(Received for publication, February 25, 1997, and in revised form, April 30, 1997)
From The most abundant chlorophyll-binding complex in
plants is the intrinsic membrane protein light-harvesting complex II
(LHC II). LHC II acts as a light-harvesting antenna and has an
important role in the distribution of absorbed energy between the two
photosystems of photosynthesis. We used spectroscopic techniques to
study a synthetic peptide with identical sequence to the LHC IIb N
terminus found in pea, with and without the phosphorylated Thr at the
5th amino acid residue, and to study both forms of the native
full-length protein. Our results show that the N terminus of LHC II
changes structure upon phosphorylation and that the structural change resembles that of rabbit glycogen phosphorylase, one of the few phosphoproteins where both phosphorylated and non-phosphorylated structures have been solved. Our results indicate that phosphorylation of membrane proteins may regulate their function through structural protein-protein interactions in surface-exposed domains.
Light harvesting complex II (LHC II)1
is a major chlorophyll-containing protein complex that accounts alone
for half of the pigments involved in photosynthesis in plants. It is
located mainly in appressed regions of the thylakoid membrane where it
acts as the light-harvesting antenna for photosystem II (PS II).
Reversible phosphorylation of LHC II is an established mechanism for
redistribution of absorbed light energy between PS II and PS I. Phosphorylation of LHC II (giving LHC II(P)) is triggered by conditions
where the plastoquinone pool of the photosynthetic electron transport chain becomes reduced (1). The kinase responsible for the
phosphorylation of LHC II is not yet identified, although it is
suggested that it is located in the core of photosystem II (2) or in
contact with the cytochrome b6f
complex (3, 4). LHC II(P) is found in the unappressed regions of the
chloroplast thylakoid membrane and there acts as a light-harvesting
antenna for photosystem I (PS I) (5-7). From electron crystallography
of 2-dimensional crystals, a structure for the major part of
non-phosphorylated LHC II has been described at 3.4-Å resolution (8).
This structure reveals no information regarding the N-terminal domain
that contains the phosphorylation site at position 5 (Thr); the protein
backbone was traced only to residue 26 where it ends up close to the
lipid membrane, consistent with the fact that the sequence between
residues 21 and 29 (RVKYLGPF) (9) consists mainly of hydrophobic,
aromatic, or charged amino acids. Aromatic residues are located at the
membrane surface in structures of membrane proteins (10-13), and
residues Trp-16 and Tyr-17 of LHC II may also then form a point of
contact with the membrane. LHC II has been shown to lose its ability to trimerize when more than the first 15 amino acids are removed from the
apoprotein (14). At this site, specific lipid-protein interactions
between the amino side chains and the lipid
phosphatidylglycerol are involved in stabilization of the
trimers (15), which implies that only the first 15 amino acid residues
at the N terminus may be free of competing interactions with the
membrane. This sequence (RKSAT*TKKVASSGSP, where * denotes the
phosphorylation site, Thr-5) contains numerous positive charges, which
may be compensated by the negative charge introduced by
phosphorylation. To see whether a structural change occurs within the
N-terminal domain itself, we have studied a synthetic peptide with the
N-terminal sequence normally found in pea (9) with and without Thr-5
synthetically phosphorylated. We have also studied native LHC II/LHC
II(P) from pea to see if there exists a structural analogy between the
peptides and the native protein. Our results show that phosphorylation causes a structural change both in the model peptide and at the N
terminus of LHC II itself, together with dissociation of the trimer aggregate. Specific changes in structure-dependent
protein-protein as well as lipid-protein interactions must therefore be
the basis of the mechanism by which phosphorylation controls the
functional interactions of LHC II in vivo.
The synthetic
peptides RKSATTKKVASSGSP (1585.5 Da) and the corresponding
phosphorylated form RKSAT(PO3)TKKVASSGSP (1664.5 Da) were
synthesized as described earlier (16). For the Fourier transform
infrared (FTIR) and circular dichroism (CD) measurements, full-length
proteins of LHC II were isolated from pea leaves (Pisum sativum L.) according to standard protocols (17, 18). Normally 100 g of pea leaves were harvested, and each batch of thylakoids was then divided into two; one, giving phospho-LHC II, was then illuminated (130 µmol m Static solvent perturbation UV
absorption measurements of LHC II(P) and LHC II were performed on a
double-beam Hitachi U-3000 (Hitachi Ltd, Japan); equal concentrations
dissolved in 20 mM Tris buffer (pH 8.0) or H2O
were used as sample and reference, respectively. Ethanol was then added
to give up to 10% (v/v) in both sample and reference, and difference
spectra were measured between 200 and 300 nm at a resolution of 0.1 nm.
