Structure and Function of Chromophores inR-Phycoerythrin at 1.9 Å ResolutionTao Jiang, Ji-ping Zhang, and Dong-cai Liang*National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

ABSTRACT The crystal structure of R-Phyco-erythrin (R-PE) from Polysiphonia urceolata hasbeen refined to a resolution of 1.9 Å, based on theatomic coordinates of R-PE determined at 2.8 Åresolution, through the use of difference Fouriermethod and steorochemistry parameters restrainedrefinement with model adjustment according to theelectron density map. Crystallographic R-factor ofthe refined model is 0.195 (Rfree 5 0.282) from 8–1.9Å. High resolution structure of R-PE showed preciseinteractions between the chromophores and pro-tein residues, which explained the spectrum charac-teristic and function of chromophores. Four chiralatoms of phycourobilin (PUB) were identified asC(4)-S, C(16)-S, C(21)-S, and C(20)-R. In addition tothe coupling distances of 19 Å to 45 Å between thechromophores which were observed and involved inthe energy transfer pathway, high resolution struc-ture of R-PE suggested other pathways of energytransfer, such as the ultrashort distance betweena140a and b155 . It has been proposed that aromaticresidues in linker proteins not only influence theconformation of chromophore, but may also bridgechromophores to improve the energy transfer effi-ciency. Proteins 1999; 34:224–231.r 1999 Wiley-Liss, Inc.

Key words: R-phycoerythrin; crystal structure;light-harvesting; energy transfer; phy-courobilin

INTRODUCTION

Phycobilisomes, the light-harvesting complexes in bacte-ria and algae, are composed of phycobiliproteins and linkerproteins. Phycobiliproteins all contain different kinds ofchromophores that have similar open-chain tetrapyrrolestructures, linked via thioether bonds to cysteine residues.They are classified by their spectral properties as phycoer-ythrobilin (PEB, lmax 5 560 nm), phycocyanobilin (PCB,lmax 5 620–650 nm), phycobiliviolin (PXB, lmax 5 575 nm)and phycourobilin (PUB, lmax 5 450 nm).1–3 There arethree main kinds of phycobiliproteins, phycoerythrin (PE),phycocyanin (PC), and allophycocyanin (APC). PE is at thetip of the rod-like phycobilisome, PC is in the middle, whileAPC forms a core attached to the reaction center. Energytransfer proceeds successively from PE to PC and to APC.The three-dimensional structures of several phycobilipro-teins have been solved.4–12 All of these proteins show verysimilar structure features. Their functional unit is (ab)3 or

(ab)6 formed by two face-to-face (ab)3 trimers, (ab)3 iscomposed of three (ab)s related by a 3-fold symmetry axis.The molecule is disc-shaped with a central channel, whichhas a diameter of about 35 Å . The crystal structures ofthree PE, B-PE(2.2 Å), b-PE(2.3 Å), and R-PE(2.8 Å) havebeen solved. The subunit composition of B-PE and R-PE is(ab)6g, molecular weights of a,b, and g subunit in R- PEare 18 kDa,19.5 kDa and 33.7 kDa respectively, g subunitwas though to be a hydrophobic linker protein and situatedin the central channel of PE. Since B-PE and R-PE belongto P3 and R3 space group respectively, their centralelectron densities were averaged by three-fold symmetry,so the electron density is disordered and g subunit couldnot be located. The asymmetric unit of R-PE contains onethird of the molecule, (ab)2g1/3, two (ab) heterodimers arerelated by a non-crystallographic two-fold axis in theasymmetry unit (Shown in Figure 1). There are fivechromophores in each (ab) unit of R-PE: a84 PEB and140a PEB are linked to the a subunit; b84PEB, b155PEB,and b50/61 PUB are linked to the b subunit. The 2.8Åresolution structure of R-PE from Polysiphonia urceolatawas solved previously.12 Although valuable informationwas gained from the 2.8Å resolution structure yet, highresolution structure is needed to provide additional infor-mation of primary structure, g subunit and precise interac-tions between the chromophores and protein residues.Precise chromophore–protein interactions which canonly be seen from high resolution structure are crucialto understand the spectrum characteristics of chromo-phores.

