phycoerythrins of marine unicellular cyanobacteria

THE JOURNAL (c? 1991 by The American Society for Biochemistry and

OF BIOLOGICAL CHEMISTRY Molecular Biology, Inc.

Vol. 266. No . 15, Issue of May 25, pp. 9515-9527,1991 Printed in U. S. A.

Phycoerythrins of Marine Unicellular Cyanobacteria I. BILIN TYPES AND LOCATIONS AND ENERGY TRANSFER PATHWAYS IN SYNECHOCOCCUS SPP.

PHYCOERYTHRINS*

(Received for publication, November 5, 1990)

Linda J. OngS and Alexander N. Glazer$ From the Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720

Marine Synechococcus strains WH8103, WH8020, and WH7803 each possess two different phycoerythrins, PE(I1) and PE(I), in a weight ratio of 2-4:l. PE(I1) and PE(1) differ in amino acid sequence and in bilin composition and content. Studies with strain WH7803 indicated that both PE(I1) and PE(1) were present in the same phycobilisome rod substructures and that energy absorbed by PE(I1) was transferred to PE(1).

Strain WH8103 and WH8020 PE(1)s carried five bilin chromophores thioether-linked to cysteine residues in sequences homologous to those previously char- acterized in C-, B-, and R-PES. In contrast, six bilins were attached to strain WH8103 and WH8020 PE(1I)s. Five of these were at positions homologous to bilin attachment sites in other phycoerythrins. The additional bilin attachment site was on the (Y subunit. The locations and bilin types in these PE(s) and in the marine Synechocystis strain WH8501 PE(1) (Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9528-9534) are:

a-75 a-83 a- &50,61 8-82 8-159 140

WH8020 PE(1) PEB PEB PEB PEB PEB WH8020 PE(I1) PUB PEB PEB PUB PEB PEB WH8103 PE(1) PEB PUB PUB PEB PEB WH8103 PE(I1) PUB PUB PUB PUB PEB PEB WH8501 PE(1) PUB PUB PUB PEB PUB

Since phycourobilin (PUB) (X,,, -495 nm) transfers energy to phycoerythrobilin (PEB) (X,,, - 550 nm), inspection of these data shows that the invariant PEB group at ,842 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 a subunit, increase the efficiency of light absorption by PE(1I)s at -600 nm.

Two types of protein-pigment complexes are present in all

* This research was supported in part by National Science Foun- dation Grant DMB 8816727, National Institute of General Medical Sciences Grant GM28994, and by the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$’ Recipient of a predoctoral fellowship from the Dept. of Health and Human Services Training Grant 5 T 32 GM 07232. Present address: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305.

§ To whom correspondence and reprint requests should be ad- dressed MCB: Stanley/Donner ASU, 229 Stanley Hall, University of California, Berkeley, CA 94720. Tel.: 415-642-3126. Fax: 415-643- 9290.

oxygen-evolving photosynthetic organisms, from bacteria such as the cyanobacteria and prochlorophytes to higher plants. Reaction centers function both in the trapping of radiant energy and its conversion to electron flow, while accessory light-harvesting complexes (“antenna” complexes) absorb light over a wide range of wavelengths and convey the excitation quanta to the reaction centers. The function of the latter complexes is to increase the absorption cross-section of the reaction centers. The ratio of antenna complexes to reaction centers varies with the intensity and quality of the ambient radiation. The variation in the content of antenna complexes as well as modulation of their pigment composition permit photosynthetic organisms to adjust to and utilize efficiently light of varying intensity and wavelength distribution (1).

Whereas all reaction centers in oxygen-evolving photosynthetic organisms utilize chlorophyll a, the antenna pigments vary widely (2,3; Fig. 1). In cyanobacteria (“blue-green algae”) and in two groups of eukaryotic algae, the red algae and the cryptomonads, the antenna pigments are a family of phycobiliproteins in which open chain tetrapyrroles (bilins) are covalently attached to polypeptides (5-8). In intact cyanobacterial cells and red algal chloroplasts, the phycobiliproteins are components of a large macromolecular complex of 7 X IO6 to 15 x lo6 daltons, the phycobilisome (9-11). All phycobilisomes contain allophycocyanin (X,,, 650 nm) and some type of phycocyanin (X,,, - 620 nm). Many contain either a phycoerythrin (X,,, 565 nm), or phycoerythrocyanin (X,,, 568 nm), as well. The nature and arrangement of the bilins in the individual phycobiliproteins as well as the arrangement of the various phycobiliproteins in the phycobilisome is such as to ensure a directional transfer of excitation energy from any point within this macromolecular complex toward the reaction center (12).

Four isomeric tetrapyrroles (bilins), linked to the polypeptides through thioether linkages, function as the visible light- harvesting chromophores of cyanobacterial and red algal phycobiliproteins (see Ref. 12 for structures and modes of link- age). The spectroscopic properties of these tetrapyrroles are primarily a function of their covalent structure but are strongly influenced by their conformation and environment within the native phycobiliproteins (12). For example, phycocyanobilin (PCB)’ gives rise to an absorption maximum at 650 nm in trimeric allophycocyanin and at 620 nm in trimeric C-phycocyanin. Phycobiliviolin (PXB)-containing phycobiliproteins show a peak a t 568 nm, whereas those containing

The abbreviations used are: PCB, phycocyanobilin; PE, phycoerythrin; PEB, phycoerythrobilin; PXB, phycobiliviolin (cryptoviolin); PUB, phycourobilin; PMSF, phenylmethylsulfonyl fluoride; PTH, phenylthiohydantoin; TPCK, ~-l-tosylamido-2-phenylethyl chloro- methyl ketone; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; E, einstein.

9515

9516 Bilin Locations and Energy C ._

400 500 600 700 Wavelength(nm)

FIG. 1. The graph shows the visible absorption spectrum for highly purified water. The shaded areas rising from the water absorption coefficient curve are schematic representations (not to scale) of the prominent features of the absorption spectra of various photosynthetic accessory pigments in living cells. (Based on Fig. 3.2 of Ref. 4.)

phycoerythrobilin (PEB) have absorption maxima between 535 and 567 nm. Phycourobilin (PUB)-containing proteins show a sharp absorption peak (or shoulder, depending on overall bilin composition and content) at about 495 nm.

The pathways of energy transfer in C-phycocyanin were established by a combination of x-ray crystallographic and spectroscopic studies (see “Discussion”). C-Phycocyanin carries three PCB groups per ap monomer. A more complex situation exists in C-, B-, and R-phycoerythrins which carry five bilins per a@. Crystals of these proteins suitable for x-ray diffraction have yet to be obtained. Moreover, the presence of multiple bilins with very similar spectroscopic properties com- plicates identification of energy donors and acceptors. This difficulty is readily overcome in proteins which contain bilins with distinctive absorption spectra. Bilins which absorb at shorter wavelengths function as donors of excitation energy to those that absorb at longer wavelengths. In this study, we exploit the unusual diversity of bilin composition in the phycoerythrins of recently discovered unicellular marine cyanobacteria to identify unambiguously the terminal acceptor bilin in phycoerythrins.

B- or R-phycoerythrins are the most abundant phycobiliproteins in marine red algae (13, 14). Many areas of the oceans, particularly coastal waters, contain ill-defined yellow organic material called “Gelbstoff‘ which absorbs short wavelength visible light. When absorption by Gelbstoff and scat- tering by particulates are taken into account together with the wavelength dependence of the absorption of light by pure water (Fig. l) , transmittance through seawater is observed to be at a maximum near 500 nm. B- and R-Phycoerythrins absorb most strongly in the visible region of the spectrum between 480 and 560 nm and enable red algae to utilize green

Transfer in Phycoerythrins

light efficiently for photosynthesis. Until recently, filamentous Oscillatoria sp. (previously clas-

sified as Trichodesmium), rich in phycoerythrin, were believed to be the only major cyanobacteria in oceanic phytoplankton communities (see Ref. 15 for a review). In 1979, unicellular cyanobacteria were discovered to be widespread and significant members of the marine picoplankton community of the open oceans (16, 17). The majority of these organisms, cur- rently classified as Synechococcus spp., are small coccoid cells, 0.6-0.8 X 0.6-1.7 pm (18). The diagnostic hallmarks of these cyanobacteria are their size and their distinctive intense or- ange-red fluorescence easily distinguishable from the dull red emission due to chlorophyll-containing complexes. It was this characteristic emission, consistent with a high phycoerythrin content, which led to the detection of these organisms by epifluorescence microscopy (for reviews see Refs. 15 and 18). The contributions of the marine unicellular cyanobacteria to primary productivity in the oceans range from 5 to 25% depending on geographic location and season (18). These organisms may also be important contributors to the marine nitrogen cycle (15).

