From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China
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ABSTRACT |
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The crystal structure of allophycocyanin from red
algae Porphyra yezoensis (APC-PY) at 2.2-Å resolution has
been determined by the molecular replacement method. The crystal
belongs to space group R32 with cell parameters a = b = 105.3 Å, c = 189.4 Å, =
= 90°,
= 120°. After several
cycles of refinement using program X-PLOR and model building based on
the electron density map, the crystallographic R-factor converged to
19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 Å. The
r.m.s. deviations of bond length and angles are 0.015 Å and 2.9°, respectively.
In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers.
The chromophore-protein interactions and possible pathway of energy
transfer were discussed. Phycocyanobilin 1 Phycobilisomes are large supramolecular aggregates attached to the
stromal side of the thylakoid membrane in cyanobacteria, red algae, and
cryptomonads. These supramolecular aggregates are light-harvesting
protein pigment complexes that are composed of phycobiliproteins and
linker proteins. Based on the absorption of visible light, the
phycobiliproteins can be divided into three main groups: phycoerythrin
(PE)1 or phycoerythrocyanin
(PEC), phycocyanin (PC), and allophycocyanin (APC). With the help of
linker proteins, phycobiliproteins form the two distinct structural
domains of phycobilisome, the core and the rods. The core, which is
composed of three or more core cylinders associated by APC discs, is in
proximity of the reaction centers, whereas the rods are attached on the
core and are composed of PC discs in the middle and PE or PEC discs on
the tip. Light energy is transferred from PE or PEC via PC to APC and
finally to the reaction centers (1).
The crystal structures of several phycobiliproteins have been solved;
among them, three are PEs (2-4), three are C-phycocyanins (C-PCs)
(5-7), one is PEC:PEC from Mastigocladus laminosus (8), and
one is APC:APC from Spirulina platensis (9). All these structures are very similar. The basic building block is an The crystal structure of APC is very special compared with other
phycobiliproteins. First, the spectrum difference between APC trimer
and its monomer is very large. When APC monomers aggregate to trimer,
the absorption spectrum has a 40-nm red shift; the CD spectrum also
changes a great deal, and exiton interaction in the trimer of APC was
suggested (10), whereas the spectrum difference between C-PC monomer
and its trimer is not so large as in APC, although phycocyanin has the
same Second, the functional unit of APC was thought to be a trimer, whereas
the function unit of other phycobiliproteins were hexamer ( Third, in PE and PC, the two trimeric discs are superimposed along a
3-fold axis, but in PC and APC the two discs are connected perpendicularly. The pathway of energy transfer between PC and APC is
still unknown.
The red algae Porphyra yezoensis is an algae that exists
widely in nature. Its phycobilisomes contain R-PE, C-PC, and APC. In
this paper we report the crystal structure of APC from P. yezoensis (APC-PY) at 2.2-Å resolution. The organization of APC
trimers in the core cylinders of phycobiliproteins and the pathway of energy transfer were discussed.
Crystallization and data collection of APC-PY was reported
earlier (11). The crystals of APC-PY belong to space group R32 with
parameters a = b = 105.3 Å, c = 189.4 Å, Molecular replacement using program AMoRe (12) was carried out using
the 2.3-Å structure of APC-SP as a model. Model cell parameters were
a = b = c = 150.0 Å, The structure was refined using X-PLOR (13). The consensus sequence was
used for the initial model building. Fourier transform and electron
density were first calculated in the resolution range of 10 Å to 3.5 Å. Residues that could not be fitted into the electron density map
were omitted from the phase calculation in the next refinement cycle.
After several cycles of rigid body, positional refinement, and manual
model adjustment, the R-factor dropped to 25.4%, and a 2Fo-Fc Fourier
map looked quite good. Then the resolution was extended to 2.2 Å.
