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Biochimica et Biophysica Ac
Local protein flexibility as a prerequisite for reversible chromophore
isomerization in a-phycoerythrocyanin
Marius Schmidt a,*, Angela Krasselt a, Wolfgang Reuter b,*
a Physik-Department E17, Technische Universitat Munchen, James Franck Strasse, 85747 Garching, Germanyb Max Planck Institut fur Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany
Received 3 June 2005; received in revised form 13 October 2005; accepted 27 October 2005
Available online 21 November 2005
Abstract
Phycoerythrocyanin is the only cyanobacterial phycobiliprotein containing phycoviolobilin as a chromophore. The phycoviolobilin
chromophore is photo-reactive; upon irradiation, the chromophore undergoes a Z/E-isomerization involving the rotation of pyrrole-ring D. We
have determined the structure of trimeric phycoerythrocyanin at three different experimental settings: monochromatically at 110 K and 295 K as
well as with the Laue method at 288 K. Based on their chemical structures, the restraints for the phycoviolobilin of the a-subunit and for the
phycocyanobilin chromophores of the h-subunit were newly generated, which allows a chemically meaningful refinement of both chromophores.
All three phycoerythrocyanin structures are very similar; the subunits match within 0.5 A. The detailed comparison of the data obtained with the
different measurements provided information about the protein properties around the phycoviolobilin chromophore. For the first time, crystals of a
phycobilisome protein are used successfully with the Laue technique. This paves the way for time-resolved macromolecular crystallography,
which is able to elucidate the exact mechanisms of the phycoviolobilin photoactivity including the protein involvement.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Laue crystallography; Phycobiliprotein; Phycoerythrocyanin; Phycocyanin; Z/E isomerization; Photo-activity
1. Introduction
Phycobilisomes are high molecular fluorescent protein
complexes present in cyanobacteria and eukaryotic red algae
[1] and are mainly used as antenna pigments for photosynthetic
light collection. They absorb light in portions of the visible
spectrum poorly utilized by chlorophyll and convey the
excitation energy to the photosynthetic reaction centers. As
prosthetic groups, all phycobiliproteins contain linear tetra-
pyrroles (bile chromophores) that are covalently attached to the
proteins via thioether bonds. The spectral properties of the
different complexes are determined by the chromophore species
and the chromophore–protein interactions. Yet, five main
classes of chromophores, phycochromobilin (PAB), phycocya-
nobilin (PCB), phycoviolobilin (PVB), phycoerythrobilin
(PEB) and phycourobilin (PUB), each characterized by typical
1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2005.10.022
* Corresponding authors. M. Schmidt is to be contacted at tel.: +49 89 289
12550; fax: +49 89 289 12548. W. Reuter, tel.: +49 89 8578 2707; fax: +49 89
8578 3516.
E-mail addresses: [email protected] (M. Schmidt),
[email protected] (W. Reuter).
spectral properties, have been described [2]. PAB and PVB
attract special interest, since they are able to perform a light-
induced Z/E-isomerization within the protein environment.
The apical protein complex in the phycobilisome of
Mastigocladus laminosus is phycoerythrocyanin (PEC), and
the ring-like structure comprises three heterodimeric ah-substructures, (ah)3-PEC [3]. The h-subunit contains two
phycocyanobilin chromophores at positions Cys82 and
Cys153, whereas the a-subunit (a-PEC) bears a single
phycoviolobilin chromophore at position Cys84. To understand
the mechanism of the efficient energy transfer from the light
absorbing phycobilisomes to the photosynthetic reaction center,
the atomic structures of the phycobiliproteins and the chromo-
phores involved have to be known. High-resolution X-ray
structures of various phycobiliprotein complexes such as
phycoerythrin (PE), PEC, phycocyanin (PC), allophycocyanin
(APC) and others are presently available [4].
The light induced Z/E-isomerization between rings C and D
of the PVB accounts for the changing spectral properties of the
a-PEC [5]. At present, the molecular mechanisms, which are
important for the transformation of PVB within the protein
ta 1764 (2006) 55 – 62
http://www
Fig. 1. (a) Crystals of phycoerythrocyanin from Mastigocladus laminosus. (b)
Laue exposure on a phycoerythrocyanin crystal.