All difference spectra showed zero absorbance in the region 200-600
nm, before ethanol addition. The difference spectrum is the average of
five individual, but nearly identical, difference spectra, each from one sample preparation (five non-phosphorylated and five
phosphorylated), and the resulting spectra were then co-added and
averaged. Standard spectra of L-tyrosine and
L-tryptophan (Sigma) solutions in Tris buffer or
H2O with additional ethanol were used to confirm the origin
of the increase in absorbance at 280 nm upon solvent perturbation. Identical solutions in the sample and reference cuvettes were used to
check the samples for inhomogeneity; no spectral difference between
samples was then seen either before or after addition of ethanol.
The circular dichroism (CD) spectra were
obtained on a JASCO 720 (Japan Spectroscopic Co. Ltd, Tokyo, Japan)
spectrophotometer at 25 °C, using an 8.0 mM, 0.1 mM, or 0.8 µM protein solution in a quartz
cuvette with an optical path length of 1 mm. The scan velocity was 1 nm
s FTIR spectra were recorded at a Bruker
IFS 66 (Karlsruhe, Germany) spectrometer using a liquid N2
cooled MCT detector. 2000 scans were collected and Fourier transformed
to obtain a spectral resolution of 2 cm All nuclear magnetic resonance (NMR)
spectra were acquired at 500 MHz on a GE Omega 500 spectrometer
(General Electric, Fremont, CA). Spectra were obtained of aqueous
peptide solutions (8 mM). pH was established by addition of
small volumes of HCl or NaOH solution in the case of measurements in
H2O or of deuterium chloride or sodium deuteroxide in
D2O for measurements in deuterated solution; the peptide
solutions were self-buffering. All NMR spectra were recorded at
2 °C. To obtain a temperature dependence of the NH chemical shift
for the phosphorylated peptide at pH 5.2, TOCSY spectra were recorded
at 5, 17, and 25 °C. TOCSY spectra were acquired using the MLEV
sequence (21) with a mixing time of 120 ms, at 2048 data points with 16 repetitions and 256 All three
spectroscopic methods revealed distinct differences between the
non-phosphorylated and phosphorylated forms of a 15-residue peptide
corresponding to the N-terminal phosphorylation site of LHC II. CD
spectra clearly show differences in conformation between the two
peptides in monomeric solution (0.8 µM) (Fig. 1A). The phosphorylated peptide contains
Table I.
Showing the calculated relative amount or relative area representing
the different classes of secondary protein structures
Plant Cell Biology,
Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Peptide Synthesis and Protein Purification
2 s
1 for 20 min)
in the presence of 0.4 mM ATP (Sigma) and 25 mM
NaF (Sigma). The same purification protocol was followed for both samples, except that all buffers contained 10 mM NaF in the
purification yielding phospho-LHC II. This preparation contained a
mixture of both phosphorylated and non-phosphorylated LHC II; the
proportion of LHC II(P) was determined by mass spectroscopy to be
approximately 10%. For simplicity the phosphorylated preparation will
from herein be denoted as LHC II(P).
1 in the frequency range between 180 and 250 nm, and
each spectrum consists of eight scans. The spectra shown are the
average of four, each from an individual sample, and with the water
background subtracted. The relative contribution of each secondary
structural motif was calculated with software supplied with the
spectrophotometer (19). Solutions of LHC II and LHC II(P) were measured
between 350 and 750 nm to determine the oligomeric state (20) but were otherwise under the same conditions as those described above.
1 in the spectral
region 4000-600 cm
1. The spectra were measured using a
horizontal attenuated-total-reflection (ATR)-crystal (45°) (ZnSe).