After the chromophores were fitted in the map and followed by several
cycles of positional refinement and model adjustment, the electron
density improved further. At this stage almost all side chains were
well defined except those on the surface. Residue exchanges were
carried out at this stage according to the omit map. After several
cycles of positional refinement and model adjustment, the R-factor was converged to 24.0%, the individual B-factors were then refined, and
the R-factor dropped to 21.5%. 169 water molecules were added to the
model according to the Fo-Fc and 2Fo-Fc maps, and the final R-factor of
the model was 19.3% (R-free factor was 26.9%) in the range of 10 Å to 2.2 Å.
Amino Acid Sequence--
Because the amino acid sequence of APC
from P. yezoensis is still unknown, the following six APC
amino acid sequences were used to get a consensus sequence for model
building. Among these sequences, four are from cyanobacteria,
Anabaena cylindrica (14), Calotrix PCC7601 (15),
Fischerella PCC7603 (16), and Synechococcus PCC6301 (17), and two are from red algae, Aglaothamnion
neglectum (18) and Cyanidium caldarium (19). The
alignment of these six sequences is shown in Table
I.
Quality of the Model--
The final crystallographic R-factor for
APC-PY model is 19.3%, in the range of 10.0 Å to 2.2 Å. The Luzzati
plot gives a mean positional error of 0.26 Å (20). The r.m.s.
deviations of bond lengths and bond angles are 0.015 Å and 2.9°,
respectively. The quality of the final model is summarized in Table
II. The Ramachandran plot shows that all
dihedral angles fall into most favored or allowed regions with the only
exception of
Comparing the crystallographic sequences of the final model of APC-PY
and APC-SP, there are 37 nonidentical residues, 25 in the Molecular Structure--
The asymmetric unit of APC-PY contains
The three-dimensional structure of APC-PY
In the APC-PY crystal, two trimers associate face to face into the
(
The assembly of the hexamer in the APC-PY crystal is similar to that of
C-PC from Fremylla diplosiphon (C-PC-FR) and R-PE from
Polysiphonia urceolata (R-PE-PU) hexamers; two
(
Despite the similarity in assembly in APC-PY, C-PC-FR, and R-PE-PU
hexamers, the superposition of the C
First, the number of the residues involved in the interactions between
the two trimers in APC-PY hexamer is smaller than that in C-PC-FR and
R-PE-PU hexamers, indicating a weaker association. This is consistent
with the calculated accessible areas between the two trimers in
C-PC-FR, R-PE-PU, and APC-PY hexamers. The special polar network
present in C-PC of Agmenellum quadruplaticum (C-PC-AQ),
formed by residues 1
Despite the above difference, the interactions that maintain APC-PY as
a loose hexamer seem still to be the polar and charged interactions
between the two trimers. In APC-PY hexamer, the polar and charged
interactions are 1
Second, in C-PC-FR and R-PE-PU hexamers, the trimer-trimer association
is mediated almost exclusively by polar and charged residues (6), but
in the APC-PY hexamer, some hydrophobic residues are also involved,
such as Chromophores
Chromophores Energy Transfer--
There are 12 PCBs in APC-PY hexamer; the
arrangement of these chromophores is shown in Fig.
7. The theory of shot-distance exiton
interaction (23) and long distance dipole-dipole resonance mechanism
(24) has been used to explain the energy transfer rate between
chromophores.
Inside trimer of APC-PY, the distance between 1
The distances of chromophores between the two trimers in APC-PY hexamer
are similar to those in C-PC-FR and R-PE-PU hexamers. Based on the
1.9-Å resolution crystal structure, the possible pathway of energy
transfer within and between the two trimers of R-PE-PU were discussed
(25). There are three pairs of short distance interactions between two
trimers, such as 1
In addition to the energy pathway composed of chromophores, the
aromatic pathway formed by aromatic residues may play an important role
in energy transfer. The energy transfer from chromophores to aromatic
residues vice versa can be explained by exiton interaction mechanism, because the distances between some chromophores and aromatic
residues, such as Functional Unit--
In the core cylinders of phycobilisomes,
several APC trimers are close together, but the association manner of
these APC trimers is still unknown. Based on dissociation experiments,
it was suggested that allophycocyanin does not form hexamers (27),
because almost all the residues involved in the trimer-trimer
aggregation in C-PC-AQ and C-PC-FR hexamers are not conserved in APC.