The name Synechococcus is applied to a provisional assemblage of unicellular coccoid to rod-shaped cyanobacteria that divide by binary fission in a single plane. The marine Syne- chococcus sp. now number some 100 strains maintained primarily in the Woods Hole Oceanographic Institute culture collection (19). The strains used in this study belong to marine cluster A. This cluster includes strains isolated from both coastal waters and the open ocean with DNA G + C contents from 52 to 62 mol %. All strains in this cluster are obligate photoautotrophs incapable of using organic compounds as sole sources of carbon, and all have elevated growth require- ments for Na+, C1-, Mg2+, and Ca2+ that are characteristic of marine bacteria. The phycoerythrins in members of this strain cluster show considerable spectral diversity resulting from the wide variation in the ratio of PEB to PUB in the individual chromoproteins (18, 20-23). The spectral diversity among phycoerythrins of marine Synechococcus sp. is comparable to that seen among over 150 phycoerythrins from all of the genera of red algae studied to date (13, 14).

The studies presented in this and the companion papers (24, 25) define at the molecular level novel features whereby the phycobilisomes of the unicellular marine cyanobacteria are optimized for the absorption of green light. The marine Synechococcus strains selected for this study each produce two different phycoerythrins. The determination of the types and locations of all of the bilins in several of these proteins permits assignment of the nature and location of the terminal acceptor bilin in phycoerythrins.

EXPERIMENTAL PROCEDURES AND RESULTS’

DISCUSSION

Energy Transfer and Bilin Locations in Phycobiliproteins- Cyanobacterial and red algal phycobiliproteins are all made up of a and @ subunits. The amino acid sequences of the corresponding subunits of different phycobiliproteins are strongly homologous. The association of a@ units of different phycobiliproteins with their specific linker polypeptides (y subunits) gives rise to higher order assemblies which have similar tertiary and quaternary structures. The unique func-

Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 2-14, and Tables I-XJX) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Bilin Locations and Energy Transfer in Phycoerythrins

tional characteristics of a particular phycobiliprotein are largely defined by the type, number, and location of bilin prosthetic groups it carries (12).

The crystal structures of two phycobiliproteins, C-phycocyanin (49,50) and phycoerythrocyanin (51), have been solved at high resolution. The tertiary and quaternary structures of these two phycobiliproteins are remarkably similar. Each of these proteins carries three bilin chromophores, one on the CY

subunit, at a-84, and two on the 8 subunit at 8-82 and p-155. Detailed spectroscopic studies of C-phycocyanin established that 8-82 PCB acts as the terminal energy acceptor in this protein (52, 53). This result, together with the data on the structure of crystals of hexameric C-phycocyanin and exten- sive information on the structure of phycobilisomes, has led to a model of the energy transfer pathways in phycobilisomes (reviewed in Ref. 12). The model assumes that in the disc- shaped trimeric and hexameric phycobiliproteins, which make up the phycobilisome rods, the excitation energy is transferred from peripheral bilin chromophores such as a-84 (PCB) and 8-155 (PCB) to the terminal acceptor bilins at ,842 located near the center of the discs. Subsequently, the excitation migrates down the rod from one p-82 chromophore to the next to the acceptors in the phycobilisome core. The linchpin in such a general model of directional energy transfer is the assumption that a unique energy acceptor bilin is present in each rod component phycobiliprotein and that the location of the terminal energy acceptor is conserved among different phycobiliproteins. Whereas phycocyanins carry only three bilins, C-, B-, and R-phycoerythrins all carry five bilins per (up. The validity of the assumption of a single terminal acceptor bilin located at the position corresponding to 8-82 cannot be assumed a priori for these proteins.

There are alternative approaches to the identification of donor and acceptor bilins in phycobiliproteins. One approach is to crystallize representatives of each major class of phycobiliproteins and determine the location of the terminal acceptor bilin from spectroscopic studies on single crystals, as previously accomplished for C-phycocyanin. The assignment becomes more difficult as the number of bilins with similar spectroscopic properties per a8 unit increases to five. Thus far, attempts to obtain crystals of phycoerythrins suitable for high resolution x-ray diffraction analysis have been unsuc- cessful. The second approach is to exploit the finding that marine cyanobacteria and red algae which live in light regimes with distinctive wavelength distribution (Fig. 1) possess phycobiliproteins whose different bilin compositions lead to op- timum utilization of the available light (18, 19, 23, 54-57).

Chemically different bilins have distinctive absorption properties. Consequently, determination of the locations and types of bilins in a phycobiliprotein which carries more than one type of bilin provides unambiguous information on the positions of energy donors and acceptors. We have previously successfully exploited this approach to establish the location of the terminal acceptor bilin in the phycocyanin family of proteins whose members include C-phycocyanin, phycoerythrocyanin, R-phycocyanin, and R-phycocyanin I1 (38). As shown in Fig. 15, donor bilins can be present at either (u-84 or 8-155, or at both a-84 and p-155, but an acceptor bilin invariably occupies the 8-82 position.

The question of the terminal acceptor bilin in phycoerythrins is examined in this study by exploiting the extraordi- nary prosthetic group diversity in the phycoerythrins of the marine unicellular cyanobacteria. At the same time, this work provides valuable insights into the molecular adaptations through which the photosynthetic antenna pigments of these organisms are optimized for the absorption of -500 nm light.

PHYCOCYANINS

C-Phycocyanin

R-Phycocyanin

F'hycocrythmyanin

R-Phycocyanin I1

PHYCOF.RYTmINS

C-PE

B-PE

R-PE

WH8020 PE(Q

WH8020 PE(U)

WH8103 PE(n

WH8103 PE(U)

WH8501 PE(I1

a-84 (a-11

PCB

PCB

PXB

mB

a-75 a-83 (a-3) (a-I)

PEB

PEB

PEB

PEB

PUB PEB

PEB

PUB PUB

PUB

8-62 (3-11

PCB

PCB

PCB

PCB

a-140 8-50,61 8-82 (a-21 (3-31 (3-1)

PEB PEB PEB

PEB PEE PEB

PBB PUB PBB

PBB PEB PEB

PEB PUB PEB

PUB PUB PEB

PWl PUB PEB

PUB PWB PEB

9517

p-155 (P-21

PCB

PBB

PCB

PNB

8-159 (P-2)

PEB

PEB

PEB

PEB

PEB

PBB

PEB

PUB

FIG. 15. Location of terminal acceptor bilin in phycocyanins and phycoerythrins. The sites of attachment of the bilins in WH8020 PE(1) and PE(I1) and WH8103 PE(1) and PE(I1) are from this study, those in WH8501 PE(1) are from Ref. 24. The sources of data for the other sequences are as follows: C-PE, Ref. 45; B-PE, Refs. 46 and 47; R-PE, Ref. 48. For each protein, bilins which must serve as donor chromophores are outlined. Terminal energy acceptor bilins are shown in larger boldface font. Residue numbering is based on the DNA sequence encoding WH8020 PE(I1) (25).

a-SUBUNIT

WaSl03PE(II) - K S V I T T V V G A A D S A S R F P S A S - M E S

WE8103 P E G ) M X S V V T T V V - A A D C-PE M K S V V T T V I A A A D A A G R F P S T S D L E S

B-PE M K S V I T T V V S A A D A A G R F P S N S D L E S

8-SUBUNIT

WE8103PE(II) - L D A F S R A A V S A D S S G S F I G G - E L A

WESl03PE(I) M L D A F S R T V V S A D A X T

C-PE M L D A F S R A V V S A D A S T S T V S D I A A L

B-PE M L D A F S R V V V N S D A X A A Y V G G S D L Q

FIG. 16. Comparison of the amino-terminal sequences of the a and P subunits of WH8103 PE(1) and PE(I1) with those of C-PE (45) and B-PE (47). --, unassigned residues.

Synechococcus strains WH8103, WH8020, and WH7803 were chosen for this study because the A4~~5n,n:Ah~, ,nm ratios in the absorption spectra of whole cells and of isolated phycobilisomes (18, 23) indicated that the phycoerythrins of these strains spanned the wide range of PUB:PEB ratios available in strains assigned to this provisional assemblage of marine cyanobacteria (19).

Examination of the phycoerythrins from the three Syne- chococcus strains led to two novel observations. First, the phycobilisomes of each organism contained two phycoerythrins, in unequal amount, which differed from each other in amino acid sequence (Figs. 12-14 and 16). Second, the two phycoerythrins differed in bilin composition and content (Table I; Fig. 16). This is an unprecedented finding. C-Phy- coerythrin is a prominent phycobilisome component in many fresh water cyanobacteria (58). This protein has been purified to homogeneity from numerous cyanobacteria and the amino acid sequence of the protein from the cyanobacterium Fre- myella diplosiphon (Calothrix sp. PCC7601) has been determined (45). A single phycoerythrin is present in all in.stances. The genes encoding the N and 8 subunits of the phycoerythrins of F. diplosiphon (59), Pseudoanabaena sp. PCC7409 (60), and Synechocystis sp. PCC6701 (61) have been cloned

9518 Bilin Locations and Energy Transfer in Phycoerythrins

and sequenced. In each instance, only a single set of structural genes was found. The 01 and p subunits of the red algal B- phycoerythrin from Porphyridium cruentum have unique amino acid sequences (46,47). No evidence for multiple (Y and p subunits was seen in a detailed study of all of the tryptic bilin peptides from the R-phycoerythrin of the seaweed Gas- troclonium coulteri (48).