Peptide solution (approximately 75 µl, 0.8 µM (pH 5.2))
was spread out on the internal reflection crystal and then the sample
holder was sealed to avoid evaporation of water. All peptides were
washed repeatedly with either H2O or D2O
(Sigma), followed by rotary evaporation using a Speedvac (Savant
Industries, Farmingdale, NY) to dry the peptide between washes, to
remove traces of the trifluoroacetic acid used during peptide
purification. The H2O/D2O exchange of LHC II
was performed on a dried sample by addition of 70 µl of
D2O to the protein film. The exchange was followed by
sequential measurements of 100 scans (30 s) during a period of 2 h. The film was then repeatedly dried and rehydrated with
D2O to obtain full H2O/D2O
exchange. All the difference spectra are the average of eight
individual spectra, each from one sample preparation (four
non-phosphorylated and four phosphorylated), and each individual
spectrum is the signal average of 2000 scans. Spectral deconvolution
(LabCalc-Galactic Industries Corp., Salem, NH) and derivation were
performed. The number of bands and the peak positions thereby obtained
were used to calculate (PeakFit-Jandel Scientific Software, San Rafael, CA) a curve fit that is composed of Lorenzian bands for the original IR
absorbance band. In the case of H2O, an interactive
spectral subtraction was performed to remove the spectral influence of the
-mode of bulk water, positioned at 1645 cm
1. All
FTIR measurements were performed at 22 °C.
1 values. ROESY spectra (22) were
acquired with a mixing time of 200 ms, at 2048 or 4096 data points with
32 repetitions and 256
1 values. Nuclear Overhauser
enhancement spectroscopy spectra (23) were acquired with a mixing time
of 500 ms, at 2048 data points with 32 repetitions and 256
1 values. Sequential assignment was carried out by
methods of Wüthrich (24). One-dimensional spectra were recorded
for both peptides at concentrations 8.0, 2.0, 0.5, and 0.1 mM, and no changes were observed in line shape or line
position.
CD, NMR, and FTIR Spectroscopy of LHC II Peptides
-helix, a structural type that is absent from the non-phosphorylated
form (see Table I). The
-helix content of 12%, which
corresponds to only 2 amino residues, in the phosphorylated peptide is
not sufficient to form a complete
-helical turn. The
-structure
content is constant at around 45% for the two peptides, but the random
coil content is decreased in the phosphorylated peptide by the same
amount (12%) as the increase in
-helix. CD measurements made at the same concentration range (8.0-0.1 mM) as the NMR spectra
(see below) show almost identical spectra (Fig. 1A) for the
two peptides, both being rather similar to the spectrum of the
non-phosphorylated peptide at lower concentrations. These spectra
indicate mainly random coil structures with some
-structure
contribution. The difference between the two sets of concentrations
indicates that the peptides aggregate at higher concentrations, and
intermolecular interaction is thereby introduced.
Fig. 1.
UV-visible spectra. Panel A shows
CD spectra of the phosphorylated (a) and the
non-phosphorylated peptide (b) at 0.8 µM and
at 8 mM (c and d) in the wavelength
region 180-250 nm. Panel B shows CD spectra of LHC II(P)
(trace a, dotted line) and LHC II (trace b, solid
line) in the wavelength region 350-750 nm. The shoulder
characteristic of the trimeric form of LHC II is found at 648 nm (20).
Panel C shows the solvent pertubation difference spectrum of
LHC II(P)/LHC II in the region 250-300 nm. The proteins are
resuspended in 20 mM Tris buffer (pH 8.0), and ethanol is
added to give a final concentration of 10% (v/v). Further details are
given under "Experimental Procedures."
[View Larger Version of this Image (17K GIF file)]
Peak
assignment
CD-calculated relative amount
FTIR relative peak
area
FTIR peak position
%
%
cm
1
Aggregated strand
56
/15
52 /54
1620-1630
-Helix
0 /12
2 /23
1655
-Sheet
20
/25
38 /19
1670-1680
-Turn
21 /19
7
/3
1698
Most of the NMR spectra were taken of an 8 mM self-buffering solution, under conditions in which the CD spectra of both the phosphorylated and non-phosphorylated peptides were identical. Upon stepwise dilution down to 0.1 mM, neither the position of the resonances nor the line shape changed, which indicates that the structure remains unchanged and the eventual aggregates remain intact.
