From the Università di Verona-Facoltà di Scienze
MM.FF.NN. Strada le Grazie, 37134 Verona, Italy
The spectroscopic analysis of a recombinant CP24 complex binding eight
chlorophyll b molecules and a single chlorophyll
a molecule by Gaussian deconvolution allowed the
identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b
absorption in the antenna protein environment. Gaussian subbands at
wavelengths 666, 673, 679, and 686 nm are depleted in the high
chlorophyll b complex, thus suggesting they derive from
chlorophyll a.
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INTRODUCTION |
In higher plants, chloroplasts, chlorophyll, and carotenoid
molecules are noncovalently bound to specific transmembrane proteins to
form light-harvesting complexes called
LHCI1 and LHCII. These
antenna complexes efficiently capture the light and deliver the
excitation energy, respectively, to photosystem I (PSI) and II (PSII)
reaction centers, where electron transport occurs, yielding a
trans-thylakoid pH gradient, ATP synthesis, and
NADP+ reduction. The photosystem II light-harvesting
complex has been extensively investigated and shown to be composed of
four chlorophyll a/b proteins, the major complex (LHCII)
binding about 65% of PSII chlorophyll and three minor complexes
(called CP24, CP26, and CP29) that together bind about 15% of total
PSII chlorophyll (1). These minor chlorophyll proteins appear to be
involved in the dissipation of the chlorophyll excitation energy needed
to prevent overexcitation and photoinhibition of PS II (see Ref. 2 for a review). It was shown that more than 80% of the xanthophyll violaxanthin is associated to minor complexes in maize (3) and that
CP24 is the one with the highest violaxanthin binding capacity. This
pigment is involved in the major photoprotection mechanism in plants,
known as "nonphotochemical quenching" (4), through the operation of
a xanthophyll cycle by which it is deepoxidated to antheraxanthin and
zeaxanthin (5). The intermediate location of minor complexes between
the reaction center and the major LHCII complex (6) makes them well
suited for regulating the excitation energy supply to PSII or its
dissipation. The structural bases for the regulatory properties of the
minor chlorophyll proteins are mostly unknown due to the difficulties
in the isolation of these proteins in sufficient amounts and in their
native form. To overcome this problem, we have reconstituted the CP24
holoprotein using overexpressed apoprotein from Escherichia
coli and purified pigments. Its characterization allowed us to
obtain previously unavailable information on the number of chromophores
bound to this protein and opens the way to the mutational analysis of
this PSII subunit in both its protein moiety and the chromophores
bound. As an example of the usefulness of recombinant pigment-proteins, we have used the recombinant CP24 in order to address the problem of
Chl a and Chl b absorption in antenna proteins;
while only two chemically distinct chlorophyll species are present,
many optical transitions (spectral forms) are commonly observed in the
Qy absorption region (7-12). Lack of progress in understanding this
spectroscopic heterogeneity has been mainly due to the absence of
experimental techniques to enable selective modification of the optical
transitions. Also, it has not been possible to assign particular
transitions to Chl a or Chl b though it is
generally assumed that the shorter wavelength bands are associated with Chl b. Analysis of a recombinant CP24 complex binding mainly
Chl b allowed us to identify four Gaussian subbands peaking
at 638, 645, 652, and 659 nm, which are also present in the native
complex, thus identifying the principle components of Chl b
absorption in the antenna protein environment.
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EXPERIMENTAL PROCEDURES |
Construction of a CP24 (Lhcb6) Expression Plasmid--
To
overexpress plant CP24 in E. coli, the maize
Lhcb6 cDNA (13) was subcloned into an expression vector
of the pDS series (14). A clone was obtained by polymerase chain
reaction mutagenesis of Lhcb6 DNA. The construct pDS
12-24
(Fig. 1A) was obtained by using two primers
(5'-CCGCGCGCAGATCTTCGCC-3' (carrying the BglII site) and
5'-TCTGATCCCATGCATCCGTACGTC-3' (carrying the NsiI site)),
allowing the amplification of a 733-base pair fragment spanning the
full coding region. After digestion with BglII and NsiI, the resulting fragment was subcloned into the pDS-RBS
II expression vector. Thus, the pDS 12-24
construct codes for a protein containing four additional residues (MRIA ... ) extending the N teminus as compared with the native protein (Fig. 1B).
Plasmids were constructed using a standard molecular cloning procedures (15). Bacterial hosts were E. coli (SG13009 strain)
(16).
Isolation of Overexpressed CP24 Apoprotein from
Bacteria--
CP24 apoprotein was isolated from the SG13009 strain
transformed with the construct according to the protocols in Refs. 17 and 18.
Pigment Purification and Analysis--
Total pigment extracts
were obtained by extracting thylakoids of wild-type barley with 80%
acetone. Extracts of Chl a and carotenoids were obtained by
using thylakoids from the Chl b-less mutant chlorina
f2 (19). Chl b and carotenoids were obtained by
preparative HPLC using a reverse phase column (PHENOMENEX, Torrance,
CA) bondclone 10 C18 (7.8 × 300 mm) using 82% acetone as eluent.
Reconstituted complexes were analyzed for their pigment composition
after 80% acetone extraction as described previously (3). During all
of the procedures, care was taken to protect pigments from light and
oxygen. The concentration of pigments was determined spectroscopically
according to Ref. 20 for chlorophylls and using the extinction
coefficients given by Davies (21) for xanthophylls. The concentration
of carotenoid mixtures was estimated on the basis of an average molar
extinction coefficient of 1.4 × 10
5 at 444 nm.
Pigment composition of chlorophyll proteins was determined by HPLC
analysis according to Ref. 22. Chl a/b ratio and
Chl/carotenoid ratio was also determined by fitting the spectrum of
ethanol extracts with the spectra of purified pigments.