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–6256
environment, are almost unknown. Unlike a-PEC, the natural
occurring trimeric (ah)3 and hexameric (ah)6 phycoerythro-
cyanin complexes can only be partially shifted (approximately
10%) between the Z- and E-conformation. This behavior has
been explained by the influence of multiple interactions of the
subunits in the assembled state [6,7]. However, the possibility
exists that the spectral change happens transiently, which can
only be followed by time-resolved methods. The time-resolved
pump-probe experiments on isolated (ah)3-PEC characterized
the Foerster energy transfer between the chromophores on the
femto- and pico-second time scale [8]. However, the structural
changes usually happen on longer time scales and the time-
window must be enlarged to nano-seconds.
In the last decade, the traditional static X-ray structure
analysis has been extended by time-resolved methods [9], and
the transiently occupied structures of short-lived states can be
determined [10,11]. A prerequisite, however, are single
crystals of sufficient quality that can be investigated with
the Laue method [12]. Here, we present Laue investigations at
288 K on PEC crystals that are well suited for this type of
experiment. The structures are compared with those derived
by standard monochromatic techniques at 295 K and the
cryogenic temperature of 110 K. The results are an important
step towards the structural understanding of the Z/E-isomer-
ization of phycoviolobilin within the protein environment of
a-phycoerythrocyanin.
2. Materials and methods
2.1. Purification of PEC
The PEC used for crystallization was purified from isolated phycobilisomes
ofM. laminosus (genus Fischerella PCC7603) as developed previously [13,14].
The phycobiliproteins were separated by DEAE-Trisacryl chromatography.
Approximately 20 mL of the adsorbed phycobilisome solution was eluted with 2
mM potassium phosphate, pH 7.0 at a flow-rate of 180 mL/h until the solution
became pink. This PEC-fraction was concentrated and precipitated with
potassium phosphate, final concentration 1.8 M, and stored at 4 -C [14].
Precipitated phycoerythrocyanin was sedimented by centrifugation at 70,000�g
for 25 min at 10 -C. Subsequently, the PECwas redissolved in 20 mM potassium
phosphate and the sample was finally purified by gel filtration on a Superdex
200 pg column (Pharmacia) in 20 mM potassium phosphate, pH 8.2. The
resulting homogenous trimeric linker-free PEC-fraction was concentrated by
ultrafiltration (Amicon, Centricon), and the SDS-PAGE (18%) revealed the
purity of this PEC-solution. The crystallization was performed with a protein
concentration of 20.3 mg/mL.
2.2. Crystallization of PEC
Crystals of PEC were grown by the hanging drop method. An equal amount
of precipitant containing 5% PEG 4000 and 5 mM potassium phosphate was
added to droplets of 4 AL containing 20.3 mg protein/mL. Note, compared to
Rumbeli et al. [15], we used a three times higher protein concentration for the
experiments. The protein crystallized at pH 8.5 and 4 -C. The intensely pink
crystals reached their optimal size (0.2�0.2�0.3 mm) within ¨14 days (see
Fig. 1).
2.3. X-ray structure analysis
For the experiments at room temperature, the hexagonal PEC crystals were
sealed in glass capillaries of 1.5 mm diameter. Laue X-ray diffraction data were
collected at the BioCARS 14ID-B beamline at the Advanced Photon Source
(APS, Argonne, USA) using radiation from APS undulator A and a Mar350
image plate located 250 mm from the crystal. The synchrotron was operating in
the hybrid mode. A Laue diffraction pattern (Fig. 1b) was obtained during a
shutter opening of 21 ms. 25 still exposures with a 3- rotation between
exposures comprised a data set. Laue data were reduced to 2.8 A by LaueView
[16]. Monochromatic data were collected on an FR591 rotating anode X-ray
home source (Bruker-Nonius, Karlsruhe) equipped with a SAXI multi-wire
detector (Bruker, Karlsruhe). The data were integrated and scaled by Fsaint_
(Bruker, Karlsruhe) and further processed by the CCP4 [17] programs Fscala_
and Ftruncate_. For data collection at cryogenic temperatures the crystals were
soaked for 10 s in a solution containing 20% PEG 4000 and 15% PEG 2000
and immediately frozen at 110 K in a cryogenic nitrogen gas stream. The
typical exposure time for one image was 20 min per 0.2- rotation.