Similar conclusions were reported later (6, 9). In the APC-PY hexamer, all the interactions involved in the formation of C-PC-AQ and C-PC-FR
hexamers and all the conserved polar and charged interactions in
C-PC-FR and R-PE-PU hexamers are not present, but APC-PY can still
associate face to face to form a hexamer, which is maintained by some
polar and charged interactions, different from those in C-PC-FR and
R-PE-PU. Because the distances of chromophores between the two trimers
in this hexamer are also adequate for effective energy transfer, we
assume that the loose hexamer may represent the basic unit of APC in
physiological conditions. It is possible that linker proteins may help
to stabilize the loose hexamers.
84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2
of another monomer. In
R-PE and C-PC, chromophore 1
84 only forms 1 hydrogen bond with
2
77 residue in subunit 2
. This result may support and explain
great spectrum difference exists between APC trimer and monomer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
monomer composed of
and
subunits (R-phycoerythrin (R-PE) and B-phycoerythrin (B-PE) have a third subunit
in the center of the
molecule); three
monomers are arranged around a 3-fold symmetry
axis to form an (
)3 trimer or two
(
)3 trimers, which are assembled face to face into an
(
)6 hexamer.
84PCB and
84PCB as APC.
)6 or (
)6
. Brejc and co-workers
solved the structure of APC-SP from blue alga S. platensis
(9) in the unit cell of APC-SP crystal; two trimers are associated in a
"back to back" manner that might represent the assembly state of
APC in nature. Red alga is higher than blue alga in evolution, so it
would be interesting to know the packing of APC from red alga in the
unit cell and in nature.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
=
= 90°, and
= 120°.
=
=
= 90°, integrate radius was 30 Å, and rotation function calculation gave a rather high
coefficient solution,
= 60.07,
= 3.06,
= 88.03, Cc = 20.0. The orientations and positions of one
in the asymmetric unit were determined by the translation function with a high
correlation coefficient of 66.9%. The R-factor in the range from 10 to
4 Å was 36.1%. After rigid-body refinement, R-factor dropped to
33.1%, and the correlation coefficient increased to 71.2%. The
packing of molecules in the unit cell was reasonable.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
The sequence alignment of the APCs
and
subunit sequences is shown of APCs from
A. cylindrica (ANCY), Calotrix PCC7601 (CALO), Synechococcus
PCC6301 (SYNO), M. Laminosus (MALA), C. caldarium
(CYCA), and A. neglectum (AGNE). The consensus sequence
(CONS) was derived from the alignment. The model sequence (SEQ) is
based on the electron density. Conserved amino acids are marked in bold
letters.
77Thr (Fig. 1) (21), which has a conserved unusual dihedral angle in all known
phycobiliprotein structures. In APC-PY, the N atom of
77Thr forms a
hydrogen bond with OD (the oxygen atom in the ring D of chromophore)
oxygen atom of
84PCB. The electron density of this residue is well
defined in APC-PY. The consensus amino acid sequence (Table I) was used to build the initial model and later modified according to the electron
density map. In the 2.3-Å-resolution crystal structure of APC-SP (9),
28 residues were not well defined with 102 atoms of zero occupancy;
these residues are
25 Asp,
35Glu,
36Arg,
49Glu,
50Arg,
53Lys,
54 Gln,
76 Tyr,
79 Asp,
120Lys,
127Glu,
2
Gln,
10Asn,
17Lys,
20 Asp,
25 Gln,
35Glu,
36Leu,
39Arg,
50Asn,
58Lys,
65 Asp,
68Arg,
116Lys,
117Glu,
131 Gln,
138Glu,
150Lys. The fit of these residues
to our electron density map is better in APC-PY; most of them behave
well at 1
density level (Fig. 2);
others behave well at 0.7
density level except
76 in the loop,
which has a density at 0.5
density level.