Reconstitution of CP24-Pigment Complexes--
The reconstitution
procedure largely followed the one designed for LHCII (18, 23). In the
basic procedure, 400 µg of protein of CP24 apoprotein isolated from
bacteria was solubilized in 1000 µl of a buffer containing 100 mM Tris-HCl (pH 10), the protease inhibitors 6-aminocaproic
acid (5 mM) and benzamidine (1 mM), 12.5%
sucrose, 2% LiDS by heating to 100 °C for 3 min and sonication. After the addition of 100 mM dithiothreitol and the pigment
solution in 70 µl of ethanol, the mixture was sonicated again.
Reconstitution was achieved by three subsequent cycles of freezing (1 h,
20 °C) and thawing (30 min, room temperature). Octyl
-D-glucopyranoside was then substituted for LiDS by precipitation of
the potassium dodecyl sulfate following the addition of 4% KCl,
incubation for 15 min in ice, and centrifugation (10 min at 13,000 × g). The mixture was then loaded on a 12-ml sucrose
gradient (0.1-1 M) containing 10 mM Hepes, pH
7.6, and 0.06% DM and centrifuged for 17 h at 254,000 × g in a Beckman SW 41 rotor. The lower green band (at about
0.4 M sucrose) contained the reconstituted complex and was
harvested with a syringe. Maximal yield was obtained with chlorophyll:protein molar ratios between 40 and 80, corresponding to a
5-10-fold excess of pigments. In this work, the carotenoid concentration was maintained at 60 µg/ml.
Removal of Excess Pigments from the Reconstituted
Complex--
The green bands from the sucrose gradient were then
subjected to chromatography on a Fractogel EMD-DMAE column (15 × 150 mm) (Merck). After loading, the column was washed with 0.025% DM
(70 min at 1 ml/min). The chlorophyll-protein was then eluted by
applying a 0-500 mM NaCl gradient. The peak fractions were
concentrated by Centricon centrifugation and loaded into a glycerol
gradient (10-25%) containing 0.06% DM and 10 mM Hepes,
pH 7.6. The gradient was spun overnight in SW 60 Beckman rotor at
450,000 × g, yielding a faint upper band of free
pigments and a lower band with the chlorophyll-protein, which was
frozen in liquid nitrogen and kept at
80 °C until use.
Isolation of Native CP24--
CP24 was isolated from maize PSII
membranes as described previously (1, 24).
Electrophoresis--
Mildly denaturing electrophoresis was
according to Ref. 25 but at 4 °C and with 20% glycerol in resolving
and stacking gel. Denaturing electrophoresis was according to Ref. 26.
Protein concentration was determined by the bicinchoninic acid method (27).
Densitometry--
Densitometry was performed with a Bio-Rad 600 scanning densitometer after staining of the gel and destaining
according to Ref. 28.
Spectroscopy--
Absorption spectra were obtained using an
SLM-Aminco DW-2000 spectrophotometer at room temperature. Fluorescence
excitation and emission spectra were obtained by using a Jasco-600
spectrofluorimeter. Samples were in 10 mM Hepes, pH 7.6, 0.06% DM, 20% glycerol. Chlorophyll concentration was about 10 µg/ml for absorption measurements and 0.01 µg/ml for fluorescence
measurements. Emission and excitation spectra were corrected for
instrumental response. Analysis of fluorescence spectra was performed
according to Stepanov as previously reported (12, 29). Circular
dichroism spectra were recorded with a Jasco J-600 spectropolarimeter
as previously reported (12).
Gaussian Decomposition of Absorption Spectra--
Similarly to a
described method (11), the decomposition of the absorbance
versus wavelength was obtained by a nonlinear least squares
fitting code (OriginTM; MicroCal. Software Inc.,
Northampton, MA). Here, a linear combination of maximum 10 symmetric
Gaussians (eight absorption bands plus two border ones for tails'
adjustment) was considered in the
2 minimization (error
considered by counting statistics) by means of the Levenberg-Marquardt
algorithm (30). All of the parameters were kept free and always
unconstrained in the analysis; the fitting procedure was reproducible
when starting from a reasonable initial choice for subband FWHM (less
than 10 nm shared by all subbands) and peaks (within the 630-720
wavelength range). Less than 50 iterations were necessary for achieving
the subband positioning, and, after assigning the FWHM to the different
Gaussians, the convergence was estimated by a
2
variation down to a few percentages in successive single
iterations.
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RESULTS |
Expression of Maize CP24 Gene Construct in Bacteria and Isolation
of Overexpressed Apoprotein from Bacteria--
The E. coli
host cells SG13009 (16) are a K12-derived strain. They were transformed
with the pREP4 plasmid, which carries the kanamycin selection and the
lacI gene, encoding the Lac repressor, thus allowing a tight
control over the level of expression (Qiagen). The construct is
described in Fig. 1A, and the
sequence coded is shown in Fig. 1B. The bacterial strain
transformed with the pDS 12-24
produced, upon induction with 2 mM isopropyl-1-thio-
-D-galactopyranoside, the protein of the expected molecular weight as detected by Western blotting with a CP24 antibody (31) (Fig. 1, C and
D). The best results were obtained after 6-7 h of
isopropyl-1-thio-
-D-galactopyranoside induction in
superbroth. In all conditions tested, the expressed protein reached
10% of the total protein extract.

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Fig. 1.
Expression of rCP24 by E. coli
containing Lhcb6 expression plasmid and
isolation of rCP24 apoprotein. A, construction of
CP24-expression plasmid. B, N-terminal sequences of the
native CP24 protein and of the recombinant proteins used in this study
as deduced from DNA sequencing. The first residue of the mature protein
is labeled 1; residues labeled with negative numbers derive
from the transit peptide; C, Coomassie stained SDS-PAGE;
D, immunoblotting with antibody directed to CP24. Lane
1, BBY PSII particles; lane 2, inclusion body
preparation.