For the refinement, the PEC structure published by Duerring et al. [3] was
used as a start and initially fitted as a rigid body to the diffraction data. After a
2000 K simulated annealing protocol, the structures were iteratively refined
following model manipulation with ‘‘XtalView’’ [18] and water search. All
refinement was performed with CNS [19].
3. Results and discussion
The a-subunit of phycoerythrocyanin and the physiologi-
cally very important photoreceptors of the phytochrome-family
perform a similar reversible photochromic Z/E-isomerization
of their phycobilin chromophores [2,5]. Whereas different
authors have intensively investigated the photochemistry using
various spectroscopic methods, the involvement of the proteins
in the molecular mechanisms is almost unknown [5,7,20],
because suitable methods for such investigations are still rare.
Table 1
X-ray data collection and refinement statistics for structures PECM_110K,
PECM_295K and PECL_288K
Data collection PECM_110K M PECM_295K M PECL_288K L
Temperature [K] 110 295 288
Resolution [A] 2.85 3.0 3.2
Completeness [%] 93.4 83.0 63.0
Rmerge 9.9 13.0 16.3
Cell parametersa
a =b, c 155.0, 39.6 156.75, 40.2 156.75, 40.2
Refinement
# atoms, protein 2660 2660 2660
H2O 450 181 219
Rcryst/Rfree [%]b 22.2/27.5 19.0/26.8 20.0/27.5
M: monochromatic; L: Laue.a Space group P63, a =b =90-, c =120-.b Rfree based on 5% of the data selected by the CNS-script make_cv or the
CCP4 program freerflag.
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–62 57
Beside the NMR, which is not applicable for large proteins,
time resolved X-ray crystallography with the Laue method
offers the possibility to follow the complex interactions within
and between the proteins and the chromophores [11,21,22].
The present study is the first step towards a time resolved X-ray
analysis, which may be able to characterize the complex
protein–chromophore interactions that contribute to the re-
versible Z/E-isomerization of PEC. Five basic points should be
elucidated by the comparison of static and Laue X-ray
measurements on PEC crystals and by the data of presently
known structures of phycobiliproteins: (1) Is it possible to
collect an appropriate set of Laue data? (2) Are the structures of
the chromophores similar or even identical at the three
measurement conditions? (3) How does the space group
influence the flexibility of the protein and that of the
chromophore environments? (4) Are there any potential protein
moieties, which are candidates for the modulation of the
photochemical activity? (5) Are there particular properties of
the PVB chromophore, which are essential for the molecular
mechanism of the isomerization?
The study does not deal with the detailed molecular
description of PEC, since most details of the structure have
been already presented [3]. However, in this study the small
gaps in the amino acid sequences of the a- and h-subunits are
Table 2
Comparison of the deviations in the Ca–Ca distances and the B-factors extrac
respectively
Entries on the lower left side of diagonal: Mean Ca–Ca distance differences [A] be
difference in B-factor <DB> [A2] between the subunits, from refinement to differen
closed by the complete gene sequence [23], which was not
available for the first X-ray structure of PEC [3]. Nevertheless,
the structures of the present and of the previous study [3]
correspond very well.
3.1. Methological and structural aspects
The phycobilisomes of M. laminosus cells are well
characterized. ‘‘Maximal phycobilisomes’’ obtained at low
light and high-temperature conditions contain up to 25%
PEC [13]. Despite this relative high content, it is difficult to
prepare a homogenous phycoerythrocyanin sample which can
be crystallized successfully. This is due to the sensitivity of
PEC to artificial modifications during purification and storage.
The complex is only stable at high protein concentrations in the
presence of the respective natural linker polypeptides. In the
trimeric state without the linkers, the sample must be used
directly for the crystallization attempts. However, the crystal-
lization and the quality of the crystals are also problematic.
