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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: marius.schmidt@ph.tum.de (M. Schmidt),

w.reuter@online.de (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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