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Fractionation of the bacterial cells by the method in Ref. 17 showed
that the expression products are accumulated in inclusion bodies, as
shown for LHCII (18, 32) and CP29 (33). Repeated washings of this
pelletable fraction yielded 80% pure CP24 apoprotein as judged by
polyacrylamide electrophoresis. The protein could be easily purified to
homogeneity by preparative isoelectric focusing, but most of the
experiments here described were performed with the 80% pure
preparation without affecting the efficiency of the reconstitution. All
experiments described in this study have been performed with rCP24
purified as shown in Fig. 1C.
Reconstitution of Pigment-containing Complexes--
Triton
X-100-washed inclusion bodies (15) were extracted with 80% acetone in
order to remove residual Triton and solubilized in 2% LiDS in a bath
sonicator followed by boiling. Pigments were then added from stock
solutions in ethanol, and the reconstitution procedure was carried out
as described under "Experimental Procedures." In this experiment, a
Chl a to Chl b ratio of 1.6, similar to the ratio
in the native complex (1), was used, and the mixture contained a total
carotenoid extract from thylakoids, thus including
-carotene,
lutein, neoxanthin, violaxanthin. Following reconstitution, the mixture
was ultracentrifuged through a 0.1-1 M sucrose gradient, yielding a green band at 0.4 M sucrose, well separated from
the free pigment band on the upper part of the tube at 0.1-0.2
M sucrose (Fig.
2A). The lower green band was
harvested with a syringe and subjected to DMAE chromatography. Once
bound to the column in 10 mM Hepes-KOH, pH 7.6, 50 mM NaCl, 0.03% DM, the nonspecifically bound pigments were
washed from the complexes and the column with 10 mM
Hepes-KOH, pH 7.6, 50 mM NaCl, 0.03% DM at 1 ml/min. This procedure removed about 50% of the chlorophyll loaded in the column but not the protein. The protein was eluted at 200 mM NaCl.
When centrifuged on a 10-25% glycerol gradient, the eluted protein appeared as a single green band without residual free pigments (Fig.
2B).

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Fig. 2.
Isolation of reconstituted CP24 complex.
A, sucrose gradient ultracentrifugation; B,
glycerol gradient ultracentrifugation. The lower band from the gradient
in A was subjected to ion exchange chromatography (see "Experimental
Procedures"), and the eluate was spun into a glycerol gradient. A
single green band was detected, containing the protein while the upper,
free pigment containing band was absent.
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Influence of the Xanthophyll Availability on
Reconstitution--
When isolated from thylakoid membranes, CP24 binds
lutein and violaxanthin but not neoxanthin (3). In order to elucidate the role of different xanthophyll species in refolding of CP24, we have
carried on the reconstitution procedure with pigment mixtures differing
in xanthophyll composition. In each experiment, one xanthophyll species
was omitted from the mixture. Alternatively, reconstitution without
xanthophylls (only Chl a and Chl b) or with the
complete carotenoid set was performed. The stability of the resulting
complex was assayed at two levels of stringency: sucrose gradient
ultracentrifugation (Fig. 3A)
and mildly denaturing LiDS-PAGE (Fig. 3B). When xanthophylls
were omitted from the mixture, a green band was still obtained after
sucrose gradient ultracentrifugation, having the same mobility as the
control sample with a complete xanthophyll supply, suggesting a partial
folding was obtained even in the absence of carotenoids. However, this
complex did not survive LiDS-PAGE. The samples refolded in the presence
of two xanthophylls yielded a green band stable both in sucrose
gradients and in LiDS-PAGE, and the yields were comparable with each
other and with the control sample prepared in the presence of the three xanthophylls. Particularly interesting is the case of sample A, where a
stable complex was obtained in the absence of lutein. This result is at
variance with previous results with LHCII and CP29 (18, 33) in which
lutein was shown to be essential for stability.

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Fig. 3.
Influence of the carotenoid composition on
reconstitution of recombinant CP24. a, sucrose gradient
ultracentrifugation; b, LiDS-PAGE. The gel was
not stained. The refolding mixture contained the following pigments:
Chl a + Chl b + violaxanthin + neoxanthin (lutein
missing) (A); Chl a + Chl b + lutein + neoxanthin (violaxanthin missing) (B); Chl a + Chl b + violaxanthin + lutein (neoxanthin missing)
(C); Chl a + Chl b + violaxanthin + lutein + neoxanthin (total xanthophylls) (D); Chl
a + Chl b (without xanthophylls) (E);
native CP24 (N).
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Influence of Chlorophyll a to Chlorophyll b Ratio during Refolding
on the Pigment Binding Properties of Recombinant CP24--
The data
reported above show that CP24 can be successfully refolded in
vitro. The use of this recombinant protein for mutation analysis
is, however, more informative if its characteristics closely reflect
those of the native complex extracted from thylakoid membranes. We
optimized the conditions for the recombinant protein to reproduce the
chlorophyll a/b ratio of native CP24. For comparison, native
CP24 was purified from maize PSII membranes as described previously
(1), and it showed an a/b ratio of 1.2. A series of
reconstitution mixtures were performed with decreasing Chl a/b ratios, namely 8.0, 5.8, 2.0, 1.5, 1.0 and 0.001. The
last preparation was intended to contain only Chl b.
However, HPLC analysis of the reconstitution mixture showed 1:1000
contamination by Chl a. This experiment was performed on a
semipreparative scale, following the procedure in Fig. 2, finally
obtaining, from glycerol gradient ultracentrifugation, sufficient
amounts of the complex for spectroscopic and biochemical analysis. In
Fig. 4, a plot is shown of the dependence
of the Chl a/b ratio in the complex on the Chl
a/b ratio in the reconstitution mixture; although the reconstitution with Chl b only yielded a stable complex
having a Chl a/b ratio of 0.12, the Chl a/b ratio
of the reconstituted complex rises steeply to about 1 with increasing
Chl a availability and reaches a plateau between ratios of
2.0 and 5.8. Within this range, a ratio of 1.0 for bound Chl
a and Chl b was obtained. When a large Chl
a excess was applied (Chl a/b ratio of 8) it was
possible to obtain a Chl a/b ratio in the reconstituted
complex of 1.4. As a comparison, the data previously obtained for CP29 (12, 33) are also reported, showing that the two proteins clearly
differed in their affinity for Chl a and b. From this comparison, the two complexes with Chl a/b of 1.0 and 1.4 most closely resemble native CP24.