The two phycoerythrins, designated PE(1) and PE(II), from each marine Synechococcus strain differ from each other both in bilin composition and bilin content. In each case, PE(I), present in a smaller amount, has fewer (or no) PUB groups than the more abundant PE(I1). PE(1)s are typical of all previously studied C-, B-, and R-phycoerythrins in that they carry five bilin groups at locations homologous to the bilin attachment sites in the other phycoerythrins (Figs. 12-14). PE(I1) carries six bilins. Five of these occur at positions corresponding to those in PE(1)s and in C-, B-, and R- phycoerythrins. However, the sixth bilin in PE(I1) is attached at CY-75 (see Ref. 25). The sequence about this bilin attachment site lacks homology to other phycoerythrin (Y subunit sequences. Alignment of the sequence of WH8020 PE(I1) CY

subunit with those of the other phycoerythrins indicates that the additional bilin attachment site represents a single amino acid insertion, accompanied by a series of mutational events (see Ref. 25). The bilin content and sequence data suggest that phycoerythrins should provisionally be divided into two classes. Class I (phycoerythrins with five bilins) includes the previously studied C-, B-, and R-phycoerythrins, as well as the PE(1)s. Class I1 (phycoerythrins with six bilins) is represented by the PE(1I)s described here. A search for other examples of class I1 phycoerythrins among red algae may well prove fruitful.

In native phycoerythrins, the absorption maxima of the PUB chromophores lie at -490 nm, whereas those of the PEB chromophores lie between 540 and 565 nm. From the fluorescence emission spectra of phycoerythrin (e.g. Figs. 8-11), it is evident that excitation energy is transferred from the PUB to the PEB chromophores with very high efficiency. Fig. 15 summarizes the locations of the PUB and PEB chromophores in previously studied C-, B-, and R-phycoerythrins, in the Synechococcus spp. WH8103 and WH8020 PE(I1) and PE(1) examined in this study, and in the Synechocystis sp. WH8501 PE(1) described by Swanson et al. (24). The only invariant energy acceptor bilin position is at /3-Cys-82, which is occupied by PEB in all eight types of phycoerythrins. Since this is the only PEB group in WH8501 PE(1) (24), there is no ambiguity in the assignment of @-82 (PEB) as the conserved terminal energy acceptor in phycoerythrin. Moreover, from sequence homology, this position corresponds to the location of the terminal energy acceptor, p-82 (PCB), in the phycocyanin family of proteins (Fig. 15). These findings provide support for the two key features of the model for directional energy transfer in phycobilisome rods described above. Energy migrates from peripheral donor bilins within an .-@ domain to an acceptor bilin located near the center of the disc. The terminal acceptor bilins occupy corresponding locations in different phycobiliprotein components of phycobilisome rods.

Presence of PE(II) and PE(I) in the Same Rod Substruc- ture-Examination of the polypeptide composition of phycobilisomes by two-dimensional polyacrylamide gel electrophoresis showed that both phycoerythrins were present in these assemblies. Moreover, partial dissociation experiments on strain WH7803 phycobilisomes indicated that both phycoerythrins were parts of the rod substructures of the phycobilisome and that PE(I1) served as an energy donor to PE(1) (Fig. 11). This role is consistent with the higher content of

PEBs in PE(1)s relative to PE(1I)s and longer wavelength absorption and emission maxima in PE(1)s relative to PE(1I)s (Table I; Figs. 8-11).

Phycoerythrins in Marine Unicellular Cyanobacteria Syne- chococcus spp. Ecological Considerations-The marine Syne- chococcus spp. occur abundantly in surface waters of the temperate and tropical oceans and constitute a significant proportion of the total biomass and primary productivity in many oceanic regimes. Consequently, these organisms have been the subject of numerous ecological studies (e.g. Refs. 15, 18, and 53-57).

Flow cytometry studies show that virtually all Synechococ- cus spp. in the open ocean have PUB-containing phycoerythrins and that most of them have phycoerythrins with high PUB content (55-57,62), such as that of the phycoerythrins of strain WH8103 (Figs. 8 and 9). Strains with phycoerythrins low in PUB, such as strain WH7803 (see Fig. 11) were found only in coastal waters (57). Olson et al. (57) note that "Phy- coerythrin fluorescence per cell increased dramatically with depth in the lower euphotic zone at all stations; at some open ocean stations, very deep cells were as much as 100 times brighter than those at the surface." Phycoerythrin fluorescence per cell is a direct measure of the phycoerythrin content. Kana and Glibert (63, 64) examined in detail the effect of irradiance from 30 to 2000 FE m-* s" under continuous light on the growth and phycoerythrin content of laboratory cul- tures of Synechococcus WH7803. Phycoerythrin in high light- adapted cells represented <3% of the cell nitrogen as opposed to >20% in light-limited cells. Moreover, between 2000 and 100 pE m-* s-', phycoerythrin concentration per cell increased linearly 10-fold with the reciprocal of growth irradiance. Only a 3-fold change was seen in the phycocyanin and chlorophyll contents. This indicates that the increased capacity to absorb light at lower growth irradiance was achieved primarily by increasing the phycoerythrin content per cell and to a much smaller degree by increasing the number of phycobilisomes and cell content of chlorophyll complexes in the thylakoid membranes. Growth rate decreased by a modest amount from 1.97 day" at 2000 pE m-'s" to 1.21 day" at 100 HE m-* s-I. These results show convincingly that an increase in phycoerythrin content compensates efficiently for a decrease in irradiance.

Marine Synechococcus spp. are strict photoautotrophs and derive their energy entirely from photosynthesis. The studies cited above demonstrate that the type and amount of phycoerythrins in cells of Synechococcus spp. correlate with the distribution of these organisms in the oceans and with their growth rate. It is interesting to compare the absorption coefficient for green light (at 490 nm) of a phycoerythrin such as the PE(I1) of an open ocean marine strain such as WH8103 with that of a C-phycoerythrin of a fresh water cyanobacterium such as Synechocystis sp. PCC6701. C-Phycoerythrin carries 5 PEB groups per monomer, none on its linker polypeptides, and has an absorbance at 490 nm of 300,000 "' cm" per (a& (65). WH8103 PE(I1) contains 4 PUB and 2 PEB groups per a@ (Table I), and a bilin-bearing y subunit, and has an extinction coefficient of 2.78 X lo6 M" cm" per ( C X @ ) ~ ~ (22). As a result of three molecular adaptations, high PUB content, the additional bilin on the a subunit, and the presence of bilins on the linker polypeptides, PEW) of the marine Synechococcus WH8103 absorbs green light of the wavelength with the deepest penetration in the ocean nine times more strongly than its counterpart protein in a fresh water cyanobacterium.

Acknowledgments-We are greatly indebted to Dr. John B. Water- bury for providing us with the marine unicellular Synechococcus

Bilin Locations and Energy Transfer in Phycoerythrins 9519

strains for this study, for sharing with us generously his knowledge 32. Glazer, A. N., Hixson, C. S., and DeLange, R. J. (1979) Anal. and experience with the culturing of these organisms, and for his Biochem. 92,489-496 continued interest and helpful discussion of this work. 33. Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M.

(1984) J . Biol. Chem. 259, 5472-5480

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Ong, L. J., and Glazer, A. N. (1988) in Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial Models (Stevens, S. E., Jr., and Bryant, D. A., eds) pp. 102-121, American Society of Plant Physiologists, Rockville, MD

Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J . Biol. Chem. 266,9528-9534

Wilbanks, S. M., de Lorimier, R., and Glazer, A. N. (1991) J . Bid. Chem. 266,9535-9539

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Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856- 4863

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Berkelman, T. R., and Lagarias, J. C. (1986) Anal. Biochem. 156,

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35. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W.

36. Waterbury, J. B., and Willey, J. M. (1988) Methods Enzymol.

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38. Ong, L. J. (1988) Phycobiliproteins from Marine Cyanobacteria: Bilin Distribution and the Identification of the Terminal Accep- tor Bilin in Phycocyanins and Phycoerythrins. Ph.D. thesis, University of California, Berkeley

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(1977) Cytobiologie 16, 118-129 42. Glazer, A. N., Williams, R. C., Yamanaka, G., and Schachman,

H. K. (1979) Proc. Natl. Acad. Sci. U. S. A . 76, 6162-6166 43. Bryant, D. A,, Guglielmi, G., Tandeau de Marsac, N., Castets,

A.-M., and Cohen-Bazire, G. (1979) Arch. Microbiol. 123, 113- 127

44. Glazer, A. N. (1988) Methods Enzymol. 167, 291-303 45. Sidler, W., Kumpf, B., Riidiger, W., and Zuber, H. (1986) Bid .

