliverdin - PCB phycocyanobilin - PEB phycoerythrobilin - HPLC high performance liquid chromatography - TFA trifluoroacetic acid     

Green colouration of cocoons in Antheraea yamamai (Lepidoptera: Saturniidae): light-induced production of blue bilin in the larval haemolymph

When the larvae of a saturniid silkmoth, Antheraea yamamai, are maintained under high intensity light (5000 lux), they produce green cocoons whereas the cocoons produced under light of low intensity (e.g., 50 lux) or in darkness are yellow. The green colour of the cocoon is due to the presence of a blue bilin pigment in combination with yellow pigment, and light stimulates the accumulation of blue bilin. In the present study, we show that two blue bilins, with similar characteristics to the sarpedobilin in the green cocoon, can be induced in larval haemolymph both in vivo and in vitro. In both conditions, the amount of these bilins increased with increasing intensity or duration of light exposure. Induction also occurred at 0 degrees C. In contrast, the chromophore of the constitutive biliprotein of the haemolymph did not change depending on light conditions. Size fractionation of the haemolymph indicates that the precursor of the blue bilins induced by light is bound to a protein with a molecular mass of 5000 Da or more. Thus, in these insects, the blue bilin responsible for green colouration is facultative under photochemical stimulation.     

gamma-N-methylasparagine in phycobiliproteins. Occurrence, location, and biosynthesis

The novel post-translationally modified residue gamma-N-methylasparagine, previously detected in the beta subunit of allophycocyanin (Klotz, A. V., Leary, J. A., and Glazer, A. N. (1986) J. Biol. Chem. 261, 15891-15894), has been found in the beta subunits of a variety of other phycobiliproteins. Representatives of C- and R-phycocyanins and B-, C-, and R-phycoerythrins all contain 1 eq of gamma-N-methylasparagine on their beta subunits as judged by the presence of methylamine in acid hydrolysates. Radiotracer experiments show that the methyl group is derived from the S-methyl of methionine, implicating S-adenosylmethionine as an intermediate methyl transfer agent. Isolation of peptides from C-phycocyanins, prepared from cells labeled by L-[methyl-14C]methionine, showed that the gamma-N-methylasparagine residue is at position beta-72, within a highly conserved region in phycobiliproteins. This location corresponds to that reported earlier for the position of gamma-N-methylasparagine in allophycocyanin and R-phycoerythrin. Phycobiliprotein alpha subunits contain insignificant amounts of the adduct. Methylamine is absent from the hydrolysates of the beta subunits or alpha beta monomers of phycobiliproteins from certain organisms. These latter data indicate that the gamma-N-methylasparagine residue is dispensable in some circumstances. The function of this modification remains to be established. gamma-N-methylasparagine was also absent from several other proteins including bovine histones, porcine myelin basic peptide, and the Salmonella typhimurium aspartate chemoreceptor, all known to undergo post-translational methylations.     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.