Recently, a partial modification of the isolated a-subunit, most
probable an oxidation, during crystallization has been reported
[14]. The different qualities concerning diffraction and
mosaicity of the PEC crystals in one droplet (Fig. 1a) probably
result from this.
At room temperature, the PEC crystals show a rapid
degradation upon X-ray exposure. Due to this fact, the
exposure to X-rays was kept low enough to use only one
crystal for a data set, each. Duerring et al. [3] used four crystals
to collect a complete data set, which explains the higher
resolution of their structure analysis at room temperature.
The results from the X-ray diffraction experiments are
summarized in Table 1. At 110 K resolution and data quality is
highest as judged by the completeness and the Rmerge. The
structure (ah)3M�110K lies remarkably well in the electron
density. Only a loop consisting of the residues from a-Pro64 to
a-Ala75 shows very week density. Consequently, the B-values
refine to high values. Despite of the lower resolution, the
stretch of 12 residues, which is found rather disordered in
(ah)3M–110K, could easily be traced in the structure of
(ah)3M–295K.
The Laue-diffraction pattern in Fig. 1b shows reflections
which are spatially confined and which are not radially
elongated; a consequence of the excellent mosaicity of the
ted from the structure analyses of PECM_110K, PECM_295K and PECL_288K,
tween the a- and h-subunits. Entries on the upper right side of diagonal: mean
t experimental data.
Fig. 3. Stereo view of the chromophore structures from phycoerythrocyanin
of Mastigocladus laminosus. (a) PVB chromophore (magenta) at position
Cys84 and its surrounding residues within the a-subunit. (b) Phycocyanobilin
(PCB) chromophore (cyan) at position Cys82 and its surrounding residues
within the h-subunit. 2mFo-DFc electron density maps (blue) on 1 j level
The figure was produced with FO_ [39].
Fig. 2. Chemical structure of PVB and PCB. Blue circles mark differences at
C2, C3, C4 and C5. (a) Phycoviolobilin. (b) Phycocyanobilin.
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–6258
analyzed PEC crystals. The mosaicity of a similar crystal was
determined to be ¨0.1- on a home source. For a thorough
discussion of Laue data reduction and a discussion of assessing
the data quality see references [10,24–29]. Although reflec-
tions are detected to 2.8 A in the Laue exposures, the
completeness drops dramatically beyond 3.2 A to only 15%
at 2.8 A. Hence, the data statistics are given only to 3.2 A in
Table 1. However, all observed reflections to 2.8 A were
included in the refinement. Despite the apparent lower data
quality, the structure (ah)3L–288K fits quite well, and there are no
large differences to that of (ah)3M–295K.
The structures were superimposed and the best match
yielding minimal Ca–Ca distances was determined by
‘‘lsqman’’ [30]. The results are summarized in Table 2. The
distance deviations in the order of 0.4 A between the
corresponding Ca-atoms of the structures are very small (see
Table 2, lower left side of the diagonal bar). Somewhat larger
differences in the order of 1.7 A arise if the a-subunit is
matched to the h-subunit. Hence, the Ca-chains of the a- and
h-subunits are not identical but very closely related. Similar
results were obtained by comparing the structures from
different experimental conditions (Table 2) showing that all
data acquisition methods generate structures of comparable
quality.
Fig. 2 compares the chemical structures of the phycoviolo-
bilin (PVB) chromophore attached to Cys84 of the a-subunit
and of the phycocyanobilin (PCB) bound to Cys82 and Cys153
of the h-subunit, respectively. The chromphores are depicted in
similar orientations as found in the X-ray structures. PVB as
well as PCB are in the Z-form, which can be easily verified by
rotating ring D around the single bond between atoms 14 and
15. The configurations of PVB and PCB differ at four locations
(blue circles in Fig. 2). This affects the angles, the dihedral
angles and the improper angles within the structures of the
chromophores. If the carbon atom is sp2 hybridized (double
bond), the structure is planar with ¨120- angles between the
substituents. If the carbon is sp3 hybridized, tetraeder angles
close to 109.5- are expected. Since the structure file of PVB is
not available in the protein data bank [31], we generated the
topology and parameter files for CNS from scratch. For this
purpose, we constructed a PVB molecule in ACD/ChemSketch
according to Foerstendorf et al. [20]. This program employs a
modified Charmm force field [32] to optimize the chemical
properties such as angles and bond distances of the molecule.