Parameters of refined APC-PY model
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Fig. 1.
Ramachandran plot of the APC-PY
residues. Glycine residues are marked as squares.
Nonglycine residues are marked as crosses. 77 (277) is in
an unusual region. PSI and PHI are dihedral
angles
and
.
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Fig. 2.
Omit electron density map of residues
35Glu and
36Leu in
APC-PY.
subunit
and 12 in the
subunit (Table III). In
comparison with other APC sequences, the 37 residues of APC-PY are more
conserved than those of APC-SP. For example,
52Val and
61Gln of
APC-PY are identical to other known sequences. The electron density of these two residues in APC-PY are well defined.
Sequence comparison of APC-PY and APC-SP
and
subunit. The
subunit is composed of 160 residues, and
the
subunit contains 161 residues. Three
monomers are
arranged around a 3-fold axis to form a disc shaped
(
)3 trimer of 30 Å in thickness and 110 Å in
diameter with a cave in the center. The
and
subunits in the
monomer have similar structures, with nine
-helices (X, Y, A,
B, E, F, F', G, H) separated by irregular loops (Fig. 3).
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Fig. 3.
Ribbon representation of APC
subunit (a), APC
subunit (b).
and
subunits are very
similar to the known structure of APC-SP. The intersubunit interactions
within the (
) monomer and the (
)3 trimer are also very similar to these of APC-SP and other phycobiliproteins. In
the (
) monomer of APC-PY, the ionic- and polar-interacting residues between the two subunits are
3 Ser-
3 Asp,
13
Asp-
94 Tyr,
13 Asp-
110Arg,
17Arg-
97 Tyr,
18
Tyr-
93Arg,
13 Asp-
93Arg,
13 Asp-
97 Tyr,
18 Tyr-
89 Asp.
)6 hexamer through a crystallographic dyad
perpendicular to the triad. There are three (
)6
hexamers in a unit cell, locating at (0,0,0), (2/3, 1/3, 1/3), and
(1/3, 2/3, 2/3) (Fig. 4a). The assembly of two trimers in this hexamer is completely different from that of APC-SP. In APC-SP crystal, the two trimers are
associated loosely through
subunits in a "back to back" manner
(Fig. 4b) in the hexamer (9), but in APC-PY crystal, the two
trimers in the hexamer contact through
subunits, and the assembly
of the two trimers is much tighter than that in APC-SP.
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Fig. 4.
a, packing of APC-PY in the unit cell.
b, packing of APC-SP in the unit cell.
)3 trimers associate face to face in the hexamer.
The
subunits provide the contacting surface, and the two trimers
fit complementarily in the hexamer.
atoms of APC-PY and
C-PC-FR hexamers shows that the assembly of the two trimers in APC-PY
hexamer is obviously looser than that in C-PC-FR and R-PE-PU hexamers.
The calculated accessible areas between the trimers in C-PC-FR and
R-PE-PU hexamers are about 5900 and 6900 Å2. In APC-PY
hexamer, this value is about 3200 Å2, which is much bigger
than that in APC-SP (600 Å2); thus, the APC-PY hexamer can
be described as a "loose hexamer." The interactions between the
trimers in APC-PY hexamer are different from those in C-PC-FR and
R-PE-PU hexamers.
46 Asn-6
164 Asn-1
21 Asn-6
161 Glu-6
33
Glu-6
30 Arg (6) is not conserved in APC-PY hexamer. In addition, the
electrostatic interactions between 1
2Lys-6
23Glu, 1
17Arg-6
108 Asp, and 1
120Arg-4
174 C-terminal carboxyl
group, which were suggested to be involved in the hexamer formation in C-PC-FR (22), are also not present in APC-PY hexamer. Furthermore, the
comparison of APC-PY with C-PC-FR and R-PE-PU reveals that all the
conserved polar and ionic interactions between the two trimers in
C-PC-FR and R-PE-PU hexamers are not present in APC-PY hexamer.
