Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: Preparation, bulk and single molecule spectroscopy

Abstract

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.

Full text

Peridinin–chlorophyll–protein reconstituted with chlorophyll
mixtures: Preparation, bulk and single molecule spectroscopy
T.H.P. Brotosudarmo
a,b
, E. Hofmann
c
, R.G. Hiller
d
,S.Wo
¨rmke
b
, S. Mackowski
b
,
A. Zumbusch
e
, C. Bra
¨uchle
b
, H. Scheer
a,*
a
Department Biologie I – Bereich Botanik, Ludwig-Maximilians-Universita
¨tMu
¨nchen, Menzinger Str. 67, D-80638 Mu
¨nchen, Germany
b
Department Chemie und Biochemie – Physikalische Chemie, and Center for Nanoscience, Ludwig-Maximilians Universita
¨t, Mu
¨nchen, Germany
c
Fakulta
¨tfu
¨r Biologie, Biophysik, Ruhr Universita
¨t Bochum, Germany
d
Macquarie University, Sydney, Australia
e
Fachbereich Chemie, Universita
¨t Konstanz, Germany
Received 23 July 2006; revised 21 August 2006; accepted 24 August 2006
Available online 5 September 2006
Edited by Miguel De la Rosa
Abstract Reconstitution of the 16 kDa N-terminal domain of
the peridinin–chlorophyll–protein, N-PCP, with mixtures of
chlorophyll a(Chl a) and Chl b, resulted in 32 kDa complexes
containing two pigment clusters, each bound to one N-PCP.
Besides homo-chlorophyllous complexes, hetero-chlorophyllous
ones were obtained that contain Chl ain one pigment cluster,
and Chl bin the other. Binding of Chl bis stronger than that
of the native pigment, Chl a. Energy transfer from Chl bto
Chl ais efficient, but there are only weak interactions between
the two pigments. Individual homo- and hetero-chlorophyllous
complexes were investigated by single molecule spectroscopy
using excitation into the peridinin absorption band and scanning
of the Chl fluorescence, the latter show frequently well resolved
emissions of the two pigments.

2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Photosynthesis; Light-harvesting; Chlorophyll;
Carotenoid; Energy transfer; Single molecule spectroscopy
1. Introduction
Peridinin–chlorophyll–proteins (PCP) are water-soluble
light-harvesting complexes from dinoflagellates [1]. The native
complex from Amphidinium (A.) carterae is a homotrimer. The
boat-shaped, a-helical protomer is the 32 kDa large PCP
(l-PCP). It has an approximate C
2
-symmetry containing two
clusters of pigments, each consisting of one chlorophyll a
(Chl a) that is nested between two pairs of peridinins (Per)
[2]. The pigments in each cluster are in Van der Waals contact.
There is also contact between Per molecules from the two clus-
ters, while the center-to-center distance of the two Chl is
17.4 A
˚. Small PCP (s-PCP) of half the size (16 kDa) of the
A.carterae complex which contain a single Per
4
–Chl acluster,
has been found in several species including Heterocapsa pyg-
maea [3]. These s-PCP dimerize, resulting in a similar topology
as the monomeric protomers from A.carterae [4]. An analo-
gous aggregate is also formed by the 16 kDa N-terminal
domain of PCP from A.carterae (N-PCP) reconstituted with
Chl aand Per [5]
1
.
Tight clustering, an exceptionally high carotenoid-Chl ratio,
and the presence of a highly modified carotenoid, Per, with an
unusually long-lived S
1
-state [6] render the PCPs a challenge to
understand pigment interactions and excitation energy transfer
(EET). Pairing of Per and a close spacing of the pigments had
been inferred already in 1976 from circular dichroism (CD)
spectra of PCP [7]. Moderately strong excitonic coupling
among the pigments within a cluster was also supported by
simulations of the CD and absorption spectra, which were
based on the X-ray structure [8]. However, the details are still
controversial and may require contributions from inter-cluster
interactions. Rapid fluorescence depolarization (7 ps) has been
assigned to intra-monomer EET between the Chls, a much
slower component (350 ps) to Fo
¨rster type inter-monomer
EET transfer in PCP trimers from A.carterae [9]. EET from
Per to Chl has been studied using N-PCP that have been recon-
stituted with different Chls [10], the results support theoretical
predictions [11] of a major contribution of the relatively long-
lived S
1
-state of Per [6,12,13] as donor to the Chls. The contri-
butions of the different Per excited states have been reviewed
[14]; the S
1
-state is stabilized by symmetry breaking and by
intra-Per charge-transfer [6,13,15].
The high carotenoid content and relatively small Chl–Chl
interactions also render PCP a candidate to study EET
between two Chls in a structurally well-known situation that
is relatively simple as compared to other photosynthetic sys-
tems. In this context, it is desirable to study PCP containing
two different, spectrally separated Chls [5]. Here, we report
on such complexes prepared by reconstitution of N-PCP from
Abbreviations: A.,Amphidinium; CD, circular dichroism; Chl, chloro-
phyll; EET, excitation energy transfer; l-PCP, large (32 kDa PCP);
N-PCP, N-terminal 16 kDa-domain of PCP; PCP, peridinin–chloro-
phyll–protein; Per, peridinin; s-PCP, small (16 kDa) PCP; SMS, single
molecule spectroscopy
*
Corresponding author. Fax: +49 89 17861 271.
E-mail address: hugo.scheer@lmu.de (H. Scheer).
1
The term ‘monomer’ is used here in a topological context, referring
to l-PCP from A.carterae from which the X-ray structure has been
solved. l-PCP is a 32 kDa protein, it originates from a gene duplication
and binds two Chl/Per clusters. For historical reasons, this species is
generally referred to as the PCP monomer. With respect to this l-PCP
monomer, the 16 kDa s-PCP discovered subsequently, as well as N-
PCP generated from the A.carterae protein, are topologically ‘‘half-
mers’’: they carry only a single Chl/Per
4
cluster, and dimerize to species
that are homologous to the l-PCP monomer.
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2006.08.049
FEBS Letters 580 (2006) 5257–5262

