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https://www.abbs.info ISSN 0582-9879 |
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Short Communication |
In vitro Assembly of R-phycoerythrin from
Marine Red Alga Polysiphonia urceolata
ZHANG Yu-Zhong1,2*, CHEN
Xiu-Lan1, WANG Lu-Shan1, ZHOU Bai-Cheng2,
HE Jin-An3, SHI Dong-Xia4, PANG Shi-Jin4
(1 State Key Laboratory of Microbial Technology, Shandong
University, Jinan 250100, China;
2Institute of Oceanology, the Chinese Academy of Sciences,
Qingdao 266071, China；
3Institute of Photographic Chemistry, the Chinese Academy
of Sciences, Beijing 100101, China;
4Beijing Laboratory of Vacuum Physics, the
Chinese Academy of Sciences, Beijing 100080, China )
Abstract Scanning tunneling microscope was used to
investigate the in vitro assembly of R-phycoerythrin (R-PE) from the marine red
alga Polysiphonia urceolata. The results showed that R-PE molecules assembled
together by disc-to-disc while absorbing on HOPG surface, which just looked
like the rods in the phycobilisomes. When the water-soluble R-PE was dissolved
in 2% ethanol/water spreading solution, they could form monolayer film at the
air/water interface. Similar disc-to-disc array of R-PE was constituted in the
two-dimensional Langmuir-Blodgett film by the external force. It could be
concluded that, apart from the key role of the linker polypeptides, the in
vivo assembly of phycobiliproteins into phycobilisomes is also dependent on
the endogenous properties of phycobiliprotein themselves.
Key words R-phycoerythrin； in vitro
assembly；
scanning tunneling microscopy (STM)
Phycobilisomes (PBS), the light-harvesting antennae of blue-green algae
and red algae, are supramolecular, highly structured protein complexes located
on the thylakoid surface[1]. They are primarily composed of
chromophore-bearing phycobiliproteins absorbing light over a wide spectral
range[1]. Phycobiliproteins in blue-green algae and red algae are
composed of (ab)-monomer.
During the last decade, the crystal structures of several phycocyanin (PC),
phycoerythrin (PE), allophycocyanin (APC) have been determined by X-ray
diffraction[2―7]. These results revealed that all the phycobiliproteins
have very similar three-dimensional structures. Three (ab)-monomers are arranged around
a 3-fold symmetry axis in the trimer (ab)3, while two trimers are assembled by disc-to-disc into the
hexamer (ab)6.
In addition, PBS also contain a minor portion of proteins, most of which do not
bear chromophores, and are referred to as “linker polypeptides” whose function
is to make phycobiliproteins assemble as PBS and stabilize the supramolecular
complex in vivo[1].
The assembly of phycobiliprotein discs into
phycobilisomes is dependent upon the presence of different functional linker
polypeptides. The roles of the linker polypeptides in the aggregate formation
and the fine-tuning of the absorption characteristics of phycobiliproteins have
been extensively studied[8―11], and the structure model of the
phycobilisomes in Mastigocladus lainosus was proposed[12].
However, apart from the function of the linker polypeptides, the
phycobiliprotein itself might play an important role in the assembly of the
phycobilisomes. Because, although there were phycobiliproteins in the
Cryptophyceae and some of the Dinophyceae, phycobolisomes did not exit, and the
subunit composition of the phycobiliproteins was different from that of the
blue-green algae and red algae[1].The observation of interaction
between the phycobiliprotein molecules could give the evidence for the above
suggestion. The three dimensional structure of the C-phycocyanin (C-PC) and
phycobilisomes in Spirulina platensis was observed with STM[13,14].
In this paper, STM was used to investigate the spontaneous behavior of pure
R-phycoerythrin (R-PE) without linker polypeptides while adsorbing on the newly
cleaved highly oriented pyrolytic graphite (HOPG) surface, and then, we report
the abilities of R-PE to form two-dimensional orderly films by
Langmuir-Blodgett (LB) technique and their structural observations.
1
Materials and Methods
Materials and Methods
1.1 Isolation of R-PE and
absorption on the HOPG surface R-PE was purified from marine red alga Polysiphonia
urceolata. The phycobiliproteins were extracted by autolysis in distilled
water, and then by fractional precipitation with ammonium sulphate from high to
low concentration (55%, then 50%―45%). The crude sample was separated through a
hydroxylapatite column, washed with 30 mmol/L phosphate buffer (pH 6.8, 0.2
mol/L NaCl). The collected R-PE was finally purified through a Bio Gel P-300
column (Bio-Rad, Richmound, UA), washed with 50 mmol/L sodium-phosphate buffer
(pH 6.8, 0.1 mol/L NaCl). The absorption spectrum was determined with Shimadzu
UV-240 spectro photometer at room temperature.