Recently, the crystal structure of R-phycoerythrin (R-PE) from Polysiphonia urceolata has been refined to aresolution of 1.9 Å, based on the atomic coordinates ofR-PE determined at 2.8 Å resolution, through the use ofdifference Fourier method and steorochemistry param-eters restrained refinement with model adjustment accord-ing to the electron density map.

Abbreviations: Phycoerythrobilin (PEB), b- Phycoerythrin (b-PE),Phycocyanobilin (PCB), B- Phycoerythrin (B-PE), Phycobiliviolin-(PXB), R- Phycoerythrin (R-PE), Phycourobilin (PUB), C- Phycocyanin(C-PC), Phycoerythrin (PE), Phycocyanin (PC), Allophycocyanin(APC)

Grant sponsor: Chinese Academy of Sciences; Grant number:85KZ04-40; Grant sponsor: National Natural Science Foundation ofChina; Grant number: 39470152.

*Correspondence to: Dong-cai Liang, National Laboratory of Biomac-romolecules, Institute of Biophysics, Chinese Academy of Sciences,Datun Road 15, Chaoyang District, Beijing 100101, China. E-mail:dcliang@sun5.ibp.ac.cn

Received 4 June 1998; Accepted 28 September 1998

PROTEINS: Structure, Function, and Genetics 34:224–231 (1999)

r 1999 WILEY-LISS, INC.

MATERIALS AND METHODS

The crystals of R-PE were formed by the vapor diffusionmethod.12 The crystal belongs to the R3 space group withcell dimensions of a 5 b 5 189.8 Å and c 5 60.1 Å. Thediffraction data were collected at room temperature usinga Siemens (Munich, Germany) X-200B Area Detector witha three-axis camera driven by a PCS computer. The X-raysource was an RU300 (Tokyo, Japan) Rigaku rotatinganode generator operating at 12KW. The distance betweenthe crystal and detector was 16 cm. Crystals were mountedin random orientations. Data were collected in the v rangeof 0 to 360° with a scan (Dv of 0.15°. Data processing wasperformed with the XENGEN package (V1.3). Three setsof data were collected and combined. There were 54,504unique reflections with completeness of 86% from 10 to 1.9Å and 45% from 1.95 to 1.9 Å.

Refinement and Quality of Model

The starting model was the R-PE structure determinedat 2.8 Å resolution and X-plor program13 was used forrefinement. Before refinement the scale factor and thermal

factor were calculated by a statistical method. The struc-tural model with overall B-Factor of 19.6 was refined in thefirst stage. During the refinement, the resolution wassuccessively extended from 2.8 Å to 2.2 Å and 1.9 Å.Tightly constrained stereochemical parameters were usedto maintain the precision of the original model. 2Fo-Fc andFo-Fc electron density maps were observed on a SGIgraphics workstation using the Turbo-Frodo14 program.The positions that deviated from the electron density mapwere adjusted. After individual B factor (Bj) refinementthe R-factor was 22.7%. Three hundred and eighty seven(387) water molecules were introduced into the final modelto give an R-factor of 19.5% and an R-free factor of 28.2%.The r.m.s deviations of bond distances and bond angleswere 0.017 Å and 2.9° respectively (Table I). The Ramach-andran plot of the R-PE model was fairly reasonable,95.1% of residues were in the most favored region and themodel (including 10 chromophores) fit the electron densitymap very well.

Fig. 1. Nomenclature used for subunits 1–6.

Fig. 2. Electron density map of a162Tyr (thick line) with 2Fo-Fc omitmap (thin line).