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Fig. 4.
Plot of the dependence of the Chl a/b
ratio in rCP24 on the Chl a/b ratio of the
reconstitution mixture during refolding. In this experiment, all
carotenoids were present in excess.
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Biochemical Characterization of Recombinant CP24--
In order to
further characterize the recombinant CP24 complex
(a/b = 1.4 and a/b = 1.0),
we determined the chlorophyll to protein stoichiometry. Toward this
end, we used a highly purified LHCII preparation (24) as a standard
protein binding 12.6 ± 0.1 chlorophylls/polypeptide (1, 34). The
chlorophyll concentration of the LHCII and of CP24 samples was
carefully determined by HPLC analysis, and aliquots of the samples
corresponding to different amounts of chlorophyll were loaded on an
SDS-PAGE gel. After running, the gel was stained for quantitative
analysis according to Ball (28), and the Coomassie Blue binding to CP24
and LHCII gel bands was determined by densitometry and by elution of
stain and spectrophotometric determination. The Coomassie Blue-stained
gel and the resulting plot is shown in Fig.
5, B and C. Pigment
binding to rCP24 was 0.79 with respect to LHCII. Once corrected for the
sequence-specific difference in Coomassie Blue binding between CP24 and
LHCII (1), a value of 10 chlorophyll (a plus b)
mol per mol of CP24 apoprotein was obtained.

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Fig. 5.
Pigment binding properties of rCP24.
A, HPLC analysis of rCP24 1.0 and native CP24 with native
LHCII as a reference. Note the absence of a neoxanthin peak in rCP24
and nCP24. B, SDS-PAGE analysis of the protein:chlorophyll
ratio. LHCII, used as a reference, was loaded in different amounts in
lanes 3-7. Numbers above refer to the
amount of chlorophyll loaded on the individual lanes. rCP24 1.4 and
rCP24 1.0 (0.37 µg of Chl a + Chl b) were
loaded respectively on lanes 1 and 2. After
running, the gel was stained with Coomassie Blue. C, plot of
the data obtained by densitometry of the gel of B. ,
LHCII; , rCP24 1.0 (0.37 µg of Chl); , rCP24 1.4 (0.37 µg of
Chl).
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The results of HPLC analysis of pigments extracted from native and
recombinant CP29 proteins are reported in Table
I. Both the native and the recombinant
protein contained, besides Chl a and b, the
xanthophylls lutein and violaxanthin but not neoxanthin and
-carotene, although the latter carotenoids were present in the
reconstitution mixture in the same amounts as lutein and violaxanthin. Per 10 chlorophylls (a plus b), two xanthophylls
were found per rCP24 polypeptide. This is consistent with the value of
two xanthophyll molecules per polypeptide found in the homologous
protein CP29 (35). Table I also reports HPLC analysis of the rCP24
proteins with Chl a/b ratio of 0.12, obtained by refolding
with Chl a/b = 0.001. It is shown that the specificity
of carotenoid binding is retained irrespective of the binding of Chl
a or Chl b, since neoxanthin was never present in
the proteins. If a value of two xanthophyll molecules per polypeptide
is assumed (as in the rCP24 1.4 and rCP24 1.0), then rCP24 0.12 binds
one Chl a and eight Chl b as shown by the
Chl:xanthophyll ratio, which is 4.5, thus speaking for nine bound Chl
rather than 10. These data are summarized in Table
II.
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Table I
HPLC analyses of nCP24, rCP24 1.0, rCP24 1.4, rCP24 0.12, and LHCII
Values are in mol/100 mol of Chl a. Data are the average
from three replicates.
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Table II
Stoichiometry of pigment binding to nCP24, recombinant CP24 1.0, recombinant CP24 1.4, recombinant CP24 0.12, and native LHCII
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Spectroscopic Characterization of Recombinant CP24--
The
absorption spectrum of native CP24, which is significantly different
with respect to other antenna complexes (10), is shown in Fig.
6A. It is characterized by a
675-nm red absorption peak and a 654-nm shoulder. Of the absorption
spectra for rCP24 0.12, rCP24 1.0, and rCP24 1.4, only the last two
closely resemble nCP24; both the 675-nm peak and the amplitude of the
654-nm shoulder were reproduced in the recombinant protein, while the
ratio of the absorption in the Soret region versus that in
the Qy transition is the same, i.e. indicating a similar
binding of xanthophylls, relative to Chl, in native and recombinant
proteins (see above). The absorption spectrum of rCP24 0.12 was very
different; the 675-nm peak disappeared, while the 654-nm form becomes
the absolute maximum in the Qy absorption region. Two broader
absorption features with low amplitudes were evident between 590 and
630 nm in the spectrum of nCP24, rCP24 1.0, and rCP24 1.4 (arrows in Fig. 6A). Of these, the red-most one
(centered at 622 nm) disappeared in rCP24 0.12. Circular dichroism
spectra are shown in Fig. 6B. In the case of nCP24, the Qy
region is characterized by two negative signals at 679 and 649 nm,
while in the Soret region it is negative between 490 and 465 nm with a
minimum at 481 nm and a shoulder at 497 nm. Recombinant CP24 had a
similar general shape; however, while rCP24 1.0 very closely fitted the
nCP24 spectrum, rCP24 1.4 showed a 2-nm red shift in the position of
the red-most signal (681 versus 679 nm) and in the relative
amplitude of the signals in the Soret region. rCP24 0.12 strongly
differed in the Qy region, showing a 17-nm blue shift in the red-most
band. Moreover, the amplitude of the signal of this sample in the Soret
region was much increased and slightly shifted to peaks at 476 nm (
)
and 463 nm (+).

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Fig. 6.