The NMR spectra of the non-phosphorylated peptide are largely
independent of pH, whereas the spectra of the phosphorylated peptide
change significantly in the pH range 4.0-7.5 (Fig. 2). We analyzed the spectra corresponding to residues 2-14 taken at pH
values 4-5.6 and residues 3-14 at pH 6.2. At higher pH, the exchange
rate of the backbone NH protons was too fast to enable analysis of the
spectrum other than to assign the spin systems on the basis of their
analogy with the resonances in the spectrum taken at lower pH.
In all NMR spectra, the number of spin systems exceeds the expected
number deduced from the primary structure. In the non-phosphorylated peptide (Fig. 3A), doubling occurred of the
spin system of Gly-13, whose NH protons appear at 8.45 and 7.9 ppm. In
the corresponding ROESY spectrum, a strong sequential cross-peak for
Gly-13(8.45)-H-Ser-14-NH is present, whereas only a Gly-13(7.9)-NH
Ser-14-NH cross-peak can be observed, and not the corresponding
H-NH
cross-peak. The Lys-7(8)
H-Ser-14-NH cross-peak can also be
identified for the non-phosphorylated peptide.
For the phosphorylated peptide we confine our discussion to the spectra measured at pH 6.2, the highest pH at which a spectrum could reasonably be interpreted (Fig. 3B) and the pH closest to the physiological value (pH 8). In addition, the spectra at pH values in the range 4.2-5.6 are complicated by the existence of a number of minor spin systems that probably arise from minor structures in slow exchange. Many spectral features of the non-phosphorylated peptide are seen also for the phosphorylated peptide. A new position appeared for the Gly-13 spin system resonances which is indicated by the cross-peak with coordinates 8.12 and 3.45. We could observe only a weak Gly(8.12)-NH-Ser-14-NH sequential cross-peak in the non-phosphorylated peptide. Distinct differences were found, not surprisingly, for the residues Thr-5 and Thr-6.
The NMR spectra of the phosphorylated peptide contained many unique
cross-peaks. These are difficult to assign unambiguously, since the
peptide obviously adopted more than one conformation (Fig.
4).
Fig. 5 shows the FTIR spectral region of the C=O stretch
vibration of the peptide backbone, normally denoted as the amide I band
(1580-1750 cm1) or amide I
when studies are performed
in deuterated solvent. The dihedral angles of the peptide backbone
determine the geometry of the backbone. Different backbone geometries
thus imply different lengths and strengths of the hydrogen bonds
involving C=O groups. The different characteristic amide I frequencies
arise from the variation in length and direction of these hydrogen
bonds correlated with the different structures. Different peak
positions have been assigned through both empirical and theoretical
work to different structural motifs of the peptide backbone (25). Bands
located around 1650-1658 cm
1 correspond to
-structures (26) or, as found in some cases, loops (27), whereas
bands centered around 1620-1640 and 1680-1689 cm
1 are
due to
-structures (28, 29), and those at around 1690-1700 cm
1 are due to
-turns (30). Fig. 5 shows the FTIR
spectra of the non-phosphorylated (panel A) and
phosphorylated (panel B) peptide (trace a in
H2O, trace b in D2O), together with
the 2nd derivative spectrum (trace c), the Fourier
self-deconvoluted spectrum (trace d), and the individual
spectral components (traces e) from the curve fitting
procedure, respectively. As can be seen, deuterated and
H2O-subtracted spectra are in a very good agreement.
Furthermore, both the Fourier self-deconvoluted and the 2nd derivative
spectra indicate identical numbers of bands and band positions. Table I
lists the relative band areas and peak positions obtained for the
different spectral components and their correspondence to different
structural classes. The relative area of an infrared absorption band
can, as a first approximation, be assumed to be a measure of the
relative amount of that particular component. However, this assumption
does not take into account absorption by amino acid side chains, or the
slightly different extinction coefficients of different structural
motifs (31-33). The most conspicuous difference between the FTIR
spectra of the phosphorylated and the non-phosphorylated peptides is
that the phosphorylated peptide contains a definite contribution from
- structure that is absent from that of the non-phosphorylated
form (Table I). Furthermore, the large contribution of
-turn
structure in the non-phosphorylated peptide is much lower in the
phosphorylated form.
Structural Effect of Phosphorylation of LHC II by FTIR, CD, and UV Solvent Perturbation Spectroscopy
In addition to information
obtained from synthetic peptides corresponding to the LHC IIb N
terminus, we have performed studies of the native protein. Fig.