TABLE I. Parameters of the Refined R-PE Model (a2b2 inAsymmetric Unit)

Number of protein atoms 5110Number of chromophore atoms 430Number of water molecules 387r.m.s. deviation of bond length (Å) 0.017r.m.s. deviation of angle (°) 2.9Resolution (Å) 8–1.9Number of reflections 54504Completeness (%) 86R-free (%) 28.2R factor (%) 19.5

TABLE II. R-PE Sequence in 1.9Å Resolution†

a subunitMKSVITTTISAADAAGRYPSTSDLQSVQGNIQRAAARLEAA-

EKLGSNHEAVVKEAGDACFSKYGYLKNPGEAGENQEKINKC YRDIDHYMRLINYTLVVG-

GTGPLDEWGIAGAREV YRTLNLPSAAYIASFVFMRDRLC IPRD MSAQAGVEYCAALDYL-

INSLS

b subunitMLDAFSRVVVNSDSKAAYVSGSDLQALKTFINDGNKRLDA-

VNYIVSNSSC IVSDAI SGMICNPGLITPGGNCYTNRRMAAC LRDGEIILRYVSYALLAGDA-

SVLEDRCLNGLKETYIALGVPTNSTVRAVSIMKAAAVCFITNTASQRKVEVAEGDC SALA-SEVAS YCDRVVAAVS†Residues changed in 1.9Å structure are emphasized by bold charac-ter. C indicates cysteins bound to chromophores.

225STRUCTURE AND FUNCTION OF CHROMOPHORES IN R-PHYCOERYTHRIN

RESULTS AND DISCUSSIONSequence

The sequence of R-PE from Polysiphonia boldii15 wasused for model building of R-PE at 2.8 Å. The resolutionincrease to 1.9 Å and further refinement provided a morereliable determination of the R-PE sequence. Six residuesin each (ab) unit of R-PE were changed according to theelectron density map, a132Ala = Ser, a136Thr = Met,a162Phe= Tyr, a164Thr= Ala, b145Ser = Thr, andb151Ile= Ala. The revised sequence is shown in Table II.

Figure 2 shows the good fitting of residue a162 to theelectron density map.

Interactions Between R-PE Subunits

High resolution structure of R-PE provides more preciseand reliable information of all kinds of interactions, suchas the interactions between different subunits, differentchromophores and interactions between the chromophoresand protein residues.

Fig. 3. Chemical structure of chromophore PEB (a) and PUB (b).

TABLE III. Interactions Between the Chromophores and Protein Residues

a84 a140a b84 b155 b50

O1D a80 Lys NZ 3.23 a139 Arg NH1 3.20 b79ArgNH2 2.90 b148A Ser N 3.39a139 Arg NH2 3.12 b80ArgNE 3.09 b148A Ser OG 2.73

O2D a80 Lys NZ 2.83 a139 Arg NH1 2.88 b79 Arg NH1 2.76 b147 Thr O 3.38b79ArgNH2 3.20 b148A Ser N 2.92

O1A a86Arg NH1 2.78 a140DArgNH2 2.69 b86ArgNE 2.72 b131 Arg NH1 2.84a86Arg NH2 2.78 b86ArgNH2 2.77 6a147 Gln NE2 2.88

O2A a83Lys NZ 2.62 a140DArg NH1 3.32

NA a86 Arg NH2 3.19 a140DArg NH2 3.32 b86Arg NH2 3.05 b39 Asp OD2 2.91 b54 Asp OD2 2.69a87 Asp OD1 2.90 b87Asp OD1 2.80a87 Asp OD2 3.32 b87Asp OD2 3.10

NB b35 Asn ND2 2.85NC a74 Ala O 3.41 b72Asn OD1 3.04 b151 Ala O 2.80ND a87 Asp OD1 2.85 a139Arg NH2 3.46 b87Asp OD1 2.74 b39 Asp OD2 2.91 b54 Asp OD2 2.81OB 2b77 N 3.00 a47 Asn ND2 3.00 a28 Gln OE1 2.85OC a74 Ala O 3.50 b75Cys S 3.55 b153 Gly N 2.87 b148B Gln N 2.84

a75 Gly N 3.50 b153 Gly O 3.12

226 T. JIANG ET AL.

The asymmetric unit of R-PE contains four subunits,1a,1b,6a, and 6b,1(ab) and 6(ab) are related by a non-crystallographic two-fold axis. The rms deviations be-tween 1a and 6a is 1.040 Å, between 1b and 6b is 1.062 Å.The r.m.s deviations between chromophores are 1a84/6a84(0.215 Å), 1b84/6b84(0.223 Å), 1a140a/6a140a(0.962

Å), 1b155/6b155(0.698 Å), 1b50/6b50(0.236 Å). The r.m.sbetween 1a84/6a84 and 1b84/6b84 are smaller than otherchromophores because they are on the inside surface ofmolecule and have less flexibility.