A.carterae with Chl aand Chl b. The procedure yields dimeric
aggregates that correspond topologically to l-PCP monomers
1
,
and are mixtures with respect to their pigment composition:
besides homo-chlorophyllous Chl a/Chl aand Chl b/Chl b
complexes, the preparation yields hetero-chlorophyllous com-
plexes containing Chl ain one cluster and Chl bin the other.
While these complexes could not be separated by chromato-
graphy, identification of the hetero-chlorophyllous complexes
and their investigation was possible by difference spectroscopy,
and by single molecule spectroscopy (SMS).
2. Materials and methods
2.1. Materials
Trimeric PCP was isolated from A. carterae [2]. The N-terminal do-
main of the apoprotein (N-PCP) was obtained as published [5]. Per was
extracted from A. carterae as published [6]. Chl awas extracted from
spray-dried Spirulina geitleri, Chl bfrom the Chl a/bmixture extracted
from frozen spinach, and purified over DEAE cellulose [16].
2.2. Reconstitution
Reconstitution followed the protocol of [5]. 625 ll apo-N-PCP
(20 lM) in tricine buffer (50 mM, pH 8.0) were combined with
225 ll tricine buffer (50 mM, pH 7.6) containing KCl (10 mM). An
ethanolic solution (150 ll) of Per (80 lM) and Chl a(or b) (20 lM)
was added, the mixture incubated at 4 C for 48 h, and the crude
reconstitution product purified over a small DEAE Tris–acryl col-
umn (5 ·25 mm, tricine buffer (5 mM, pH 7.6, 2 mM KCl), NaCl
step gradient 100, 200 and 500 mM). Fractions containing the recon-
stituted complex (100 mM NaCl) were dialyzed against the starting
buffer.
2.3. Spectroscopy
Absorption spectra were recorded with a Lamda 2 spectrophotome-
ter (Perkin–Elmer), bulk fluorescence excitation and emission spectra
with a model LS55 spectrofluorimeter (Perkin–Elmer), and circular
dichroism spectra with a model J810 spectropolarimeter (Jasco). All
measurements were done at room temperature. Data were transferred
to Origin 6.0 (Microcal) for further analysis. Chl aand Chl bextinction
coefficients in the reconstitution system were determined relative to the
known ones in acetone [17]. The respective pigment was dissolved in
acetone, and an absorption spectrum recorded. The solvent was then
evaporated in a stream of Ar, the pigment re-dissolved in the same vol-
O
COCH3
o
OO
O
OH
R
Mg
O
N N
N N
H
H
COOCH3
H
COOC20H39
Fig. 1. Molecular structures of the cofactors of the PCP complex. Top: Per and Chl ain the two pigment clusters of a PCP monomer from A.
carterae ([2], pdb entry 1PPR) are shown as orange and green bold stick models, the two lipids as wire models, using DS ViewerPro V5 (Accelrys).
The center-to-center (Mg–Mg) distance of the Chls is 17.3 A
˚. Chemical structures of Per (center) and of Chl a(R = CH
3
) and Chl b(R = CHO)
(bottom).
5258 T.H.P. Brotosudarmo et al. / FEBS Letters 580 (2006) 5257–5262