Optical spectroscopy of nCP24 and recombinant
complexes with different a/b ratio. A,
absorption spectra. Optical pathlength was 1.0 cm. B,
circular dichroism.  , native; - - -, rCP24 1.0;
· · · ·, rCP24 1.4; ·-·-·-, rCP24 0.12. The samples
were on Hepes-KOH, pH 7.6, 0.06% DM, 15% glycerol.
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Fluorescence spectroscopy was then performed in order to verify the
functional connection between pigments bound to the protein in native
and recombinant CP29. Fluorescence excitation spectra (with 680-nm
emission) are shown in Fig.
7A. Chl a and Chl
b contributions are clearly detected at 440 and 465 nm,
indicating efficient energy transfer between Chl b and Chl
a. In pigment-proteins, both vibrational relaxation within
pigment-excited states and energy transfer between pigments are rapid
with respect to the excited state lifetime. Therefore, thermal
equilibration between all inter- and intramolecular energy levels is
rapidly attained. The actual occurrence of this thermal equilibration
can be verified by applying the Stepanov analysis of the steady-state
fluorescence (29, 36) by which the fluorescence spectra, expected on
the basis of complete thermal equilibration within the complexes, can
be calculated from absorption spectra. Measured and calculated spectra
will be coincident for fully equilibrated pigment-proteins. When
Stepanov analysis was applied to native CP24, quite good correspondence
between calculated and measured emission spectra is observed over most
of the emission band (Fig.
8A). In the case of rCP24,
this correspondence was good for rCP24 1.0 and rCP24 1.4 (Fig. 8,
B and C), while in the case of rCP24 0.12, a
distinct deviation was observed (Fig. 8D); two emissions
were present in both the measured and the calculated spectra at 663 and
679 nm, respectively, corresponding to Chl b and Chl
a emissions as indicated by the relative amplitude of these
two components upon 475- and 440-nm excitations. The emission spectrum
obtained with 440-nm excitation was similar to the calculated spectrum
but showed a higher amplitude of the 663-nm component, thus implying a
small excess of excited states on Chl b.

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Fig. 7.
Fluorescence excitation spectra of native and
recombinant CP24. Emission was at 681 nm; bandwith for excitation
end emission was 2 nm. Sample concentration was less than 0.1 µg of
Chl a + Chl b/ml. Other conditions as in Fig.
6.
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Fig. 8.
Comparison between calculated (dashed
lines) and measured (dotted lines) steady state
fluorescence spectra for native and reconstituted CP24 complexes with
different Chl a/b binding ratios. Emission spectra
were calculated from absorption spectra (solid lines) using
Stepanov's relation (26). D, emission spectra with 475-nm
excitation (·-·-·-) in addition to the 440-nm excitation
(dotted line in panels
A-D).
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Gaussian Deconvolution Analysis of Native and Recombinant
CP24--
The results reported above show that rCP24 complexes can be
obtained in vitro, and their chromophore content can be
modulated for obtaining a stable complex with altered Chl a
to Chl b ratio. By reconstituting CP24 with a large excess
of Chl b over Chl a (Chl a/b = 0.001) a CP24 binding eight Chl b and one Chl a
was obtained. Such a complex is ideal for the purpose of addressing the
problem of correspondence between chemically distinct chlorophyll species and absorption forms in the protein, since chlorophyll b absorption is greater than is the case for proteins
extracted from thylakoids and therefore can be analyzed with little
interference by Chl a absorption. In Fig.
9, the Qy transition region is shown for
the absorption spectra, together with the results of Gaussian deconvolution, from native CP24 and three different recombinant complexes (rCP24 0.12, rCP24 1.0, and rCP24 1.4), while the
characteristics of this analysis are summarized in Table
III. The spectra were deconvoluted as
linear combinations of symmetric Gaussians whose parameters (peak
wavelength, full-width at half-maximum, and percentage amplitude to the
630-720-nm spectrum integral) were kept free. The only constraint
considered was the final control of the total bandwidth in the 9-11-nm
range, as expected from the analysis of electron-phonon coupling and
site-inhomogeneous broadening in a variety of antenna chlorophyll
proteins (10, 12, 37, 38) in the accepted solutions. For the sake of
model simplicity and reducing the arbitrariness of the fitting, the
minimal choice of adjustable parameters was always used,
i.e. both the minimal number of Gaussian forms and the curve
symmetry for a unique bandwidth; in this sense, at room temperature the
absorbance fine structure, like inhomogeneities in bandwidth, are
hardly recognizable in the spectra due to the smearing by thermal
broadening.

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Fig. 9.
Gaussian subband analysis of absorption
spectra of native and reconstituted CP24 measured at 300 K. Lower left, nCP24 (native); upper
right, rCP24 1.0; lower right, rCP24
1.4; upper left, rCP24 0.12. The residual plot is also shown
below each plot for fitting accuracy (see also
"Experimental Procedures").
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Table III
Gaussian decomposition parameters for native and reconstituted CP24
complexes with different Chl a/b ratios
See "Experimental Procedures" for method of analysis. The data are
graphically represented in Fig. 9. The temperature was 300 K.
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Eight significant Gaussians were needed in order to fit the spectra of
the native complex and of the rCP24 1.0 and rCP24 1.4, consistently
with the results previously reported for CP29 (12) and LHCII (38). As
pointed out above, although the Chl a/b ratio changed, the
total number of Chl molecules bound per CP24 polypeptide remained
essentially constant at 10 in the different reconstitution conditions,
except for the case of rCP24 0.12, which had an empty site. This
implies that several Chl binding sites can either be occupied by Chl
a or Chl b. As can be clearly seen in Table III, the wavelength of the subbands did not change significantly in most of
the decompositions. Thus, it should be relatively easy to identify the
subbands associated with Chl b or with Chl a
absorption by determining those in which the absorption intensity
respectively increases or decreases in the protein with higher Chl
b binding upon normalization of the spectra. For
normalization, we used the measured Chl a/b stoichiometry
assuming a relative Chl b/Chl a extinction ratio
of 0.7, based on solvent values (12, 39). In this respect, the total Qy
absorption areas for the different complexes were rescaled to the
calculated Chl a plus Chl b total extinction. The
subband amplitudes are then compared with the normalized area and
reported for the different complexes in the histogram of Fig.