The interactions between the a and b subunits of C-PC,B-PE, and APC have been studied previously.8,9,11 In R-PE,in addition to the conservative hydrogen bonds a13Asp-b93Arg, a13Asp-b94Tyr, a13Asp-b110Arg, a17Arg-b97Tyr,a3Ser-b3Asp, we found another hydrogen bond betweenthe N atom of a1Met and the O atom of b1Met (3.0 Å). Thishydrogen bond does not exist in APC since compared to PE,APC has two residues deleted in the N-terminal.

The functional unit of R-PE is (ab)6g , with two (ab)3

assembled face-to-face. Analysis of the interactions be-tween these two trimers shows that although thereare more than 200 hydrogen bonds and more than 1000VDW interactions, they exist almost only between aand a subunits or between a and b subunits, but notbetween b and b subunits, indicating that the a subunitand the b subunit have different roles in hexamer as-sembling.

Microenvironment and Conformation ofChromophores

The energy transfer mechanism was explained mainlyby dipole-dipole resonance and exciton interaction theory.16

The formula Ket 5 k2/t0(R0/g)6 is usually used to calculatethe energy transfer rate where: Ket is the energy transferrate, k2 the orientation factor; R0 5 50 Å the Forsterresidue, t0 the fluorescence lifetime, and g the distancebetween two chromophores. These factors are influencedby interactions between the chromophores and proteins.

Fig. 4. The (b50/61) PUB electron density map (thick line) with the2Fo-Fc omit map (thin line).

Fig. 5. Packing of chromophores in the crystal cell.

TABLE IV. DihedralAngles of Chromophores†

f1 f1 f2 f2 f3 f3

a84 PEB 1.61 2176.5 21.94 8.47 87.7 52.9a140a PEB 3.55 2175.2 1.23 3.70 58.4 76.8b84 PEB 23.37 162.2 26.10 19.4 48.5 70.2b155 PEB 28.00 157.0 2.64 1.21 89.8 51.7b50/61 PUB 245.8 2101.7 14.9 5.27 63.6 63.5†The dihedral angles f1, f1, f2 . . ., are defined by the atomsNC-C(4)-C(5)-C(6), C(4)-C(5)-C(6)-ND, ND-C(9)-C(10)-C(11) . . ., etc.

227STRUCTURE AND FUNCTION OF CHROMOPHORES IN R-PHYCOERYTHRIN

It is well known that in phycobiliproteins chromophoreswith the same chemical structures have different spectralcharacteristics because of their different microenviron-ments. The interactions between the chromophores andprotein residues in R-PE are shown in Table III, whichreveal some interesting phenomena.

First, almost all nitrogen atoms ND (in ring B) and NA(in ring C) form hydrogen bonds with Asp or Arg andoxygen atoms O1A (in ring C) form hydrogen bonds withNH1 and NH2 of Arg. Since ring B and ring C areconnected with double bonds to form a conjugate planewith rigidity, the strong interactions between Arg and theatoms in ring B and ring C of chromophore imply thatresidue Arg in R-PE plays an important role in maintain-ing the orientation of chromophores. The orientation factork2 of chromophore may be greatly influenced by Arg. Sincering A of PEB and the rings A and D of PUB combine withthe remaining part of the chromophore through a singlebond (Fig. 3), the flexibility of chromophore and the fluo-rescence lifetime t0 are influenced by the interactionsbetween OB (in ring D), OC (in ring A), and proteinresidues.