Chem. Hoppe-Seyler 367,627-642 46. Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M.

(1984) J. Biol. Chem. 259,5472-5480 47. Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and

Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370,115-124 48. Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-

4863 49. Schirmer, T., Bode, W., Huber, R., Sidler, W., and Zuber, H.

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53. Schirmer, T., and Vincent, M. G. (1987) Biochim. Biophys. Acta

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(1988) Deep-sea Res. 35, 425-440 56. Li, W. K. W., and Wood, A. M. (1988) Deep-sea Res. 35, 1615-

1638 57. Olson, R. J., Chisholm, S. W., Zettler, E. R., and Armbrust, E. V.

(1990) Limnol. Oceanogr. 35,5845-5854 58. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and

Stanier, R. Y. (1979) J. Gen. Microbiol. 11 1, 1-61 59. Mazel, D., Guglielmi, G., Houmard, J., Sidler, W., Bryant, D. A,

and Tandeau de Marsac, N. (1986) Nucleic Acids Res. 14,

60. Dubbs, J. M., and Bryant, D. A. (1987) in Progress in Photosyn- thesis Research (Biggins, J., ed) Vol. 4, pp. 765-768, Martinus Nijhoff, Dordrecht, The Netherlands

61. Anderson, L. K., and Grossman, A. R. (1990) J. Bacteriol. 172,

62. Wood, A. M. (1985) Limnol. Oceanogr. 30, 1303-1315 63. Kana, T. M., and Glibert, P. M. (1987) Deep-sea Res. 34, 479-

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(1985) J. Mol. B i d . 184, 257-277

and Hackert, M. L. (1986) J. Mol. Biol. 188,651-676

(1990) J. Mol. Bid. 21 1, 633-644

Sect. C. Biosci. 42, 258-262

893,379-385

8279-8290

1297-1305

495

516

5495

Continued on next page.

9520 Bilin Locations and Energy Transfer in Phycoerythrins SUPPLEMENTARY MAERIALTO:

Phymcrythrinr of Marine Unicellular Cyanohmria

1. BUUiTfPES ANDLOCATTONS ANDENERGY TRANSFER PATHWAYS

IN SYNECHLXOCCUSSPP. PHYCOERYTHRINS.

Linda J. Ong and Alcrandu N. G l a r s

!XiPEF3?vtENTALPROCF,DURES

Maids . Hydmxylapstia was prepared by the method of Sicgelman e l al. (26). Rerwollen m i n o p u l a r DEAE-cellulose DE-52 WBS fmm Whatman. Bio-Rex 70 resin was obtained from BioRad and SP-Sephadex C-25 and Scphacryl S-203 horn P h m i a .

- Absorption spectra were determined with a Beckman model 25 recording rpc-lutions whoy absabancc acceded 1.5Icm at a,. light pathlengths shorter than I cm were used. The absorbance shown in the figures was calculated for a lightpath of 1 Em. C o m c t d fluorescence emission spccw were d c t e h n c d with a Perkin-Elmer MPFMB rpectrofluorimcter equipped with a Hamamam R926 phototube and a DCSU differential corrected rpcctra unit. For fluorercsncc ~pectros~opy, pmlsin rolutionr were in 50 mM Na-phosphafe, pH 7.0, and lhc excitation and emission dill wsrn set at B 4 am bandpass.

. The bilin types and contents ofthe various phycobilipmlsinr. subunits. and bdin pcptidss were determined fmm their absorprion spccm in 8M tuea at pH 1.9 or 3.0. Extinction coefficients under these conditions a1 490 and 550 nm are 94,ox) and 0 M-1 cm-' for PUB and 18,260 and 53.700 M" Em-' for PEB, respectively (27).

containing polypeptides w u c idsmified by formation of fluorescent Znz+ complcxcs as described by Bcrkdman and . SDS-PAGE was p e r f o d as described by Yamanaka SI al. (28). Bdin-

La anas (29). Zinc BCCBIS (1 mM) was added to both the rrackng and rcparaung gc1 solutions and m the running

Polypptides conraining PEB or PCB fluoresced mange-rcd PI repmed earlier (29), whereas thow cmtaining mostly buffer. Aflsr electrophorcsir. thc gels were examined under illumination from a Fotodyne UV-light box.

PUB unittcd a yellow-pen fluornxencs. B i l i n - M n g polypeptides m i g r a t e d fas ter in Lhc premce of tine ions. To eliminals the pawbation of lhc mobility of bilin-bearing polypptidcr by Znz+ionr, SDS-PAGE was paformed in the abwncc of zinc aceate. Afrcr electrophoresis the gels were soaked for 15 mi" in 1 0 0 ml of running buffer containing 10 mM zinc a c e t m and 0.01 % ( w v ) -onium prsulfale. Under lhcsc conditions. bilin-containing p o l y p ~ p t i d ~ ~ also fluoresced under UV illumination. However, the fluorexcncc inrcnriry of polypeptides bearing only PUB was lower than that sten in gels slccuophphorersd in 1hs pre~cnec of Zn2+. Two-dimensional PAGE was prformed as described by O F m l l ( M ) .

. . . . i " ~ ~ f 12.4 ml of 5.20% ( w h )

- Pmrein solution (1501ll)

sucrosc in the same buffer. The gradients were centrifuged in a Beck- SW41 mtor for 22 h 81 259.aX)g The

absorbance meaSunmenls ac 220 nm and at the visible maxima of standard pratcins and of pmlcins under study. as gradients were then fractionated fmm the bollom into 0.5 ml fncrionr. The pmlsin distribution war detcmined fmm

wcU as fmm SDS-plyanylamids gel elsErmphmris of every fraction.

. .

. . Model 510 pumps. P Model 680 aummatic gradient CMIWI~, and a Krslm Spectroflow 773 absorbance detccto1. A

- HPLC was pcrfmed with a Waten system consisting of 1wo

Supelco LC-I8 preparative mlumn (5 Ilm; 10 x 250 mm) or analy%ed column (5 lun, 4.6 x 250 mm). or a BieRad

described below WVCI p e r f o m d on the analytical C O I Y ~ ~ S . The solvent system used was 0.1 M NaHzPOd. pH 2.2. W304 C 4 analyiical column (5 um; 4.6 x 250 mm) wuc uxd. Unless othcnvix specified. the HPLC separations

with the specified concenw$ion(r) of acstomdle. In the descriptions below only the acetonitrile conccnmnon~ M specified; LC I- buffs was used throughout.

F a desalting. the peptides wen adSorbcd on a 4.6 x 40 mm C-4 guard column @io-Rad, Hi-Porn Rp) equilibrated in IO mhl trifluomacctic acid (TFA). The column was then washed with 6 volumes of 10 mM TFA and the peptide clvted frontally with 60% ( v k ) acctonitrilc-IO mM TFA. Bilin pcpcides of less than ten residues w e n desalted in the - manneron C-18 reverie phase Columns.

IlO-I150C for 22 h. Bilin-linked cysrsinyl reSlduCs were convsncd to cyrmie acid by hydmlyrir in 6N HCI . Peptides were hydmlyzd in 6N HCI containing 0.1 % ( d v ) phenol undcr vacuum PI

eonuining 0.21 M dimcthylrulfoxids (32). Amino acid analy~cs were p c r f o d on a Durmm amino acid analyrcr with ophthalal&hydcposlcolumndcrivati~ti.donand fluorexcnccdsrection.

. .

20 n~n0m01c.s of peptide. Manual Edman degradation was carried out by the 4 , ~ d i m c l h y l a m i n o a r n b e ~ ~ ~ ~ ~ 4- - Subtrsrivc Edman degradation was pcrfomd by the pmcedure of Lundcll ct al. (33) on IO-

isorhiocyanarclphcnylisothiocyan~,~ coupling method of Chang et al. (34) on 2 10 10 nanomolsn of peptide. For automated Edman dcgradation. pepl~dcr (250.500 pcomoisr) were rquened in an automated gas-liquid phasc sequcnator (Applied Biosysmms. Inc., Fostcr City. CA) (35). The FlX dsrivativss of the ammo acids were identified by HPLC in a dedicated analyzer (Applied Biolyst~ms, he.) and the data manipulated using lhc manufacturer's sofrwm.

0.6.6.8. and 1.0 M sucmss in 0.75 M'NaK-phosphatl- 1 mM W T A ai dH 7.0.

0.2.0.4.0.6. and 0.8 M S Y ~ E C in 0.75 M NaK-phosphate . I mM EDTA, pH 7.0. and 7.5 ml of I M su~mso in the Larger scale prnpantions wue performed in P Beckman SW 28 mtoron sucrow dcnriry gradknls of 6.5 ml u c h

m e buffer. Five ml of ~ u m a t a n l were loaded on each m d i m and ccntrifusalion was Dcrfmcd at 18.500 mm

. .

for 17-18 h a 18oC.