6 shows the amide I band region of the ATR-FTIR spectra
of LHC II and LHC II(P), together with the difference spectrum. These
absorbance bands are the sum of absorbances from each amino acid in the
protein, and the first 15 amino acid residues of the N terminus
contribute only as a part of the full structure of 234 amino acids. The
main absorbance band is located at 1653 cm1, indicating
mainly an
-helical structure, which is in agreement with the model
based on electron diffraction (8). A second band is also shown in the
figure at around 1550 cm
1 and is assigned to the
delocalized amide II vibration. This band has a more complex origin and
is therefore not interpreted here. Even though the individual spectra
seem to be identical, the difference spectrum shows significant
changes. The positive peaks in the difference spectrum reflect
structures more abundant in the non-phosphorylated protein than in
the phosphorylated protein, and vice versa. For the samples measured in
H2O, the positive band located at around 1625 cm
1 is assigned to
-strand structures, whereas the
negative bands at 1678 cm
1 and 1652 cm
1 are
assigned to
-turns and
-helices, respectively.
H2O/D2O exchange experiments (Fig.
6B) confirm that the 1652-cm
1 band originates
from an
-like structure and not from a random or unordered structure
(25), since deuteration has no effect on this band even after 2 h,
indicating no direct contact with the surrounding solvent. Furthermore,
the deuteration effect on the amide II band (delocalized C---N---H
bending mode) at 1550 cm
1 confirms the assumption that
part of LHC II is exposed to the surrounding medium and is not embedded
in the membrane. After exposure to D2O for only 5 min,
approximately 25% of the total area of that band is shifted to 1465 cm
1, corresponding to 25% of the protein accessible for
rapid H/D exchange. The hydrophobic segments inside the membrane and
coiled structures outside the membrane are prevented from such
exchange, and only a minor increase of exchanged protons can be found
even after 2 h exposure. Interestingly, the negative 1652 cm
1 band in the D2O difference spectrum is
the only one of the three major bands found in H2O
difference spectrum that remains at the same positions in both
solvents. The absolute area of the difference bands in this region is
approximately 4% of the total area in the absorbance spectrum.
The FTIR results demonstrate that LHC II(P) has a higher content of
- and
-turn structures and a lower content of
-stranded structures than the non-phosphorylated form of LHC II. The protein segments that cause the 1625- and 1678-cm
1 bands are
located outside the membrane domain of non-phosphorylated LHC II. The
protein segment that causes the 1652-cm
1 band originates
from an
-structure present only in LHC II(P). The total number of
amino acid residues participating in these changes is in the order of
5-10 amino residues.
CD in the wavelength region 350-750 nm has been suggested as an assay for the oligomeric state of native protein (20): the trimeric form of LHC II has a characteristic negative shoulder in the CD spectrum at 648 nm, and further but less significant differences were found at 412 and 478 nm between the monomeric and trimeric forms of LHC II. LHC II(P) CD spectra measured here have a less pronounced shoulder at 648 nm than the LHC II spectra (Fig. 1B) together with spectral features typical for the monomeric form of LHC II at the other two wavelengths. Studies of the minor light-harvesting chlorophyll-a/b-binding protein CP 29 (Lhc b4) have shown that increased chlorophyll b content will enhance a negative signal at 648 nm in the CD spectrum (34). Our findings thus indicate that dissociation of the trimer and phosphorylation may perturb the chlorophyll b2 or b3 (8) in the same way. This suggests that phosphorylation induces dissociation of LHC II trimers. The samples here denoted LHC II(P) contain around 90% of non-phosphorylated LHC II (see "Experimental Procedures") and would therefore be expected to show only 10% of the decrease of the 648 nm signal of LHC II, as observed (Fig. 1B).
Aromatic amino acid residues (Trp, Phe, and Tyr) absorb light in the UV region. Their absorption spectra may be perturbed by change in the polarity of the environment, e.g. by adding glycerol or ethanol. Membrane-embedded, or otherwise buried, residues will, however, not be affected to the same degree by changes in the solvent. Such solvent perturbation was used here to study the differences in number of buried aromatic residues between the LHC II and LHC II(P). After an addition of 10% (v/v) ethanol, to samples of LHC II and LHC II(P) at equal concentrations, a positive peak is found in the difference spectrum LHC II(P)/LHC II at around 280 nm (Fig. 1C). Both Tyr and Trp groups have stronger absorbance at 280 nm when dissolved in ethanol than when dissolved in H2O. This implies that the phosphorylated samples of LHC II have more aromatic amino acid residues exposed to the surrounding medium than the non-phosphorylated samples.