Secondly, almost all the chromophores form hydrogenbonds with residues in the same (ab) unit. An exception isthe OB atom (in ring D) of a84PEB, which forms ahydrogen bond with the N atom of b77 of another (ab)subunit. In all phycobiliproteins only the dihedral angle ofb77 is in the unreasonable area of the Ramachandran plot,indicating that the free energy of residue b77 is very high,so this hydrogen bond is not stable and gives ring D of a84PEB some flexibility. Since the conformation of ring Dinfluences the absorption characteristics of a84 PEB, theflexibility of ring D may be necessary for the energyabsorbing and releasing capabilities of chromophore. Inaddition, since this hydrogen bond is formed between twodifferent (ab) units, it is an important factor in the spectralchange when (ab)3 is dissociated to (ab).17–19

Phycourobilin(PUB)

The conformation of PUB joined to b50 and b61 was welldefined in the 1.9 Å resolution structure of R-PE. The fourchiral atoms were identified as C(4), C(16), C(21)-S, andC(20)-R, omit electron density of PUB is shown in Figure 4.The fitting of other chromophores to the electron density isalso satisfactory. The PUB dihedral angle were calculatedand showed that rings A and D deviated from the B, Cplane (Table IV) greater than rings A and D in otherchromophores, so PUB can absorb light of shorter wave-length than other chromophores.

The chromophore linked to b50/61 in B-PE is PEB andthe chemical structural difference between PEB and PUBis shown in Figure 3, where the carbon atom C(4) (in ringA) is a chiral atom in PUB. Compared to b50/b61PEB,b50/b61PUB forms an additional hydrogen bond, which isbetween the OC atom (in ring A of b50/61 PUB) and the Natom (in b148b Gln) with a bond length of 2.84 Å. Thishydrogen bond will influence the conformation of ring A ofPUB and may cause the spectral difference between PEBand PUB.

Energy Transfer

Distances between five chromophores (a84, a140a, b84,b155, b50/61) in a special (ab) unit and other chromo-phores in (ab)6 (Fig. 5) were calculated and shown in TableV. Distances shorter than 30 Å were indicated by boldfigures, suggesting energy transfer pathways as shown inFigure 6.

Within one (ab)3 trimer of R-PE, b50/61 PUB is couplingwith a84, b84, and b155 and plays a role in transferringenergy from the outside surface to inside surface of themolecule and finally to the terminal energy acceptor b84.a84 PEB play a role in passing passes the outside energyfrom b50/61PUB and a140aPEB to b84PEB.

Between two (ab)3 of R-PE, there are only three couplingdistances shorter than 30 Å, i.e. 1a84 = 4a84 (28.7 Å),a140a = b155 (26.5 Å), b155 = b155 (25.9 Å). Althoughthe coupling between 1a84 and 4a84 suggests a possiblepathway of energy transfer on the inside surface of mol-ecule, it should be more complicated because of the exis-tence of g subunit. Early work indicates that linker

TABLE V. Distances Between Chromophores WithinR-PE (Å)†

1. ln (ab)3

1a84 1a140a 1b84 1b155 1b50

1a84 28.2 50.1 49.5 58.51a140a 28.2 63.2 41.8 61.71b84 50.1 63.2 39.5 23.91b155 49.5 41.8 39.5 26.11b50 58.5 61.7 23.9 26.12a84 66.7 92.3 52.0 87.3 75.72a140a 74.3 101.1 76.0 106.2 99.42b84 19.4 43.2 33.6 47.2 47.72b155 40.2 65.9 68.3 84.5 86.62b50 19.0 46.0 57.0 66.1 69.93a84 66.7 74.3 19.4 40.2 19.03a140a 92.3 101.1 43.2 65.9 46.03b84 52.0 76.0 33.6 68.3 57.03b155 87.3 106.2 47.2 84.5 66.13b50 75.7 99.4 47.7 86.6 70.0