7 - Csllr w s s stored as fmzen pellets at -209- and were thawed immedistsly prior 10 u s . Cells were suspended a1 0.1-0.15 g we1 wcighf pa ml in 50 mM N w h o l p h m - I mM NaNj. I mM PMSF at pH 7.0, and lhc ruspcnrion passed three times lhmugh B French p n u v r s cell as dsscribcd above. The solution was centrifuged at 16,wO# for30 min 81 18% lk phycobiliprorsinconlaining supanatan1 was saved and brought to 65% of r a m t i o n of (WhSO4. Recipitalion was allowed 10 pmeed overnight. Symchocoeew strains WH8103. WH8020. and WH7803 u f h contained a majm phycarythrin [designated PE(1I)I. a lcss abundant phycarythfin [designated PE(I)I. a R-type phycocyanin (37). and an allophycmysnin. The purification of the phycarythrins fmm strain WH8103 is described fully bdow. Thc corresponding proteins from m a i m WH8020 and W 8 0 3 wcre pufified by rlrmlar pnxedures. A delailed accmnt of the pwificalion of all of the= pmteins i s given in ref. 38.

N a m C I , pH 7.0. and applied m a hydroxylapatite column (4.5 x 5.0 cm). All of IhC adsorbed -The (NhhS04 prs lpiralc fmm 16 gm wet weight of ECIIS was dtalylsd against I mM

~ l u a t e was bmught.to 65% of saturation wirh solid (Nh)zSOd and kept overnight at 4°C. T h e precipitate was phycobil~pmtcins were then fmnmlly sluLcd from !he column with 0.15 M Na-phosphaa - 0.1 M NaCI. pH 7.0. The

collecred by centrifugalion. rcsuspsndcd and dialyzed preparator/ 10 hydmxylaparils chromatography as dsxribcd above. The solution was applied Io a hydrorylapaiilc column (4.5 x 5.0 cm). developed rrcpwisc wlth 5, 35.50, and 120 mM Na-pborphalc buffcn conmining 0.1 M NaCl a1 pH 7.0. Fraction 1. elulcd wnh 35 mM buffer. contained most of the PE(II) and R-PC11 (38) and was brought IO 65% of satumion with (N&)zS04. The precipitate was dirrolvcd in 25 mM N a - x e ~ ~ k . pH 5 .5 , and dialyzed exhaustively against the same buffer at 4oC. T h e solution was applied ID a column of Whatman micropnular DE=-ccllulow DE42 (4.0 x 9.0 cm). The column was developed wilh a linear pdicnt of 150 ml of 0.025 M Na-acerate. pH 5.5, in the miring chamber and 150 m10.25 M in the rersrvdu. 21 I ~ ~ n s t a n l flow raw of 17 muh. Fractions wnh A l w nm:Aw ,,,,, 5 1.9 were combined and pICEipilaled

purified PE(I1). The pmlsin was stored as a p m i p a a s in 65% saturation of (Nh)zSO4 ar 4 T .

m i n a n d allophycocyanin. Ths (NH4)2SO4 prrcipirac war diwlvcd ~n 25 mM Na-acetate, pH - Fraction Ill. eluted with 0.12 M Na-phOsphsIC. pH 7.0 contained primarily PEU) and

5.5 and dialylcd against the same buffer a1 4oC. The solution was applied 10 B Column of DEAE-cellulose DE-52 (4.6 x 1.5 cm) and rhc column dcvcloped With a linear gradient of 30 ml Of Na-acctalc. pH 5.5. 0.025 M m Lbc mixing chamber and 0.25 M in the rescrvoLT. 81 B flow n f c of 3 mVh and 0.5 ml fncciona were collccled. Fractions wlrh Ayjl nm:&9~ "~ 2 1.14 were pooled. Thcrs rcprcscntcd purified PE(1).

. . p n x e d m of Glazer and Fang (39). The (NH&S04 precipira8s (ea. 5 mg) of purified PE(I1) was resuspended in a

- The subunit scparakmr w a r perfomxd by the

minimum volume of 1 mM Na-phosphate. pH 7.0. and dialyzed against the same buffer. The solution was acidified by the sddition of 6N HW 10 20 mM. A ~olution of I O M una-0.4% (vh) acetic acid-IO mM 2-mrcap~thanol. pH 3.0. was then added 10 give a final urea conscnmtion of 8 M and the mixture allowed IO s m d for I5 mm. An qual Volume of 0.4% (vlv) acetic acid-10 mM 2-mcnaptocthanol was then added and the solution mixed with 0.6 ml of

6 . 7 , and 8 M urea - 10 mM 2-mcnaptosthano1, pH 3.0 @H adjdlusted with HCI). Fractions sluted with 6 7 M urea Bio-Rex 70 (minus 4w mesh; Bio-Rad Laboraroner, Richmond. CA). The column was dcvslopcd ~ tspwiw with 4,

with &go n m : A ~ ~ ~ nm 2 18.7 (a subuniz) and thox sluwd with 8 M u n a with &w n m : A ~ j ~ nm = 1.3-1.4 (P subunit) were pooled rcpanialy. dialyzed exhaustively againrc u) mM HCI and s m d at 4oC.

B and B P resuspended in B minimum volume of 1 mM Na-phosphate. pH 7.0, and dialyrcd against t h c same buffer. Solid m a

- The (NHg)zS04 prccipilate of pure PEW was

(uluz pure) was added 10 8 M and lhc pH was adjurred 10 pH 2.2 with 6 N HCI. An equal volume of 0.4% (vlv) acetic acd-IO mM 2-mcrcapcosthanol was then added and the solution applied IO a column (1.5 x 1.2 cm) of Bio-Rex 70 presqullibratcd with 4 M ures-0.4% ( v h ) acetic acid-I0 mM 2-mctraptalhanol, pH 3.0. After application of IhC sample the resin was rluriijcd and !he slumy was applied to a column of BloRcr 70 (1.4 x 3.6 cm) cquilibraled with the 6- SOIVS~L The column was dsvelopcd stepwire with 4.6,7.8. and 9 M urea - 10 mM 2-mercapfosthanol. pH 2.2 (pH adjusted with HCI). Fractions cluled wilh 7 M m a with ",+A 550 "~ 21.9 (a subunig and those clurcd

double-distilled water and storcd at 4oC. with 8-9 M urea with 4 9 5 n m : A ~ ~ ~ "~ s 1.3 (P subunit) wcrc pooled rcparamly. dialyzed cxhaurtivcly againrl

p f i m lhc sumw density gradienL eonmining ' . - Strain WH7803 phycobdlromcs wsrc

appmrimaLcly 0.5 mg phycobiliromer per ml, was conecnmtcd three-fold by d idydr against sohd Aquacide I L 4

solution was then dialyrcd against 0.4 M NaK-pbosphalc. pH 7.5 for 16 h 81 4% and 0.5 ml aliquotr wsrc layered (BioRad. Richmond, CA) for I2 hovn at 4oC. To achicvc panid dissmiauon oi the particles, rhc phycobiliromc

on I2 ml 5.20% wh linear I U C ~ I C density gradients m 0.4 M NaK-phosphao. pH 7.5. The gradicnls wcrc centrifuged in a B e c b SW41 mor at 30,wO rpm for 21 h. Six colored bands were resolved on Ihc &em$. There were collcctsd and their absorption and fluorexcncc emission rpccm determined in Ihc gradrcnl buffer. The polyppride canpos~tion of each band was c r a m i d by SDS-polyaqlamids gel elstrophassir on a 14% gel.

- Frozen EFUS of main WH7803 (IO g we1 wcrghr) were I N a - p h o s p h a t e . I mM Na N, - I mM PMSF. at pH 7.0. The rurpcnsion was passed t h e times through an Aminco-French pressure cell 81 18,000 P.I.I. ar 4OC. The rurpcnsion was ccnmfuged at 16,000 r g for 30 mi" ac 18oC and (N&)zSOd added 10 the supernatan1 Io 65% of raturslion. After 6 h a t 441. the precipitate was collccled. resuspended and dialyzed overnight against 0.001 M Na-phosphalc- 0.1 M NaCI. pH 7.0. The solution was applied IO a column (1.9 x 6.5 em) of hydroxylapalirc cquilibrarcd in the dialysis buffer. The column was dcvcloped step-wac with 10,25,55. and 1 I 5 mM Na-phosphate-0.1 M NaCI. pH 7.0. Thc fractions were pooled as Indicated in Fig. 4, precipitated by the addition of solid ( N ~ ) z S O I to 65% of ~aluration. and $lored at 4DC.

. . .