10 as a ratio of specific subband
chloropyll content to total chlorophyll pool. It can be noticed that
nCP24, rCP24 1.0, and rCP29 1.4 are rather similar to each other,
although rCP24 1.0 most closely fits the values of nCP24. The rCP24 1.4 complex (slightly higher Chl a content with respect to nCP24
and rCP24 1.0) had increased absorption at 666 and 673 nm, while the amplitude decrease at 645, 652, and 659 nm suggests that these absorption forms are related to Chl bound to the less selective sites.
The case of rCP24 0.12 was the most informative; the 686-nm component
was completely absent, and those peaking at 666, 673, and 679 nm were
strongly decreased (by 32, 73, and 85%, respectively). On the other
hand, the shortest wavelength forms at 638, 645, 652, and 659 nm had
their amplitude increased by 13, 35, 54, and 38%, respectively.

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Fig. 10.
Histogram of areas for the different
subbands as obtained from Gaussian deconvolution. Spectra were
normalized for the same total Chl a plus Chl b concentration
as determined by HPLC analysis (see also "Experimental
Procedures").
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As for the blue-most form at 638 nm, all of the complexes, either
reconstituted or native, have a chlorophyll ratio of about 1 per site,
and (within ±10% of overall error associated with this extrapolation)
this ratio suggests the constant filling of this binding site and its
unfavored affinity to Chl a.
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DISCUSSION |
The publication of a near atomic structure of LHCII has been a
major event in the study of chlorophyll a/b proteins (34), since this structure gives the first insight into pigment and protein
organization not only in the major LHCII complex but also in the
homologous proteins that contribute to the organization of the PSI and
PSII antenna complexes (40, 41). Nevertheless, a number of questions,
important in order to understand the relation between structure and
light-harvesting and energy transfer functions of each individual
chlorophyll protein and of the whole antenna system need to be
addressed. Thus, an assignment of spectroscopic characteristics to the
various chromophores in the crystal structure as well as the transition
dipole orientations of the single chlorophylls would be very useful. It
is hoped that this information can be obtained by a combined effort of
spectroscopic and molecular genetic approaches (11, 10, 18, 42), as
proven effective in prokaryotic systems (43-45). In the case of minor
chlorophyll proteins, besides the obvious function of light harvesting
and energy transfer, a variety of regulatory mechanisms are associated
(3, 46-48) whose structural frameworks need to be elucidated. As an
example, a single point mutation of the Glu166 residue of
CP29 abolished dicyclohexylcarbodiimide binding, thus allowing
identification of a protonable residue potentially responsible for
triggering of high energy quenching by low luminal pH (35). This
approach needs to be extended to the other antenna proteins. In this
work, we have reconstituted a CP24 complex from pigment extracts and
the apoprotein overexpressed in E. coli as a step toward
both the crystallization of the pigment-proteins and mutational analysis.
Native Versus Recombinant CP24: Is Recombinant CP24 "Better"
Than the Complex Extracted from Leaves?--
Our procedure yields
recombinant CP24 proteins that exhibit many features of the native
protein extracted from thylakoids: apparent molecular mass in green
gels and density gradients, specificity of xanthophyll binding,
characteristics of absorption, and circular dichroism spectra.
Reconstitution of pigment-proteins from recombinant apoprotein and
isolated pigments was previously performed in the case of the major
LHCII protein (32, 42), of two LHCI subunits (49), and of the minor
PSII subunit CP29 (33). In the latter case, it was possible to
reproduce all of the biochemical and spectroscopic features of the
native complex, thus allowing mutation analysis of chlorophyll-binding
sites (12, 35). While the characteristics of CP29 are well known from
previous studies (1, 10, 11), those of CP24 are ill defined. Since the
first report (50), CP24 has been reported to have different chlorophyll
a/b ratio, ranging from 0.9 to 1.6, depending on plant
material and purification procedures (for a review, see Ref. 2), thus
suggesting that this protein is unstable and loses pigments once
extracted from thylakoids. This is possibly due to the lack of the
short amphiphylic helix located near the C terminus of most Lhc
proteins (40), as suggested from the results of C-terminal deletions in
LHCII with impaired Chl binding (32, 51). Because we could not rely on
the correspondence of the nCP24 preparation to the protein inserted in
the thylakoid membrane, we refer to the case of CP29 as a model. As
shown in Fig. 4, a family of rCP29 proteins was generated by refolding
in the presence of pigments mixtures with different Chl a/b
ratios (12, 33); increasing Chl a/b ratio yielded proteins
with lower Chl b content, thus reaching the Chl
a/b ratio of 3.0, corresponding to the ratio in the native protein, with an 8-fold excess of Chl a over Chl
b during refolding. A rCP29 complex with increased Chl
a content could only be obtained by using a Chl
a/b ratio >20 during
folding2; therefore, a
plateau was obtained in the plot of Fig. 4, showing that between 8 and
20 a rCP29 with a Chl a/b ratio of 3 was obtained. This
complex was carefully examined and found to be identical to the native
complex by analysis with a number of spectroscopic and biochemical
methods (12, 33, 35), thus suggesting that this protein's conformation
is intrinsically more stable as compared with others with altered Chl
a versus Chl b binding. Following this
example, we could identify a similar behavior in the case of rCP24; Chl
a/b in the complex rapidly increased from 0.12 to 1.0, and
this value was maintained with an increasing Chl a/b ratio
in the folding mixture between 2.0 and 5.8. rCP24 1.0 has a CD spectrum
almost identical to nCP24, while other products show distinct
differences in their CD such as blue or red shifts in the wavelength of
the red-most negative signal. A similar behavior was previously
recognized in the case of CP29, where products with altered Chl
a/b ratio showed red or blue shifts in their red-most
negative signal (12, 33). On the basis of the CD spectra and of the
plot of Chl a/b ratio in the refolding mixture versus that in the complex, we propose that rCP24 1.0 corresponds to the undenatured CP24, while the nCP24 is partially
denatured as a result of the purification procedures. This is not
surprising, since purification of CP24 requires a preparative
isoelectric focusing step in which complexes are subjected to low pH
and high voltage during several hours, while the refolding procedure
involves milder steps such as rapid FPLC separation and glycerol
gradient ultracentrifugation. It appears that the more stable
CP29 protein is not affected by purification procedures, since its
spectroscopic properties remain the same irrespective of the procedure
used (6, 52), while CP24 is quite sensitive to harsh purification steps
as discussed above.