2. ln (ab)6

1a84 1a140a 1b84 1b155 1b504a84 28.7 38.2 60.3 58.7 71.44a140a 38.5 54.7 79.1 83.2 93.04b84 60.2 78.8 47.1 72.4 68.54b155 58.8 82.9 72.3 94.1 93.84b50 71.7 93.1 68.8 94.3 91.25a84 74.9 96.2 58.7 89.4 81.75a140a 96.6 117.7 66.8 102.6 88.35b84 58.8 66.8 32.8 46.0 44.55b155 89.4 102.3 46.0 74.8 59.45b50 82.0 88.5 44.9 59.6 52.16a84 69.6 72.4 36.1 40.9 37.66a140a 72.4 65.0 48.1 26.5 33.86b84 35.8 47.6 46.9 52.5 60.76b155 40.8 26.5 52.7 25.9 49.06b50 37.4 33.3 61.1 49.1 66.9†The distance between two C(10) atoms of the chromophores is takenas the distance between two chromophores. Distances shorter than30Å are marked with bold figures.

228 T. JIANG ET AL.

proteins affect the spectral properties of phycocyanin.20 In2.5 Å resolution crystal structure of C-PC,5 ring D ofchromophore b84 is somewhat mobile and is assumed to belack of linker protein.

The electron density of chromophore b84 ring D iswell-defined in 1.9 Å resolution structure of R-PE. Acluster of electron density is near to b84 ring D andextends into the molecule center. It obviously belongs tothe g subunit and is well- defined in Fo-Fc or 2Fo-Fc map(Fig. 7). The confirmation of b84 ring D was restricted bythis density and indicates that g subunit or other linkerproteins can influence the spectral property through chang-ing the microenvironment of chromophores in a and bsubunits. It is interesting that the number of aromaticresidues in g subunit is much higher than other kinds ofresidues and if a phenylalanine residue can be put into theelectron density at the position close to b84 PEB .Weassume that another function of linker proteins is forming

Fig. 6. Possible pathway of energy transfer in R-phycoerythrin.

Fig. 7. Chromophore b84 PEB and the electron density map near thering D of b84 PEB.

229STRUCTURE AND FUNCTION OF CHROMOPHORES IN R-PHYCOERYTHRIN

the aromatic pathway to bridge the chromophores andinfluence the spectrum.

On the outside surface of R-PE, a140a = b155 (26.5 Å),b155 = b155 (25.9 Å) can be regarded as two possiblepathways of energy transfer. Calculation of the distances

between chromophores belonging to different (ab)6 re-veals that these distances are very short. Distance be-tween the nearest two atoms of chromophore b155 anda140a is only 3.4 Å (Fig. 8). Such a short distance makesit possible that the two chromophores form a conjugatesystem which means that the energy transfer betweenthem will be very quick. The arrangement of phycobilipro-teins in phycobilisomes shown by electron microscopy2, 21

are similar to their packing in the crystal. The functionof b155PCB of PC, b155PEB, and a140a PEB of PE wereless known in early studies, now we assume thata140a PEB of R-PE may transfer energy rapidly to b155PCB of PC or to b155 PEB of PE located in the adjacent rod(Fig. 9) because chromophore b155 has the same positionin R-PE and C-PC, it provide energy pathways betweentwo different phycobiliproteins or between two trimers ofR-PE .

CONCLUSION

In addition to the coupling distances of 19 Å to 45 Åbetween the chromophores which were observed and in-volved in the energy transfer pathway, high resolutionstructure of R-PE suggested other pathways of energytransfer, such as the ultrashort distance between a140aand b155. It has been proposed that aromatic residues inlinker proteins not only influence the conformation ofchromophore, but may also bridge chromophores to im-prove the energy transfer efficiency.Fig. 8. Distances between chromophores a140a and b155.

Fig. 9. Sketch of phycobilisome.

230 T. JIANG ET AL.

ACKNOWLEDGMENT

Depositing of the atomic coordinates and structurefactors of R-PE to PDB is in progress.

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231STRUCTURE AND FUNCTION OF CHROMOPHORES IN R-PHYCOERYTHRIN