ume of the buffer system by first adding the appropriate amount of eth-
anol and the aqueous components, and then the absorption was re-
corded again (see Fig. 1).
Single molecule spectra were obtained using a modified scanning
confocal microscope (ZEISS LSM 410). Fluorescence spectra of single
immobilized complexes were recorded after excitation into the Per
absorption (532 nm). Fluorescence emission in the spectral range from
575 to 725 nm was dispersed with a Amici-prism and projected onto a
CCD camera (Princeton Instruments). Typical integration times were
0.3 s with a spectral resolution of 1.5 nm. Further details will be de-
scribed in a paper dedicated to single molecule spectroscopy of PCP
[24].
3. Results and discussion
Reconstitution [10] of N-PCP from A.carterae with Chl a
(1.1 moles/mole protein) and Per (4.4 moles/mole protein) re-
sults in hetero-chlorophyllous complexes as evidenced by their
optical spectra (see below). Quantitatively, the pigment ratios
in the reconstituted and purified complexes were determined
spectroscopically [18], after dilution into acetone to a 20:80
water/acetone ratio. The Chl a/bratio is decreased from 1:1
in the reconstitution mixture to 43/57 in the purified complex,
indicating a slight preference for the non-natural pigment, viz.
Chl b. Chls with C‚O – groups at C-3 and C-7 have been
bound before to N-PCP [5]. In the X-ray structure, the region
around C-7
1
carrying the carbonyl-oxygen in Chl b, has two
hydrophilic groups nearby that could assist binding: a ring-N
of His66 (3.8 A
˚) and a backbone C=O of Ala-63 (5.1 A
˚)[2].
Binding of Chl bis, however, even possible in relatively hydro-
phobic environments [19,20].
Absorption and circular dichroism. The homo-chlorophyllous
N-PCP complex containing only Chl ahas absorption and CD
spectra (Fig. 2B) that are similar to those of native l-PCP from
the same organism (Fig. 2A), the spectra of the latter are iden-
tical to the ones of PCP reported before [8]. However, the
negative CD-band of Per peaking around 530 nm is slightly
red-shifted and broadened in Chl a–N-PCP as compared to
native l-PCP. The CD-spectrum of both complexes is domi-
nated by an intense, s-shaped band system peaking at 530
() and 445 (+), with the zero-crossing near the absorption
maximum of peridinin. Superimposed on the positive lobe of
this signal is a much smaller and narrower, s-shaped band sys-
tem that is centered near the absorption maximum of Chl a. Its
negative lobe shows as a dip in the intense positive Per band,
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.00
0.05
0.10
0.15
0.20
0.25
0.00
0.05
0.10
0.15
0.20
0.25
400 500 600 700
0.00
0.05
0.10
0.15
0.20
0.25
-20
-15
-10
-5
0
5
10
15
20
669
474
437
437 474
670
460
648
460
668
427460
444
529
673
427458
443
531
672
443
485
467 525
649
Absorbance [a.u.]Absorbance [a.u.]Absorbance [a.u.] Absorbance [a.u.]
A: l-PCP
B: Chl
a
-N-PCP
C: Chl
b
-N-PCP
Wavelen
g
th [nm]
441
650
D: Chl
a/b
-N-PCP
AL-AR [Milidegrees]
-30
-20
-10
0
10
20
30
AL-AR [Milidegrees]
-30
-20
-10
0
10
20
30
AL-AR [Milidegrees]
400 500 600 700
-30
-20
-10
0
10
20
30
AL-AR [Milidegrees]
Wavelen
g
th [nm]
428
441
447
468
483
530
649
661
669
Fig. 2. Absorption (left) and circular dichroism spectra (right) of (A) native PCP, and of RPCP reconstituted with Chl a(B); Chl b(C) and a 1:1
mixture of Chl aand Chl b(D).
T.H.P. Brotosudarmo et al. / FEBS Letters 580 (2006) 5257–5262 5259