The optimized molecule was read into xplo2d [30], which
created the desired files. Since only minor changes were
required, we repeated this procedure for PCB although PCB is
available from the pdb data bank. With restraints adopted from
methionin, the chromophores were attached to the cysteins, and
these constructs could be treated in the refinement as if they
were amino acid residues.
3.2. Functional aspects
In principle, the backbones of both subunits are segment-
ed into nine a-helices, the two N-terminal helices H1 (X)
.
Table 3
Chromophore–protein interactions
aPVB84 d [A]
Ring A No hydrogen bonds
Ring B Propionyl-O1-h..Arg57Nh2 3.4
Ring-N-aTyr129OD 3.5
Ring C Propionyl-O1-aLys83N~ 2.9
Propionyl-O1-aArg86ND1 2.9
Ring-N-aAsp87Oy2
Ring D Ring-N-h..Gln79O(1 3.5
Ring-Carbonyl-O-h..Gln79O(1 3.3
Ring-Carbonyl-O-h..His75N 3.0
bPCB82Ring A No hydrogen bonds
Ring B Propionyl-O1-hAsn78Ny2 3.5
Propionyl-O2-hArg77ND1 3.0
Ring-N-hAsp85Oy1 3.1
Ring C Propionyl-O1-hArg84ND2 2.8
Ring-N-hAsp85Oy1 2.7
Ring D No hydrogen bonds to the protein;
exposed to the water space
bPCB153Ring A Ring-N-hThr149O 3.0
Ring-Carbonyl-O-hGly151O 2.6
Ring B Ring-N-hAsp39Oy1 2.6
Ring C Ring-N-hAsp39Oy1 2.7
Propionyl-O1-hThr149Og 2.9
Ring D Strongly sandwiched between
residues aPhe28, aLeu24, hAsn42,h..Ser154; no direct hydrogen bonds
Hydrogen bonds between polar groups with distances smaller than 3.6 A are
listed. The double dagger (..) indicates inter-subunit contacts between
symmetrically equivalent heterodimers that assemble to the (ah)-trimeric
PEC ring.
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–62 59
and H2 (Y) and the following seven (six) globin-like folded
helices H3–H9 (A, B; E–H). The nomenclature is adopted
from the pdb data bank. The letter code in parentheses
originates from one of the first X-ray structures of
phycobiliproteins [33]. Generally, H1 and H2 are responsible
for the assembly of the a/h-heterodimers and the first
chromophore-binding site is always located at helix H5
[3,14,34].
Fig. 3 compares the refined Cys84-PVB and Cys82-PCB a-
and h-chromophores embedded in their electron densities. The
Fig. 4. Comparison of the temperature factors of the different experiments on phyco
PECM_295K; light grey: PECL_288K K. Arrows mark the binding sites of the chromo
chromophores are refined in the Z-configuration (compare
Figs. 2 and 3). Both ‘‘monochromatic’’ structures as well as the
structure obtained from the Laue data are very similar,
therefore only the chromophore structures from the 110 K
data are displayed in stereo. The chromophore stabilizing
amino acids a-Lys83, -Arg86, -Asp87 and -Tyr129 for PVB as
well as h-Arg77, -Arg84, -Asp85 and -Tyr117 for PCB are also
shown. With the exception of the tyrosins, these amino acids
are located in the N-terminal part of helix H5. They interact via
hydrogen bonds with the propionic acid group of the pyrrole-
ring B and the nitrogens of the rings B and C. This arrangement
is a common principle at least in PEC- and PC-subunits [3]. In
addition to the very similar interactions of both chromophores
with their own subunits, the PVB chromophore is stabilized by
H-bonds to the neighboring h..-subunit, which is only present
in the trimeric assembled state of PEC. Hydrogen bonds
between h..-Arg57 and the propionic acid group of ring C
were identified. Ring D is stabilized by interactions with h..-
Gln79O(1 (3.3 A to the ring O and 3.5 A to the ring N) and by a
hydrogen bond to the backbone N of h..-His75 (3.0 A). Most
probable, the latter bonds account for the strongly depressed
photoactivity of the PVB chromophore within the trimeric
assembly of PEC [5,6]. Since the chromophore conformations
shown in Fig. 3 are quite similar within the protein
environment, the large spectral differences between PVB and
PCB mainly derive from the reduced conjugation of the k-electrons in phycoviolobilin.