Chlorophyll Binding--
Porphyrin binding of rCP24 1.0 has been
determined; it binds 10 chlorophyll molecules per polypeptide and
therefore five Chl a and five Chl b. This value
of 10 is intermediate between those of LHCII (seven Chl a
plus five Chl b (1, 34)) and of CP29 (six Chl a
plus two Chl b (1)). Comparison of cDNA-deduced sequences shows that at least seven of eight residues reported to
coordinate porphyrins in LHCII (34) are conserved, thus setting at
seven the lower limit for Chl binding in CP24. The previously reported
value of five chlorophylls per CP24 polypeptide (1) is therefore to be
ascribed to pigment loss, since the same measurements yielded
eight Chl in CP29 with both the native and recombinant protein
(12, 33).
Carotenoid Binding and Specificity: Lutein Is Not Essential for
Folding of CP24--
Two xanthophyll molecules have been found in
rCP24, irrespective to the Chl a/b ratio. This is in
accordance with the case of CP29 (3, 12, 33) but different with respect
to LHCII, where three rather than two xanthophyll molecules per
polypeptide were found (53), suggesting that the presence of two
xanthophyll binding sites might be a general feature of monomeric
antenna complexes. Accordingly, monomerization of LHCII has been
reported to be accompanied by carotenoid loss (54). A peculiar feature of CP24 with respect to the other PSII antenna proteins is the lack of
neoxanthin (3). This feature was reproduced through in vitro
reconstitution, further indicating that the procedure here described
yields antenna proteins closely resembling their native state. LHCI
proteins also lack neoxanthin when isolated from thylakoids (55), but
Lhca1 and Lhca4 proteins, reconstituted by a different procedure, bound
substantial amounts of neoxanthin (49). The functional reason for the
absence of neoxanthin in CP24 and in LHCI proteins is presently
unknown. CP24 can only be reconstituted in the presence of carotenoids
consistently with previous reports with LHCII (18, 23, 32) and CP29
(33). However, there is only limited specificity in carotenoid binding. Any one of the carotenoids (neoxanthin, violaxanthin, or lutein) can be omitted from the reconstitution mixture in agreement
with previous findings with LHCII from delipidated thylakoid
proteins (23).
Xanthophyll Cycle Carotenoids in CP24--
Recombinant CP24 does
not bind neoxanthin; however, it forms a stable complex in the absence
of lutein and therefore binds only violaxanthin. This result is
particularly interesting, since it is different from what has been
found with LHCII or CP29. The latter have an absolute requirement
for lutein, although they can be interchanged with other xanthophylls
(18, 23, 32, 33). Analysis of Arabidopsis mutants showed
that increased amounts of other carotenoids can compensate for the
absence of lutein (56).
It is tempting to correlate the capacity of violaxanthin to stabilize
the pigmented complex of the CP24 apoprotein with the dominant role
that CP24 is thought to play (together with CP29 and CP26) in the
xanthophyll cycle (57, 58). These three complexes bind most of the
violaxanthin in dark adapted plants (3) and of the zeaxanthin after
exposure of leaves to excess light (57, 59). However, the violaxanthin
to zeaxanthin conversion is more rapid and complete in CP24 and CP26 in
comparison with CP29 (59, 60). Recent results with CP26 suggest that it
shares the plasticity with respect to carotenoid binding described here
for CP24 when the proteins are refolded in vitro (61). These
data suggest that the ability of antenna proteins to participate in the
xanthophyll-dependent light regulation mechanism correlates
with the ability of the protein to accommodate violaxanthin within the
carotenoid binding sites. The involvement of xanthophylls in the
dissipation of excess light energy includes the deepoxidation of
violaxanthin to yield zeaxanthin. This reaction is not yet fully
understood (for a review see Ref. 2) particularly with respect to the
location of the violaxanthin that forms the substrate to the
deepoxidase, situated in the lumen.
Chlorophyll a and Chlorophyll b Contributions to the Absorption
Spectrum--
On the basis of the determination of the chlorophyll to
protein stoichiometry, it is shown that each native monomer complex binds on an average five molecules of Chl a and five
molecules of Chl b. When the a/b binding ratio is
decreased, the amount of bound Chl b increases while that of
Chl a decreases. It is important to note that the total
average number of bound chlorophylls tends to remain constant at 10. In
the extreme case here investigated, consisting of eight bound Chl
b molecules and one bound Chl a molecule per
polypeptide, a slight decrease of Chl binding of 0.9-1 molecules per
polypeptide is detected, thus indicating that ectopic Chl b
binding is only moderately defavored. This result suggests that CP24
has 10 Chl binding sites, several of which may be occupied by either
Chl a or Chl b. This statement has some limitations, since at least one site remains largely empty if Chl
a is not available, and another shows a very high affinity for Chl a, since it binds Chl a even where there
is a 1000-fold excess of Chl b.
In order to reconstitute spectroscopically useful complexes, it is
necessary to demonstrate that pigment binding occurs in a specific and
"correct" way and that the complex is functionally competent. To
this end, we performed two distinct kinds of experiment. First, the
absorption/fluorescence relationship was analyzed by means of the
Stepanov expression. This analysis demonstrated that thermal
equilibration is essentially attained in both native and reconstituted
complexes, which means that all pigments are coupled energetically even
in the case of rCP24 0.12 and that the deviation from equilibration is
rather small (possibly due to the a missing chlorophyll site).