. . against 25 mM Na-acetate. pH 5.5, and the solution applled 10 a column 01 Whatman micmgranular ccUuloD DE-52

. The fracmn I precipitate (Fig, 4) was dlrsolved in and dialyzed

(1.9 x 6.5 cm) equilibnred with !he same buffer. The ~olumn was developed with 60 ml oi 25 mM N a - a ~ ~ l a l ~ . pH 5.5, in the mxing chamber and 60 ml of 250 mM Na-acetate, pH 5.5. in the reservoir. PE(Il)-conminxng fractions (Ay5 nm:&9s nm P 2.2) were p m l e d and prscipicated by the addition of solid (NH4)zSO4 Io 65% of salvration at 4oC. The pmipmtc was dissolved in 0.001 M Na-phoaphalc-0. I M NaCI. pH 7.0, and dialyzed a g a n x rhc 6 m c buffcr. The solution was then applied to a column (1.9 I 1.8 cm) of hydroxylapatits equilibrated with the same buffer. The column was develop4 with 1, 15, and 20 mM Na-phosphate-0.1 M NaCI. pH 7.0. Pure PE(II1 cluted a1 20 mM Na-phosphate and fractions with AsI5 nm:A195 " ~ - 2.2 were pooled and stored as a prscipitats in (N&)>SOd at 65% of Sawration at 4oC.

7.0. and dialyzed against the same buffer. The solution was then appltcd to a column (1.9 x 1.5 Em) of For purification of PE(0. fraction 111 (Fig. 2) was collccred dissolvcd in 0.001 M Na-phosphate-0.1 M NeCL pH

hydmrylapatilc equilibrated with the same buffer. The column was developed with 15. 25. 50. and 100 mM Na- phosphate-0.1 M NaCI. pH 7.0. PE(1) eluted primarily at I00 mM Na-phosphate. PUre fractions wllh A-5 M1:A195

Z 4.4 were p l e d and rmred as a precipitate in (NH4)2SO4 at 65% of ~ a t ~ r a f m n a! 4oC.

ND;phosphatc. pH 7.0. To denature the phycobilrpmsin. the solution was - The following general pmccdure was uxd. The phycobrlipmtein solution

acidified to pH 2.0 by the addition of 1 N HCI to 20 mM. TPCK-trypsin was then added to 2% wiw cnzymelsubrtrate. and solid NbHCO3 to 0.1 M. and !he pH was adjusted to 8.0 with 2.0 M NaOH. The mixture was scaled u n d s Nz and incubated 81 309- in the dark. Aflcr 2.5 h. a Second aliquot of uypsin was added and ths incubation continued for another 2.5 h. The reaction was s!oposd by rhs addition of HIPQ LO 0.2 M or of laci id

. . . . .

coUu'ted (Fig. 5). The bilin ~@Acs in thew paLs w a r punfisd 80 homogeneity by HF'LC on I rewm phars C-4 550 nm indicating sbssncc of PEB peptides. Thrrs pcslrs. A. 8. and C. with maximum absorbance at 495 nm w m

column under i-tic conditions with 17%. 18%. and 19% ncetonitrilc for peaLr A , 8 , and C. rcrpcctivcly. The bilin m t i d c s in A. B. and C were identified as WH8103 PE(II) a-l PUB. WH8103 PE(I1) 0-2 PUB. and WH8103


the dark. The nacaon was smppcd by the addition of 2-mercaptocthanol to 5% by volumc and of glacial acetic and 10 30% by volumr. The ~ o l ~ f l o n was applied IO a Sephadex G-50 column (1.4 I 70 cm) equilibrated and eluted with 30% m u c o u ~ acetic acid. Frxdons with &M., 5 0.05 w m m l c d and coneenmred by nlw cvaooration. The

33% Eetonimik. P m l U was coneenwrrd by r o t a r y evaporation and subjected to HPLC on a C-4 r e v e x phars column with a

5adicnt of 25 IO 50% acetonimle. A major bilin peptide-containing pee was collcclcd (Fig. 6, A). The p l e d matcrial was conccnmrcd by flushing with N2 and diluted 1:3 by volumc with HPLC p d e walcr. Peptide W H 8 103 PE(0 8 2 PEB was purilied w hnmgsncily by nchmmalogmphy of this mataial on a C 4 rev- phase column.

Pool In was rnncmmwd by mtaq emperation and diluted I:I with M mM Na-phosphate. pH 2.5. The solution

column was dcvslopcd with a linear gradient Of 1W ml M mM Na-phosphalc. pH 2.5, in the mixing chamber and was applied 10 B wlumn (0.6 x 16.5 m) of SP-Scphadcx C-25 cqui l ibmd in 50 mM Na-phoqhate. pH 2.5. The

w r e collected Two colored p k r , A and B, were resolved. The cluafes w m p l e d and the two fractions desalted 1W m10.6 M NsCl - 50 mM Na-phosphate, pH 2.5 in the nsemir. a1 B flow m e of 15 mvhr and 2.0 ml fractions

separalelyonC-18nvcrwph~columnr. PmlAwntainedaPUBpeptideandaPEBpeptide dcrignatedWH8103 PE(1) 0-2 PUB and WH8103 PE(1) p-I PEB, respectively. The two peptides were p u r i f i i to homogeneity by HPLC on C-I8 revc~sc phsw column^ under iwmatic condition^ 81 19% acctonidls for WH8lO3 PE(1) e-2 PUB and 27% acewnidlc for WH8103 PE(I) p-1 PEB. A single PEE peptide. WH8l03 PE(1) a-1 PEB, conrained in mi B WPI similarly unfed under i rmat ic conditions st 23% acetonidle. Solutions of the lhree ~urified ca t ides

i s m t i c dution step was p r . P m l 11 was concenmted by mlary evaporation under Y ~ C Y U ~ and dilulcd I:I by volume with 50 mM Na.

phosphate, pH 2.5. The solution was applied lo I SP-Scphadcr C-25 c o l u m n equilibrated in 0.05 M Na-phosphate, pH 2.5. The col~rm~ was developed with a lincar sadicnr of 225 ml 0.05 M Na-phosphate. pH 2.5 in the mixing chamber and 225 ml 0.05 M Na-phosphate. pH 2.5. in the reservoir a1 a flow rate of 20 mVhr and 2.0 ml fractions

desalted on individual C-I8 reverse phase columns as previously d s h b e d . P w l A contained B bilin peptide. were collcclsd. Three bihn-containing p k r . A. B, and C. wen separated. The pe&s were each pooled and

WH8020 PE(n) (IJPUB. which was purified to homogeneity by HPLC on a pIsparative reverse phaw C-I8 column by i smat ic elution with 23% accraniuilc. Pool B contained two bilin peptides, WH8020 PE(I1) a-2 PEB and WH8020 PE(I1) p:l PEB. which wcrc similarly punfied to homogatcity by HPLC by stepwise dcvclopment of the c o l m initially wnh 25% and then with ?€I% accwniuilc. Pml C mntained a single bilin peptide. WH8020 PE(n) a- 1 PEB, wYch was pvrificd ID homogeneity by i-tic cludOn with24% aalonidlc.

. . . . . .

PEB BS the sole bilin peptide Pml In was conccnmted by mlary cvaporation and dilulcd 1:l. by volume. with 50 mM Na-phosphate, pH 2.5.

The solotion WBS applied to B column of SP-Scphadex C-25 (0.6 x 16.5 cm) equilibrated in 50 mM Na-phosphate. pH 2.5. The column was developed with a linear 5adienr of 1M ml 50 mM Na-phosphate. pH 2.5, in the miring chamber and IM m10.6 M NaCl - 50 mM Ns-phmphalc. pH 2.5. in the rcscrvoir. at a flow ntc of IS ml per hr and 2 ml fractionr wtre coUs~ted. Two solored p&s, A and B, wen resolved and pooled separately.

isocratic elution with 22% and 25% Bcswniuilc. rcrpectivcly. P w l B contained a single PEB pcptids, a-I, which P m l A conmined Two bilin peptides, a-2 and p1, which wen purified by chmmarography on C-lU column^ with

was @ed LD homogeneity by chmmawpphy with i-tic elution wirh 23% accwniuile.