A detailed description of the chromophore protein interac-
tion is necessary to understand why the PCB chromophores
appear photo-inactive, whereas the PVB chromophore shows
10% photoactivity in trimeric PEC and even 100% in isolated
a-subunits [6]. In principle, a Z/E-isomerization of PCB by a
rotation around the double bond between the atoms 15 and 16
is conceivable (see Fig. 2). In the hPCB-153 chromophore the
pyrrole-ring D and the entire chromophore are strongly
restrained by protein interactions (see Table 3). Therefore, a
large displacement of this chromophore is unlikely. In contrast,
the hPCB-82 D-ring shows no interactions with the protein
(see also Table 3). Motions of this ring would be allowed
within the water space. However, an E-configuration of hPCB-82 cannot be found in all available X-ray structures of PEC and
PC from cyanobacteria and rhodophyta. An explanation for this
erythrocyanin crystals from Mastigocladus laminosus. Black: PECM_110K; grey:
phores. (a) a-subunit. (b) h-subunit.
Fig. 5. Comparison of the mainly deviating structural details of a-phycocyanin
from Thermosynechococcus vulcanus (PDB access. No. 1ON7) and a
phycoerythrocyanin from Mastigocladus laminosus. The figure displays the
helices H4 and H5 with their connecting loop. Only the amino acids between
AA53 and AA 90 are shown in detail with their side chains. The figures have
been produced with the Swiss-PdbViewer [40], which is available at the
‘‘ExPASy Proteomics Server’’ (http://www.expasy.org). The H-bonds, drawn in
black dashed lines, have been calculated without hydrogens in the range from
2.2 to 3.3 A. The rings D of the chromophores and several amino acids have
been marked for comparison. (a) ‘‘H4-Loop–H5’’-region of a-PEC from
Mastigocladus laminosus. (b) ‘‘H4-Loop–H5’’-region of a-PC from Thermo
synechococcus vulcanus.
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–6260
fact would be a rapid thermal relaxation from a merely
stabilized E-state back to the stable Z-configuration. Hence, a
short-lived E-configuration, which is transiently present in the
h-subunits of PEC and PC, cannot be completely excluded.
Finally, in the trimeric state of PEC, the aPVB-84 chromo-
phore motions are strongly restrained by the interaction of the
D-ring with the neighboring h..-subunit (Table 3). Despite this
interaction, a reduced but significant photoactivity takes place
within the PEC trimers [6]. However, it is by no means clear
how the E-configuration is built up, and how it is stabilized to
avoid quick thermal re-isomerization. We, therefore, postulate a
local protein structure in the a-subunit that allows a rapid built-
up of the E-form and, in addition, enables its subsequent
stabilization.
A possible candidate can be identified by comparing the
temperature factors (B-values) of the structures. It should be
mentioned that an absolute determination of B-values at a
resolution around 3 A is questionable, but the relative changes
of individual, properly restrained B-values are comparable. If
the B-values between the a- and h-subunits are compared,
large differences in the range of 25 A2 to 30 A2 are observed
(Table 3, upper right side of the diagonal). As already observed
[3], the a-subunit appears much more disordered than the h-subunit. Large B-values can be found in subunit a and haround AA60–80 (Fig. 4). These amino acids correspond to
solvent exposed loops, which are different in the a- and h-subunits but present in all phycobiliproteins. At ambient
temperatures, the structures of these loops can be changed
easily for example by the interaction of the naturally occurring
linker proteins with the h-subunits [35] (W. Reuter, unpub-
lished data).
However, in the a-subunit, the B-values around AA60–80
are much larger than in the h-subunit. This flexible loop may
move to a new position and interact with the photo-isomerized
chromophore, hence stabilizing the E-form. Our results
correspond well with results from a theoretical [36] and a
NMR study [14] on isolated a-PEC, where the a-PEC in the Z-
form is more flexible than the protein with the chromophore in
the E-configuration.