Secondly, by the fluorescence experiments, the excitation spectrum
reveals that chlorophyll b is able to transfer excitation
energy to Chl a. These observations, together with the
similarity of rCP24 and the native complex in respect to many optical
properties, strongly indicate that the pigment binding occurred
"correctly" during the present reconstitution experiments.
In the remaining discussion, we will address the relevance of the
present experiments to an understanding of the absorption characteristics of Chl a and Chl b in the antenna
proteins of higher plants. In order to accurately describe room
temperature absorption in the Qy region, taking into account reasonable
values for the broadened bandwidths (9-11 nm at room temperature, 6-7 nm at 71 K) (62), eight Gaussian subbands are required. In the 630-665-nm region, where Chl b absorption is expected, four
subbands, with wavelength positions at 639, 645, 653, and 659 nm were
obtained. These values were previously reported in the case of the
homologous proteins CP 29 and LHCII (12, 38), suggesting that the
conclusions reached in the case of CP24 can be extended to the
absorption of the whole Lhcb proteins. Independent evidence for the
648- and 655-nm transitions comes from photo-oxidation experiments in
CP29 (12). In rCP24 0.12, all four subbands increase in intensity, while those at higher wavelengths are strongly decreased. It is clear
that these former four subbands are associated with Chl b
absorption. An interesting point that should be emphasized is that we
detected only small changes in the wavelength positions of these four
Chl b transitions by Gaussian analysis or second derivative
analysis (not shown), although the average binding stoichiometry for
Chl a changes from about 5 to 1 molecules per polypeptide.
This was also true for the four Gaussian subbands in the 660-690-nm
range (peaking at 666, 673, 679, and 686 nm) that are strongly
decreased or absent in rCP24 0.12, and they can be associated to Chl
a absorption. It should be noted that, although in rCP24
0.12 a single Chl a molecule is present per polypeptide, its absorption can be deconvoluted into three Gaussian subbands, i.e. 666, 673, and 679 nm. It follows that the
number of spectral bands associated with Chl a in rCP24 0.12 is greater than the average number of bound Chl a molecules.
This clearly indicates Chl a binding heterogeneity at the
level of the different complexes. We therefore envisage each CP24
preparation to represent a mixed site population of bound Chl
a molecules. A similar conclusion was obtained for Chl
b in CP29 (12). This is in line with the conclusion
discussed above that most Chl binding sites (all except for two) may
bind either Chl a or Chl b.
The above conclusions, although mostly evident from the analysis of
rCP24 0.12, which is partially affected in its pigment-pigment energy
transfer due to an empty Chl site, are consistent with the
characteristics of the fully functional rCP24 1.0, rCP24 1.4. The exact
population binding ratio is expected to be determined, at least in
part, by the Chl a/b ratio present during
pigment-protein folding both in vivo and in
vitro. This is in agreement with a previous work on intermittent
light-grown plants in which lower Chl b availability caused
a higher chlorophyll a/b ratio in CP29 and CP26 proteins
(63). It is clear that sites have different binding affinities for Chl
a versus Chl b; absorption forms
described by Gaussian subbands peaking at 666 and 673 nm are increased
in their amplitude in rCP24 1.4 (six Chl a versus
four Chl b) with respect to rCP24 1.0 (five Chl a
versus five Chl b), implying that there are
binding sites that have a low selectivity against Chl b.
Another observation is that the amplitude of the 639-nm Chl
b subband increases by 13% in rCP24 0.12 with respect to
rCP24 1.0, while the increase in other Chl b forms ranges
from 30 to 55%; this suggests that the 639-nm absorption is produced
by a rather unique Chl b site and that the Chl a
sites, where ectopic Chl b may be bound, do not provide the
basis for such a strong blue shift as observed in Chl b
absorption (Chl b spectrum in ether peaks at 645 nm).
Charged groups interacting with
-orbitals might be responsible for
this absorption (45).
In the end, protein-pigment interaction in different sites seems to
play a dominant role in determining the absorption energies. That
excitonic interactions, although weak, exist in CP24, as can be clearly
observed by the behavior of the positive 428-nm (+) signal and of
the 477 (
) signal in the CD spectra. This structure, attributed to
Chl a-Chl b interaction (64), is present in
native complex, and its amplitude is greatly increased in the complex binding one Chl a and eight Chl b molecules.
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CONCLUSIONS |
In this study, we have reconstituted the CP24 pigment-binding
subunit of photosystem II from the overexpressed apoprotein and
purified pigments. The recombinant protein binds five Chl a,
five Chl b, and two xanthophyll molecules per mol of
polypeptide and appears to be more stable with respect to the protein
extracted from leaves, which loses pigments during purification.
Recombinant CP24 has a different specificity in xanthophyll binding
with respect to other members of Lhc family that can be important in
the xanthophyll cycle photoprotection mechanism. This recombinant
protein can be used for mutational analysis of both the polypeptide
sequence and of the chromophore moiety. As an example of the latter
approach, we show, by using recombinant proteins with altered
chromophore composition, that the principle components for chlorophyll
absorption can be described by four Gaussian subbands (639, 645, 653, and 659 nm) for chlorophyll b and four more (666, 673, 679, and 686 nm) for chlorophyll a in the CP24 spectrum.
We thank Harald Paulsen (Botany III, Munchen,
G.) for helpful suggestions and the kind gift of the pDS-RBS II
plasmid. Dr. Dorianna Sandonà is thanked for discussion and
advice on DNA work, and Roberta Croce is gratefully acknowledged for
performing HPLC, FPLC, CD spectroscopy, and analysis of fluorescence
spectra. David Simpson, (Carlsberg Laboratory-Kopenhagen) is thanked
for the kind gift of chlorina f2 barley mutant.
Professor Andrea Melandri is thanked for encouragement and help.