25 Asp-6
37Arg, 1
22 Gly-6
26Arg, 1
25
Asp-6
161Glu, 1
25 Asp-6
165 Tyr, and 1
28Lys-6
147 Asp. In
APC-SP, only a few polar and charged interactions (<4 Å) exist between the two trimers, such as 1
65 Asp-6
131 Gln and
1
120Asn-6
120Asn, indicating a very loose packing.
21 Pro,
22 Gly,
104 Val,
164 Phe,
42 Ala, and
46 Ala.
84PCB and
84PCB--
In APC, two
phycocyanobilins are covalently bound to cysteine residues at position
84 and
84 (Fig. 5). Both
chromophores are well defined in APC-PY (Fig.
6). The geometry and protein environment
of these two chromophores resemble those of APC-SP. The
84 PCB
chromophores have a protein environment similar to that of
84PCB.
The polar and ionic protein-chromophore interactions in
84PCB and
84PCB are shown in Table IV.
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Fig. 5.
Chemical structure of PCB.
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Fig. 6.
The coincidence of chromophores in APC-PY
with 2Fo-Fc electron density map 84PCB
(a),
84PCB
(b).
The polar and ionic protein-chromophore interactions in APC-PY (Å)
84PCB and
84PCB have similar hydrophobic
environment; there are three aromatic residues close to
84, such as
90 Tyr,
91 Tyr, and
119 Tyr, and three close to
84, such as
90 Tyr,
91 Tyr, and
119 Tyr. In C-PC-FR,
90 and
91 are all Tyr, and
90 and
91 are all Ile. In R-PE-PU,
90 and
91 are His and Tyr, respectively, and
90 and
91 are all Ile. But in
all known APCs,
90,
91,
90, and
91 are all Tyr. So the microenvironment of
84 and
84 in APC-PY is similar to that in C-PC-FR and R-PE-PU, indicating that
84PCB and
84PCB have similar conformation and spectrum character.
90 Tyr and
91 Tyr stabilize the
84PCB ring D conformation, which may make PCB have different spectrum characteristics in APC and C-PC.
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Fig. 7.
The chromophores of APC-PY hexamer.
84PCB and 2
84PCB
in APC-PY is about 34 Å and that between 1
84PCB and 2
84PCB is
about 21 Å. These values are similar to those in C-PCs. The chromophores are too far away to have exiton interaction. It is also
difficult to explain why exiton interaction exists in APC but not in
C-PC. Our study of chromophore-protein interactions and comparison of
microenvironments in R-PE-PU, C-PC-FR, and APC-PY show that almost all
the chromophore-protein interactions exist within the same monomer
(
), the only exception being
84PEB in R-PE-PU, which forms a
hydrogen bond with
77 Thr in another monomer. In C-PC-FR, the
situation is the same as in R-PE-PU. However, it is different in
APC-PY; its
84PCB forms five hydrogen bonds with the residues in
other monomer, such as
84PCB O2B-2
62 Tyr OH, O1C-2
62 Tyr N,
O1C-2
67 Thr OG1, O2C-2
67 Thr OG1, and OD-2
77 Thr N (see Table
IV). We believe this difference may explain why the spectrum of APC
changes greatly when its monomers associate to trimer. In APC-SP,
distances of
84PCB O2B-2
62 Tyr OH, O1C-2
62 Tyr N, O1C-2
67
Thr OG1, O2C-2
67 Thr OG1, and OD-2
77 Thr N are all within the
distance of hydrogen bond formation. As we know,
62 Tyr and
67
Thr are close to chromophore
84PCB and may control the conformation
of chromophore and bridge between
84 PCB and
84 PCB to make the
exiton interaction occur.