The B-factors of crystal structures depend on both, the
individual flexibility of the protein subunits and the interactions
of molecules in the particular space group of the crystal [37].
Because of the importance of the B-values determined in this
study, all phycobiliprotein entries of cyanobacteria and rhodo-
phyta in the protein data bank were inspected. All structures
show more or less higher B-values of the a-subunits, regardless
of the space group and the phycobiliprotein class. Nevertheless,
only in the a-subunit of the PC from Thermosynechococcus
vulcanus (PDB access. No.1ON7) the B-values are comparable
to those of PEC. Interestingly, the parameters of the unit cell
are nearly identical to those of this study. The crystals have also
been measured at 100 K and the final resolution of the PC
structure [38] corresponds well with that of PECM–110K;
therefore, the data are directly comparable. Additionally, a
sequence alignment of a-PEC with all known phycobiliprotein-
subunits from cyanobacteria and red algae revealed the highest
homology to a-PC from T. vulcanus. It should be mentioned as
a footnote that the sequence homology of a-PEC of M.
laminosus with its own a-PC is very low, much lower than that
with the a-PC of T. vulcanus. Even the a-PCs of several red
algae match much better. This fact may be very interesting for
the search in the a-PEC’s evolutionary origin.
To corroborate our suggestion that a local protein moiety
may enable the formation of a stable E-form, we examined the
structural differences between the a-PEC of M. laminosus and
a-PC of T. vulcanus, since the latter is very similar to a-PEC
-
-
M. Schmidt et al. / Biochimica et Biophysica Acta 1764 (2006) 55–62 61
but is not photoactive. The overall structures of T. vulcanus PC
and M. laminosus PEC fit very well on top of each other. The
number and quality of interactions by H-bonds and aromatic
amino acids of the proteins with the chromophores of the a-
and h-subunit, respectively, are nearly identical. Only minor
differences in the chromophore environments of PVB and PCB
within the a-subunits of the PEC or PC of the two
cyanobacteria, respectively, can be observed. Hence, the
structures and interactions of the two chromophores offer no
serious explanation for their markedly distinct photochemical
behavior [5,7,14,20]. Differences, however, can be found in the
aforementioned large, flexible loop connecting H4 and H5
(Fig. 5). The loop of a-PC of T. vulcanus comprises 15 amino
acids, and it is stabilized by a well-developed network of 12
hydrogen bonds. In addition, the ‘‘H4-Loop-H5’’-arrangement
is stabilized by a strong H-bond between Tyr60 and Lys81
(Fig. 5b). Such an H4–H5 interaction cannot be found in the
structure of a-PEC from M. laminosus. The complete loop of
a-PEC consists of 19 amino acids and it contains a small
helical turn formed by Tyr65, Thr66 and Thr67. This turn does
not appear in the a-PC structure, and it results from the
exchange of Thr68 and Ser72 in a-PC against Gln68 and Pro72
in a-PEC. Despite the extended length of the loop, only nine
hydrogen bonds can be detected in a-PEC (Fig. 5a) leaving a
highly flexible and structurally fragile moiety. The compara-
tively high structural flexibility of the loop in the Z-form is
proposed to be important for both, the rapid stabilization of the
E-form as well as for the reversibility of the photochemical
reaction. In addition, the particular physical properties of the
PVB chromophore, e.g., the Fshort wavelength_–Fhigh energy_absorbance and the allocation of the k-electrons within/aroundthe chromophore, have to be considered for the reversible
photochromic shifts.
The coordinates of the structures determined at 110 K
(PECM–110K), at room temperature (PECM–295K) and with the
Laue method (PECL–288K) are deposited in the protein data
bank with accession codes 2c7l, 2c7j and 2c7k.
Acknowledgements
MS and WR were financially supported by the Deutsche
Forschungsgemeinschaft, Sonderforschungsbereich 533 (TP
B10 and TP A2). Vukica Srajer and Reinhard Pahl are
acknowledged for their assistance in the Laue data collection
and Benjamin Lehne for technical assistance.
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