1 PEB. p.2 PEB and p 3 PUB Vsblc IU). Thc data Which led to the assignment of the squcncc to each of these Five bilm pcptidu wcrc isolatcd from the ayptic digest of WHU103 PE(I): WH8103 PE(1) (1.1 PEB. a-2 PUB, 8-

pepudcs m summanzed m Tables VII-X. The squcnccr are shown in Figs. 12-14, Peptide p-1 PEB war the only mypypUc bilin peptidc with >dentical amino acid rcqucncc and bilin rypc in bath WH8103 PE(I1) and PE(I). From the comparison of the IequenCCI in Flgs. 12-14. iI is evident that WH8103 PE(I) contains the five bilin atlachment sites common to C-, B-. and R-phycosrythrinr. but lacks the nwcl a-3 anachment sits p n ~ m in PE(Il).

with WH8103 wen used 10 dsrcrmme rhs amino acid Ecqucnesr and bilin types in q p t i c peptides fmm WH8020 Ssqucnccr of Typtic Bitin pcptidcs M v c d horn WH8020PE(II) and PE(Q - Rocedurcs s~milar 10 those uwd

PE(0) and WH8020 PE(1). Six unique W t i e bilm peplidsr a-l PEB, a-2 PEB. a-3 PUB. p- I PEE. p.2 PEB. and

pmwnred in Tables XI-XV. Thc rqucncc of peptide p-1 PEB was dstsmincd by manual Edman dcgradallon. Thc p3 PUB (Table 111) wen irolared fmm WHSOZO PE(1I). Data srlrblirhing the wqucncer of !he% pepudcr BR

W H W O PEflll b i l i peptides show rmmg homology w the six b i l i pcptides from -8103 PE(U) (Figs. 12-14). Five "ypflc b i h peptides WCIS rrolared fmm WH8020 PE(I) (Table nI) and thcir ammo acid ~ q u c n c e dslcrmined

Pables XVI-XlX). A compdson Of rhc sequences of these pcpridcr with those derived from !he other phyclnryIhnns is given in Figs. 12-14

FRACTION NUMBER

Fig. 2. Separation of Synechococcus sp. WH7803 phycobiliproteins on hydroxylapatitc. For dctails see "Experimental Fmcedures.'' Fractions were p l e d as indicated.

I Tryptic digest 0; WH8103 PE (11) , , Pellet fra;i,, of digest, Soluble fraction 01 digest

I Sephaiex G-50 c-4 HPLC

PEB peptide 8.2 I A1 A2 Bt 82

27 residues C-4 HPLC PUB peptide 8.3

40 restduel

PUB pep t ide pep l ide 5-2

3 res idues ',".," 1 5 residues 1

P U B P U B peptide 5.1 peptide 5 - 3

7 residues

peptide p-1 P E B

6 residues

Fig. 3. Flow sheet of the purification of uyptic bilin peptides from Synechocmcus sp. W 8 1 0 3 phycarythnn, PE(I1).

9522 Bilin Locations and Energy Transfer in Phycoerythrins

0 10 20 RETENTION TlME Irnlnl

4 0 %"I .50

.25

0 5 IO 15

E 0 P

c

.. 0

W

z 4

II: 0 In

m

m a

0- RETENTION TIME imml RETENTlON TlME (n.nl

RETENTION TIME (rnin)

Fig. 5. Separation Of thc bilin peptides fmm the cryptic digest of the a subunit of Synechaocsvs sp. WH8103 PE(II) by HPLC. For details, - 'Experimental h e d u r c r . " Bilin peptides w n e p l e d as indicated by the bus.

wa"lLENGTH1"m)

Fig. 8. A. Absorption and nuorerscncs smision spectra of WH8IO3 PE(I1). The abrorplion rpccrmm was determined on a - 0.1 mumi pmlcin ~ ~ l u t i o n in 50 mM Na-phosphate at pH 7.0. Exclralion was at 470 nm. B. Absorption rpccrra of the a (-) and p (------) subunits of WH8103 PE(1I) i n 8 M urea - 0.4% (vlv) acclic acid. 10 mM pmrcaploe!hanol. pH 3.0.

Fig. 6. Purification of tryptic bilin pepcides WHSI03PE(I) p-2 and F3 by high PRllUreliquidchmmalography. Panel A shows the chmmalo phy of Fracdon I1 from the Scphader 0-25 gcl tilaation of rhe rryptic digest. Bilin peptide WH8103 PEU) E P E B was p l e d as indicated by the bar. P m d R shows thcshmmarography of Fmtion I horn the urns Sephadcr 0.25 E O I Y ~ . Bilin peptide WH8103 PE(1) p.3 PUB was p l e d PI indicated hy the bar For demils. re "Enpetimcntal Rmdurcs."

Bilin Locations and Energy Transfer in Phycoerythrins 9523 I I I

WAVELENGTH (nm)

determined on a - 0.5 mghnl protein wlvtion in M mM Na-phosphate a1 pH 7.0. Excitation was 1 4 9 0 om. me Fig. 10. Absorpuon and tlvorsrccncc emission rpccrra of WH8020 PE(I1). 7hc absorption spectrum was

specmum of WH8020 PE(1) was identical IO *at of swan WH7803 PE(0 shown in pmnsl B of Fig. I I).

WLYLLENCTH Inml

a-1 a-2 a-3

WH8103 PE (11) PUB PEB

C-K-R RB

Gc-A-P-R K-C-A-T-EGK

WE103 PE(1) PFB Eim

C-Y-R A€-A-P-R

WH8020 PE(I1) PEB PEQ PZTB

C-K-R N-D-G-C-S-P-R E / W - (A, T) - (E/Q, G) -K

Ww8020 PE(1) PFB

C-Y-R AX-A-P-R PEB

C-PE PEB

C-A-R PEB

Gc-A-P-R

EPE PEB PEB

C-Y-R LC-V-P-R

R-PE PEB

C-Y-R PEB

L-C-V-P-R

9524 Bilin Locations and Energy Transfer in Phycoerythrins 8-1

WH8103 PE (11) , Mi8103 PE (Y) PEB WH8020 P E W ) , WH8020 P E W C-PE, B-PE, 8-PE

M-A-A<-L-R

8-2

WH8103 P E U I ) M-P-V-T-T~-S++I-A-GE-A-A-S-Y-F-D-M-V-I-S-A-I-S E m

WHB103 P E W M-E-T-TQG-DCS-A-L-V-S-E-A+S-Y-F-D-D- A8

WH8020 P E W ) A-A-V-T*-S-S-L-A-G-E-A+S-Y-F-D-A-V-I- -A- Pm

pE8 WH8020 P E W L-ESE-T-T+G"GA-A-L-K-A-E-A+ -Y-F-D-

Pm C-PE GT-P-V-V-EORC-A-S-L-V-A-E-A-S-S-Y-F-D-R-V-I-S-A-L-

PQ) B-PE K-K-S-F-A-AGD-C-T-S-L-A-S-E-V-A-S-Y-F-D-R-'?+A-A-

€m R-PE I-2-I-A-Aq7D-C-S-A-L-S-S-E-V-A-S-Y-COR

8-3

I P U B 1

I P U B 1

- P U B 1

WH8103 PE(II) L-nA-V-N-A-I-T-S-N-A-S-C-I-V-S-D-A-V-A-G.1. -C-E-N-T-G-L-T-A-P-N-G-G-V-Y-

WH8103 PE(I) L-D-A-V-N-A-I-T-S-N-A-Y-C-I-V-S-D-A-V-T-G-M-I-C.E.N-T~-Irl-~A~.G-N*-E.

WH8020 PE(II) L-D-A-V-N-A-LrS-S-N-A-A-C-I-V-S-D-A-V-AG. - -E-N- -G-L-T-A.P-

I P E B l

- P E E 1

I P E B I

- P U B 1

WH8020PE(D L - D - A - V - N - A - I - T - S - N - A - S - C - I - V - S - D - A - V - T O Y .

C-PE L-D-A-V-N-A-I-A-S-N-A"CM-V-S-D-A-V-A~-M-I-C~E-N~-L-I-Q~A~G-G

B-PE I r D - A - V - N - S - 1 - V - S - N - A - S - C - M - V - S - D - A - V - S - G ~ -

R-PE L - D - A - V - N - S - I - V - C - N - A - S - C - I - V - S - D " V - S G -

TABLE II

Aufomarsd Ed- Dsgradation of the a and 8 Subunits of WH8103 P E O and PEma

PE(II) a subunit PE(ID 8 subunit PE(0 a subunit PEm p subunit

Cycle Amino Yidd Cyslc Amino Yieid Cyde Amino Yield Cycle Amino Yield acid @mol) acid @mol) acid @mol) acid @mol)

separation by SDS-PAGE in the pmcncc of Zn2+ (wc rcf. 29). each of the -30 ma y subunits emits p n fluaswsnce indieadng the preence of ewalcnlly atwhchod bilins.

1 2 3 4 5 6 7 8 9 IO 11 12 13 14 IS 16 17 18 19 20 21

23 22

24 25

27 26

28 29 30 31 ~~

117

197 177

1MI 135

109 131 160 24

77

I5 57 36

38

21 9

1 2 3 4 5 6 7 8 9 10 I I 12 13 14 15 16 11 18 19 20 21

23 22

25 24

21 26

28

-

1 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 I 6 17 18 19

50 37

24

9

8

5

I 2 3 4 5 6 7 8 9 IO I 1 12 13 14 I 5 16 17 18 19

Mcl I34 Le" 55 Asp 61 Ala 67 Phe 47

~Scqucncingwarperformedon-250pmoiofcachPE(II)aand~rubunir.-ISOpmolofPE(I)aand-25(lpmol~ subunit.

bnd. not daemincd.