84
4
84,1
140a
6
155, and 1
155
6
155.
84PEB is on the inner
surface of R-PE-PU, and 1
84PEB
4
84PEB may be the
dominant energy transfer pathway between the two trimers. Similar
energy pathways (1
84PCB
4
84PCB) also exists in
C-PC-FR hexamers (26). In C-PC-FR, R-PE-PU, and APC-PY hexamers, the
distances between 1
84 (C10 atom) and 4
84 (C10 atom) are 27.5, 28.7, and 30.3 Å, respectively, which are comparable. Therefore, the
distance between the two chromophores of APC-PY seems adequate for
effective energy transfer.
84PCB-
90 Tyr,
84PCB-
91 Tyr,
84PCB-
90 Tyr,
84PCB-
91 Tyr are very short (~4 Å).
Förster's dipole-dipole resonance transfer can occur between
different aromatic residues rather than between chromophores and
aromatic residues, because the overlap integral between chromophore
absorption (
max
650 nm for fluorescence spectrum)
and aromatic residue emission (
max
300 nm for
fluorescence spectrum and
max
400 nm for
phosphorescence spectrum) is quite small. In APC-PY there are two areas
abundant in aromatic residues as shown in Fig.
8. One is close to chromophore
84PCB
and composed of
164 Phe,
165 Tyr,
166 Phe,
168 Tyr,
90
Tyr,
91 Tyr,
97 Tyr,
119 Tyr, and
18 Tyr. Another is close to chromophore
84 and composed of
165 Tyr,
166 Phe,
168
Tyr,
90 Tyr,
91 Tyr,
94 Tyr,
97 Tyr,
119 Tyr, and
18
Tyr.
164 Phe is involved in the hydrophobic interactions between the
two trimers. The aromatic residues in the
subunit and the
subunit have high homology and similar locations. Other aromatic
residues are on the periphery of the disc, such as
76 Tyr,
60
Phe,
76 Tyr,
62 Tyr,
30 Tyr,
31 Phe,
30 Phe, and
81
Tyr; among them
76 Tyr and
62 Tyr may mediate the energy transfer
between 1
84PCB and 2
84PCB. Because PC and APC are connected
perpendicularly in vivo, aromatic residues on the periphery
may mediate the energy transfer from the chromophores of PC to those of
APC.
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Fig. 8.
The location of aromatic residues in
APC-PY.
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ACKNOWLEDGEMENT |
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We thank Professor Lu-Lu Gui, Institute of Biophysics, Chinese Academy of Sciences and Professor You-Shang Zhang, Institute of Biochemistry, Chinese Academy of Sciences for their support and concern.
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FOOTNOTES |
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* This work was supported by Chinese Academy of Sciences (KJ85-04-40) and the National Natural Science Foundation of China (39630090).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: National Laboratory of
Biomacromolecules, Institute of Biophysics, Chinese Academy of
Sciences, 15 Datun Rd., Chaoyang District, Beijing 100101, China. Tel.:
86-10-64889867; Fax: 86-10-64889867; E-mail:
dcliang{at}sun5.ibp.ac.cn.
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ABBREVIATIONS |
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The abbreviations used are:
PE, phycoerythrin;
APC, allophycocyanin;
PC, phycocyanin;
C-PC, C-phycocyanin;
PEC, phycoerythrocyanin;
APC-PY, allophycocyanin from P. yezoensis;
APC-SP, APC from S. platensis;
C-PC-AQ, C-PC
from A. quadruplaticum;
C-PC-FR, C-PC from F. diplosiphon;
R-PE-PU, R-PE from Polysiphonia urceolata;
PCB, phycocyanobilin;
, 1
, 2
, 2
, ... 6
stand for
the individual subunits of different monomers within the hexamer
(according to Schirmer et al. (6));
r.m.s., root mean
square.
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REFERENCES |
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