that is located 7 nm to the red of the Chl aabsorption max-
imum (437 nm). The positive lobe is discernible as a distinct
peak in the largely unstructured Per band, located 10 nm to
the blue of the Chl aabsorption maximum. There is no such
splitting for the Q
Y
-band of Chl aat 672 nm. The major
band of Chl ain the Soret-region is the B
x
-band [21,22]. Since
the orientation factors are similar for the x- and y-polarized
transitions, the lack of a split Q
Y
CD-signal indicates that
the split Soret CD-signal is not due to excitonic interactions
among the Chls of adjacent pigment clusters, but results
mainly from coupling with Per within a single cluster [4,8].
Reconstitution with Chl binstead of Chl ayields a complex
with a blue-shifted Q
y
band of the Chl and a red-shifted Soret
band (Fig. 2C), these shifts are similar to those of Chl avs. Chl
bin acetone. The red-shifted (460 nm) and more pronounced
Soret band reflects the larger extinction coefficient of Chl b
as compared to Chl a[23]. Accordingly, the absorption of
the Chl bcomplex is dominated by the latter (460 nm). The
s-shaped CD signal in the Soret region is also much more
prominent than in the Chl a-containing complex, as shown
already by Miller et al. [5]. The zero-crossing of the intense,
s-shaped feature is close to the Chl babsorption maximum.
The Q
y
-band shows, like in the Chl acomplex, no splitting;
this further supports [4,8] that excitonic interactions of the
Chls occur mainly with Per.
The spectrum of the hetero-chlorophyllous complex
(Fig. 2D) is superficially a mixture of the ones of the homo-
chlorophyllous complexes. However, a closer inspection re-
veals differences, that are likely due to Chl a/Chl binteractions.
(i) The Chl b-related Q
y
-CD band at 649 nm is stronger than
that of Chl aat 669 nm, while based on the homo-chlorophyl-
lous complexes (Fig. 2, see also [5]) and the Chl a/b-ratio of 43/
57 (see above), at most equal intensities are expected. (ii) The
red absorption and CD-bands of Chl aare blue-shifted by
2 nm in the Chl a/bcomplex as compared to the Chl a– only
sample (Fig. 2A). (iii) Subtraction of the absorption spectra of
the homo-chlorophyllous Chl a–N-PCP or Chl b–N-PCP com-
plex from that of the hetero-chlorophyllous complex always
gave additional bands (Fig. 3). It should be noted that the sit-
uation could be further complicated by the possibility that
there are mono-chlorophyllous complexes, in which one bind-
ing site is empty, and by the presence of ‘‘half-mers’’
1
carrying
only a single Chl/Per
4
cluster. However, these are unlikely
based on the tight pigment binding and dimerization of indi-
vidual N-PCP, and were not considered here. Fluorescence
spectroscopy of bulk samples gave likewise inconclusive results
(data not shown).
The emission spectra of the hetero-chlorophyllous complex
show one major peak at 673 nm and a smaller one at
652 nm, corresponding to emissions from Chl aand Chl b,
respectively (Fig. 4). The excitation spectrum for the shorter-
wavelength emission shows, accordingly, mainly contributions
of Per and Chl b. The excitation spectrum for the long-wave-
length peak has contributions from Per (k
max
505 nm), Chl
b(k
max
= 463 nm) and Chl a(k
max
= 437 nm). This clearly indi-
cates inter-cluster energy transfer from Chl bto Chl ain the
hetero-chlorophyllous N-PCP complex. Assuming that the
pigment geometry is similar to that in the l-PCP monomer,
and the separation of the two emission bands corresponds to
the equilibrium energy difference between Chl aand Chl b; rel-
ative contributions of 6.1:1 can be estimated for the emissions
of Chls aand b, respectively. This value was estimated in the
following way: Boltzmann equilibrium (n
Chl a
/n
Chl b
=
exp(-DE/kT) between the two emittors (673 and 651 nm,
respectively) results, at a temperature of 298 K, in relative pop-
ulations of 10.1:1. Since the fluorescence results from a mixture
of species consisting of the homo-chlorophyllous complexes,
(Chl a)
2
–N-PCP and (Chl b)
2
–N-PCP, and the hetero-chloro-
phyllous complex, Chl a/b–N-PCP, contributions from the
three components have to be summed. Their relative popula-
tions were estimated assuming a random distribution of Chls
aand bin the experimentally determined pigment ratio of
43:57 (see above), resulting in relative populations of 18.5%,
32.5% and 49%. Finally, the emission of Chl bwas corrected
by the relative fluorescence yield of 0.45 [5] of Chl b–N-PCP
compared to Chl a–N-PCP. The experimentally obtained Chl
a/bemission ratio, determined from the data in Fig. 4 by
0.00
0.02
0.04
0.00
0.02
0.04
600 650 700 750
0.00
0.02
0.04
Absorbance [a.u.]
C
B
Absorbance [a.u.]
A
Wavelength [nm]
Absorbance [a.u.]
Fig. 3. Comparison of homo- and hetero-chlorophyllous N-PCP
complexes. Absorption spectra of Chl a/b–N-PCP (—) and absorption
differences (- - - -) with the spectra of Chl a–N-PCP (A), Chl b–N-PCP
(B) and the sum of Chl a–N-PCP and Chl b–N-PCP (C). The
subtracted homo-chlorophyllous spectra are shown as dotted lines,
they were scaled to give minimum deviation in the region of the
respective Chls.
400 500 600 700
0
3000
6000
9000
Intensity
Wavelength [nm]
λem = 650 nm
λem = 670 nm
λexc = 500 nm
Fig. 4. Fluorescence of reconstituted Chl a/b–N-PCP complex; emis-
sion (k
exc
= 500 nm, ) and excitation spectra (k
em
= 650 nm,
;k
em
= 670 nm, ). Excitation spectra were normalized
to the Per maximum near 505 nm.
5260 T.H.P. Brotosudarmo et al. / FEBS Letters 580 (2006) 5257–5262