The R-PE sample solution was dialyzed against 5
mmol/L phosphate buffer (pH 6.8) for 24 h and diluted with distilled water.
Then, 5 ml
diluted sample solution was dropped on freshly cleaved HOPG surface, and then
stayed on it for 20 s for Fig.2(A) and 1―2 min for Fig.2(B) in air at room
temperature. The excess solution was removed with filter paper. The
concentration of R-PE was approximately 5 mg/L in Fig.2(A) and 20 mg/L in
Fig.2(B), respectively.
1.2 Preparation of LB
films All monolayers
were prepared on Sixing Film Deposition System (Jilin University, China) with a
surface area of approximate 648 cm2. Triple distilled and adjusted
water (pH 5.6) was used as subphase. p-A curve measurements were carried out by spreading a 2% ethanol/water
mixture containing about 0.3 g/L R-PE onto the subphase surface, and ethanol
solvent was allowed to evaporate for a period of 15 min before the compression
of the monolayer at a rate of 0.5 cm2/s. Surface pressure was
measured with Wilhelmy plate. Monolayer was compressed to a pressure of 15
mN/m, and was allowed to stabilise for a period of at least 40 min before
dipping down the mica matrix. The lifting speeds were 5 mm/min upward and 15
mm/min downward. The first layer was allowed to dry for 50 min to ensure good
contact between the mica matrix and the monolayer. For the subsequent layers,
the time for standing at the down and up positions were 1 and 12 min,
respectively. The transfer ratio in the upward collection was approximately
0.95, and no deposition took place during downward motion. Newly cleaved mica
were used as the matrix for the preparation of LB films of R-PE. In order to
enhance the electroconductivity of the films, the matrix onto which R-PE
monolayer was deposited was coated with gold. The thickness of gold film was
controlled as thinner as possible so as to minimize the interference of gold
film, however, a continuous gold layer should meanwhile be formed so as to have
good electroconductivity. In order to obtain good STM images, three layers of
R-PE were transferred onto the mica.
1.3 STM experiments STM experiments were
carried out in ambient environment with a domestic STM set- up CSTM-9100
(manufactured by Institute of Chemistry, the Chinese Academy of Sciences). STM
measurement was performed with normal STM constant current mode, using tungsten
tips made by electrochemical etching. All STM images presented here were raw
data images without any smoothing and filtering.
2 Results and
Discussion
Fig.1 was the absorption spectrum of R-PE isolated
from marine red alga Polysiphonia urceolata. The major absorption peaks
were located at 498 nm, 545 nm and 565 nm, which was associated with the
reported results of R-PE[1]. The absorbent ratio of A565/A280>4
suggested that the purity of isolated R-PE was good. It was confirmed by the
SDS-PAGE electrophoresis that there was no linker polypetide in the isolated
R-PE solution.
Fig.1 The absorption spectrum
of R-PE from marine red alga Polysiphonia urceolata
The
actual distribution of the sample on substrates greatly depends on the
concentration and size of biological objects, as well as the time allowed for
adsorption. We have succeeded to observe the structure of individual C-PC
molecules with STM. The concentration of C-PC solution was 5 mg/L, and the time
for adsorption was 20 s. Here the concentration of R-PE in the solution was
raised to 20 mg/L, R-PE solution was dropped on HOPG surface and stayed on it
for 1―2 min. In this case, all most of R-PE molecules aggregated together, and
few individual molecules were found on the HOPG surface from the STM image
shown in Fig.2. In some area,R-PE molecules assembled by disc-to-disc to form
more regular aggregate which was just like the native rod in phycobilisomes.
Therefore, the aggregation state in vitro verified the rod model
structure of phycobilisomes. The thickness of the disc in the rod was about 10
nm. The diameter of the rod was approximately 40 nm. The aggregation of R-PE
was (ab)6g in vivo, every one disc in the rod might
be referred as one (ab)6g.
Fig.2 STM images of R-PE
It=0.48 nA, Vbias=-235 mV. Scan area: (A) 128 nm × 128 nm, (B) 55 nm × 75 nm.
In order to further
study the self-organizing ability of R-PE in vitro, LB technique was
used to prepare LB film of the protein. As R-PE is a water-soluble protein,
small amount of ethanol was added to improve the performance of the film
deposited [R-PE was not denatured while dissolved in 2% ethanol/water solution
(data not shown)].