Bilin Locations and Energy Transfer in Phycoerythrins 9525 TABLE W

Amino Acid Composition of Tryptic Bilin Peptides Dsnved fm WH8103 PE(I1

TABLE^

Absorption Maxima in oe Visiblc Re&" of lhe S p m m 0fT1yplk Bilin Rptides ~thePhymerychnnsofS~~homccu~s~"sWHl lMmdWH8020 .

S w i n WH8103 P h y e w t h n n i Smin WHBOZO Phycatythfins

PE(l0 PE(D PE(I1) PEU)

Rptideb 1, Pep& h, nm

Peptide h, nm nm - 2

a- 1 490 II.2 490

a- I 550 a- I a-2 490

550 a-2

a- 1 550 550 a-2

a-3 550

490 0-3 492

p.1 550 p-1 550

p.2 550 p-2 550 p 1 552 p-I 550 p2 550 p.2 552 p 3 493 p 3 555 p 3 492 p 3 492

residues

nd

0.5 ( I )

1.0 ( I )

nd 3.6 (4) 1.9 (2) 1.4 (1) 0.7 (1) 0.3 (0)

1.4 (1) 1.2 (I)

0.8 (1)

1.0

nd

4.4 2.8 0.5 3.3 2.0

1.0 1 .o

rn LYS QS%H 0.69 (0.69)

1.0 (1)b 0.69 (0.69) 1.2 (0.81

1.5 (I) 1.8 (1)

Arg TYr CY%H 0.25 (0.5)

0.47 (1)b 0.46 ( I ) 0.2 (I) 0.20 (0.4) 0.47 (0.8)

TABLE V Aulmatcd Edman Dspdadatim ofTryptic Bilin Rptidu WH8103 PE(l0 a-2. m.3 and pZa

WH8103 PE(II) a-2 WH8103PE(II) a-3 WH8103 PEOI) p2

Cyck AmLa Yidd Cyck Amim Yield CYJe Amim Yield Cycle Amino Yield asid @mol) add @mol) uid @mol) add @mol)

121

122 56 14

479

271 160 337 182 114

132 64 69

43 32

15 16 17 18 19 20 21 22

18 26

8 IO 5 5 5

Ala b

Ala m

209

217 228

Ma Ala Ala

b Leu

143 165 287

241

1 2 3 4 5 6 7 8 9 10 11

531 148

107 51

173 74

89

20 71 m

12 13 14 15 16 17 18 19 20 21

52 14 22 26 36 3 17 29 4 7

9 s a IO Am 25 24 Su

23 ne 5

11 ne 25 25 Ala 5 12 Ala 22 26 lk 3 13 Gly 15 27 Scr 14 Glu

1 LC" 231 20 A!a 2 21

57 85 GlY so

Ab 22 ne 41 4 Val 80 23 nd 3 Asp 172

5 6 7 8 9 IO I I 12

1 20

179 195

116 115

24 25 26 27 28 29 30 31

12 12

8 14

5 7 3 2 6 4 2

13 14 15 16 17 18 19

104 69

31 48 48

32 33 34 35 36 37

'Scqusnsing was prfmed on -300 pmol Of peptide p-3

bA-0 acid analysis showed the pnxnec of t w o bilin-linked cpteins residues in pprids &3 (dam nm shown). A PUB-linked cyroins residue was assigned to this p i l i o n for this rem" and on the basis of the rpcsrmropic propmies of this peptide.

Bilin Locations and Energy Transfer in Phycoerythrins TABLE X

Asmated Edman Degradation ofTryptic Bitin Pcpridc WH8103 PE(1) p3a

Slcp Aminowid Yield @mol) Step Aminoxid Yicld@mol)

1 Leu 242 2 37 20 l9 Thr

Val 34 I8

3 2 13 21 2 ;: 4 Val 5

101 47

22 23 UC 52

6 7 8 9

IO I 1 12

51 81 27 9

3? 91 53

24 25 26

28 21

29 30

9 7 6 9

27 17

9

Arg 1.22 (I)b LYS

1.2 (1) 1.2 (1)

CyS03H 0.50 (0.4) e 0.92 (0.8) 1.4 (0.7) 0.0

TABLE XI1

Aulomalrd Edman Degradarion of Tryptic Biiin Rptidc WH8020 PE(II) a-2.

1 ASll 1 L.' 4 b 2 ASP 8Y 5 S U 104 3 Gly 110 6 R O 165

BScqucnsing was performed on -2% pmol of peptide a-2

shown). A PEB-Cyr rcsidve war assigned to this posilion for this reason and on the basis of the bAmino acid analyrls showed the prwnsc of MC bilin-linked cysttine residue in peprids a-2 (data not

rpccrroxopic pmpenics of this peptide.

TABLE Xlll

Amino Acid Composition and Squcnce Data on Tryptic B i h Peptide WH8020 PE(I1) a-3

. . -: Amino acid Mean nridue valus

7hr CYSOIH' 0.97

0.96 Glu 2.00

1.13 ZY 1.21

LYS 0.87

B I K M to Edman degradation.

0-3 PI glu. cy$-PUB 0-3 P2 glu (I). cyr-PUB. ala (I). thr ( I )

Both 0-3 PI and a-3 P2 were b lxkcd lo Ed- degradation.

Glx-Cyr(PUB)-(ala. thr)-(glx, gly)-lys

Cysteic acld is dcnvcd from a PUB-linked cyrrcinyl residue. Value was obtained fran d hydrolyzate prepared in 6 N HCI conmining 0.21 M dimclhyhulfoxidc.

TABLE XIV

+urnmaled Edman Degradation of Tryptic Bilin Peptide W H 8 M o PEVI) 8-2.

Step Amimacid Yicld(pmo1) Stcp Amimscid Yicld(pm1)

I 2 3 4 5 6 1 8 9 IO I1 I2 13

711 IS!

414 30

178 54 31

65 65

240 30 20

14 I5 16 17 18 19 20 21 22 23 24 25

32 17 34 62 95

4

50 12

18

4

Slep Aminoedd Yild(Fnal) Step Aminowid Y M f p d )

1 Lsu 1436 19 2 85 20 Ala

Val 74 12

21

5 6 Ala

As" I 29 23 113 24

aide b

GI"

3 2 411 4 Val 594 22 ;) 11

8 sa 75 25 26 Ann 18 7 Lsu 633 12

9 ssr 10 ASn

IO1 21 aid 54 28

11 12

Ala Ala 249

67 29 M nu 2 ;;

TABLE XV1

Ahino Acid Comporirion ofTryptic Bilin Peptides Denved from WH8020 P€(l)

residues

CySO3H' 1.3 ( I )b 0.4 1 .2 ASP

0.7 1.8 ( I ) 4.0 (5)

2.0

Thr ssr

1.5 (2) 2.1 (2-3) 0.4 (0) 2.0 (3)

Gi" 2.2(3) 1.8 (2-31 pro ndr nd (I) nd nd 9 1.8 (2-3) 3.5 (3)

nd

1.7 (2) 1.9 (2) 3.3 (4) 3.9 (5)

1.1 (I) 0.6(1) 0.4(1) 3.1 (4)

Val Ma UC

1.0 ( I ) 2.5 (3)

Leu 1.2 ( I ) 0.9 (2) 1.9 (2) TF 0.z (1) 0.7 (1) 0.8 ( I )

Arg 1.0 ( I ) 1.0 1.0 I O 1.0

Phc Methyl-c

0.7 ( I ) 0.8

TABLEXVll

Subm'accivs E d m n Dcgradvtion of Bilin Peptide WH8020 PE(I) 0-1

"Zl"OITWlC$

T F 1.0(1)* 1.7 ( I ) 1.1 ( I ) 0.8 (0.8)

CySO,Hb I 3 (1.3) 1.5 (0.8) 0.0


I A l a 659 I M S 66 I LC" 2 -6

115 2 A ! a 22 2 M a

3 A l a 572 3 A l a 25 3 GI" 199

9 4 Ro 425 4 .b 4 m I5

5 Le" 116 5 llr 6 Cln

25 36 2

9 Asp 5 10 Ala I 8 11 Ala 43 12 I N 149 13 Ma 78 14 Ala 27 I5 GI" I 1 16 Ala 18 17 Gly IO

; :y

18 (GlY) 8 : ;c ;: 21 Asp

1 LC" 28 1

2 Asp 3 Ala

34 I 6Y

4 Val 154

20 m 12 21

23 UC 35 22 E2 3:

5 AS" 125 b 6 Ah 113 25 GI" 2

24

7 I k 5 8 Thr

147 26 Am 85 27 m 2

9 41 IO Am

Scr 1 07

I 1 Ala 31 12 Ser

20 Y

13 b 4 14 UC

32 Ala 65 33 GlY

16 srr 56 35 Am'C 4 17 4 29 36 GI"

37 I 19 Val 42

TF

IS Val I 01 34 WY)

18 2 I O

phycoerythrins of marine unicellular cyanobacteria

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