Gaussian deconvolution, is 4.7. This is somewhat lower than
the estimated theoretical value of 6.1, but indicates nearly
complete equilibration between the two Chls in the Chl a/b-
complex. A more detailed analysis is difficult, however, consid-
ering the aforementioned interactions among the Chls, whose
contributions are difficult to estimate, and the assumptions
used in the analysis.
The Chl aemission of the hetero-chlorophyllous complex
(671 nm) is red-shifted by 2 nm compared to the Chl a– only
complex. Minor but reproducible shifts are also seen in the
Soret-region. Likewise there were minor shifts in the Chl b-
bands when comparing the hetero-chlorophyllous complex
with that of the one containing only Chl b. All these shifts
point to small, but distinct Chl–Chl interactions between the
Chls in the two clusters.
Proof that hetero-chlorophyllous complexes are indeed
formed can be obtained by investigations of single complexes.
Therefore we recorded room temperature spectra of individual
complexes obtained from the N-PCP reconstitution with Chls
aand b[24]. After excitation at (532 nm) where absorption is
dominated (>90%) by Per, the detected fluorescence of the
Chls (k> 600 nm) originates predominantly from energy trans-
fer. Several hundred molecules of all three reconstituted
complexes have been investigated in this fashion. Most of them
can be grouped by their emission spectra into three types of
complexes. In the case of homo-chlorophyllous complexes, a
single line was observed, as displayed in the first and second
row in Fig. 5. The complexes containing only Chl ashow nar-
row emission lines centered around 670 nm, while the fluores-
cence emission measured for those containing only Chl bis
600 650 700 750
Intensity
600 650 700 750
Chl
b
-N-PCP
Intensity
600 650 700 750
Intensity
600 650 700 750
Intensity
Wavelength [nm]
600 650 700 750
Chl
a/b
-N-PCP
Intensity
Wavelength [nm]
600 650 700 750
Intensity
Wavelen
g
th [nm]
600 650 700 750
Intensity
600 650 700 750
Chl
a
-N-PCP
Intensity
Intensity
600 650 700 750
Fig. 5. Ambient temperature single molecule fluorescence spectra. Three different complexes each (k
exc
= 532 nm) of N-PCP reconstituted with Chl a
(top); Chl b(center) and a 1:1 mixture of Chl aand Chl b(bottom). The lines at 675 nm are drawn to guide the eye. The baselines were recorded at the
same position, after bleaching of the sample.
T.H.P. Brotosudarmo et al. / FEBS Letters 580 (2006) 5257–5262 5261

characterized by somewhat broader lines centered around
650 nm. The most interesting spectra are the ones measured
for the hetero-chlorophyllous complexes, shown in the bottom
row. These systems show a broadened and often split emission
composed of two lines attributed to Chl a(670 nm) and Chl b
(650 nm). The contribution from the short-wavelength com-
ponent, associated with Chl bemission, is in many cases con-
siderably larger than expected for an equilibrated emission (see
above), it is even dominant in one of the samples shown. While
details of the energy transfer in individual complexes are cur-
rently analyzed, the observed emission of both Chl aand
Chl bfrom single complexes unambiguously demonstrates
the successful production of hetero-chlorophyllous complexes.
Acknowledgements: Work was supported by the Deutsche Forschungs-
gemeinschaft, Bonn (SFB 533, projects A6 and B7). T.H.P.B. is
indebted for a stipend from the Evangelischer Entwicklungsdienst,
Bonn. S.M. acknowledges financial support from the Alexander-von-
Humboldt Foundation, Bonn.
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