Fig.3 was surface pressure-area isotherms of R-PE
monolayers at the air/water interface. R-PE formed monolayers when they were
dissolved in 2% ethanol/water spreading solution. Due to the fact that the
interfacial concentrations of R-PE used in our experiments were far less than
the limiting interfacial concentration of proteins (0.78 m2/mg), the
desorption may be negligible. The monolayer formed under our experiment
conditions was stable. It could seen from Fig.4 that, at constant surface
pressure of 15 mN/m, the areas of the monolayers hardly changed within 3 h. In
addition, it was proved that the pretreated mica was a suitable matrix for
transferring R-PE monolayer, which might be related to the fact that the mica
has a negatively charged surface, which was similar to the thylakoid membrane
surface.
Fig.3 The -A curve of R-PE
monolayer
Fig.4 Changes in area of R-PE
monolayer with time
Fig.5 was the STM image of R-PE LB film. From
the image, it could be observed that R-PE molecules could form a regular film,
no obvious defects were found in the scan area of 480 nm×480 nm. Therefore,
under strictly controlled conditions, it was possible to prepare large area
R-PE LB film without obvious defect. From the STM image, It could also be seen
that R-PE molecules arranged on the mica surface by disc-to-disc to form
rod-likestructure, which was similar to its self-asembly while adsorbing on
HOPG surface. Then, the constituted rod arrayed together to form monolayer. The
diameter of the rod was about 50 nm, which was correspond
to that of the self assembly on HOPG surface. Every disc in the rod represented
one R-PE molecule. Thus, the aggregation form of R-PE in the LB film also
verified the rod structure of phycobilisomes in vivo. That the size of
R-PE moleculaes determined by STM was larger than that by X-ray diffraction was
mainly due to the tip-radius-induced artifacts and the effects of gold coating.
Fig.5 STM image of R-PE LB film
It= 0.99 nA, Vbias=458 mV. Scan area: 480 nm× 480 nm.
Different from all other
light-harvesting pigment complexes, which were located within the thylakoid
membrane, phycobilisomes were located on the thylakoid surface. The
phycobiliprotein discs were assembled by disc-to-disc to form aggregate, which
was the most stable state in the cell, and also helpful for the energy
transfer. The assembly of phycobiliprotein discs into phycobilisomes is
dependent upon the presence of different functional linker polypeptides, and
the roles of the linker polypeptides in the aggregate formation and the
fine-tuning of the absorption characteristics of phycobiliproteins have been
extensively studied[8―11]. Large subcomplexes containing specific
linker polypeptides have been isolated after partial dissociation of
phycobilisomes[8]. Gottsckalk et al (1993,1994)[10, 11]， Glauser et al (1993)[9] have
reconstituted (ab)3APLc8.7,
(ab)3AP・21―23
kD [the C-terminal 21―23 kD domain of the core-membrane linker polypeptide LCM],
(ab)3PCLRC29.5(ab)3APLc8.9
with purified smaller complexes throughout a complete dissociation of
phycobilisomes in low ionic intensity. Since pure phycobiliproteins seem not to
be capable of assembly into the structures characteristic of phycobilisomes in
solution, it has been inferred that the linker-polypeptides play a key role in
the phycobilisome assembly. In this paper, it was found that R-PE molecules
could self-assembly together by disc-to-disc while absorbing on HOPG surface,
just looked like the rod and core in the phycobilisomes. Therefore, it could be
concluded that the formation of the phycobilisome was dependent upon the
structure of the phycobiliproteins, and the roles of the linker polypeptides
were to locate the phyocobiliproteins in the phycobilisomes and anchor the phycobilisomes
to the thylakoid surface, which was because that all the reported
phycobiliprotein in bule green algae and red algae had similar disc-like three
dimensional structures, otherwise, the linker polypeptides in the rod, core and
core-membrane were different.
Phycobiliproteins carry covalently attached linear
tetrapyrrole pigments related structurally to biliverdin[1]. The
role of phycobiliproteins and phycobilisomes are to absorb light energy and to
transfer the energy to reaction centre of photosynthesis where photoinduced
charge separation occurs[1]. Due to the stability, high fluorescence
yield, large stokes’ shifts between absorption and emission, special property
of photophysics and photochemistry[15], and the ability of easy
preparation for two-dimensional film by LB technique, phycobiliprotein might be
acted as a kind of useful materials for crystallization electronics and
bioelectronic research.
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Received: July 11,
2001Accepted: September 4, 2001
The work was supported
by Natural Science Foundation of Shandong Province
*
Corresponding author: Tel， 86-531-8564326； Fax，
86-531-8565610； e-mail， [email protected]