Other studies of subunits of phosphoproteins or phosphopeptides (35-43) have shown local structural alteration upon phosphorylation in some cases but not in others. Of these examples, the chlorophyll protein 29 subunit of PS II is most closely related to LHC II. There is independent evidence for a conformational change upon phosphorylation of chlorophyll protein 29 (43). Previous structural studies of LHC II (8, 44) have produced no direct structural information about the phosphorylation site. Indirectly, it has been found that proteolytic removal of the first 8 amino acid (44) residues does not affect the trimerization of LHC II but removal of the first 49 does. It has therefore been proposed (44) that the segment of 8 amino acids at the N terminus is disordered and has no structural role of the formation of trimers. However, the study was carried out only on LHC II and not on LHC II(P). LHC II(P) has not so far been found in the trimeric state, which is the only state that has been crystallized. The formation of two-dimensional and three-dimensional crystals has been shown (44) to depend on specific lipid-protein interactions. Specifically, the region around residue 16 (Pro-Trp-Tyr-Gly-Pro) has been shown to interact with the lipids phosphatidylglycerol (14, 15), monogalactosyl diacylglycerol, and digalactosyl diacylglycerol. Crystal formation is also dependent on the relative concentration of the last two lipids. Monogalactosyl diacylglycerol is a non-lamellar phase-triggering lipid (for review of this area, see Ref. 45), whereas digalactosyl diacylglycerol forms bilayers or reversed hexagonal phases depending on the acyl chain composition. Furthermore, modification of the C-terminal end of LHC II (46) has been shown to be important for stabilization of the protein, particularly of the pigment-protein complex.
Our results clearly show that peptides corresponding to the N terminus
of LHC II and LHC II(P) have non-random tertiary structure although the
backbones are mostly extended. Furthermore, we found that the peptides
can form stable dimers, and the NMR data indicate that the phosphate
group of Thr-5 forms a hydrogen bond to the NH proton of Thr-6. In
glycogen phosphorylase (47), a similar hydrogen bond between the
phosphate group on Ser-14 and backbone NH of Val-15 is observed.
Surprisingly, other protons in the N-terminal region of the peptide
were affected only marginally. We also have an indication from the
other spectroscopic techniques that the phosphate group interacts with
surrounding amino acid residues and thereby alters the structure. The
phosphate group also decreases the tendency of the peptides to
aggregate. The discrepancy between the amount of the secondary
structural motif, particularly the lower values of -structure and
higher content of random coil obtained from the CD compared with the
FTIR measurements (see Table I), is similar to that described for other
proteins (27, 28, 48) and may be attributed to the different
sensitivities of the different methods and problems with structural
classification of very small peptide segments. The CD spectrum actually
reflects the asymmetric conformation of the single L-amino
acid peptide backbone, whereas the FTIR spectrum reflects the
environmental effects on the C=O bond of the peptide backbone. We
conclude that the full-length LHC II exhibits structural differences
between its phosphorylated and non-phosphorylated forms that are
similar to those of the model peptide; upon phosphorylation of LHC II, an extended structure is replaced by a short, helix-like structure and
by a
-turn. These changes are confined to parts of the protein extrinsic to the membrane.
The band at 1652 cm1, which is characteristic of a helix,
may be assigned to the local structure around Thr-5, in agreement with
our findings that the phosphorylated peptide has such a structure around its phosphorylation site. Such a structural change is similar to
that seen in the crystal structures of rabbit glycogen phosphorylase in
its non-phosphorylated and phosphorylated forms (47). The structural
information obtained from the peptides in this investigation may not
reflect a totally accurate protein backbone conformation. In the case
of glycogen phosphorylase (47) the change in local structure induces a
global structural change involving subunit interaction and cofactor
binding. Our findings of enhanced
-turn content in LHC II(P) may
thus indicate a similar global structural change upon phosphorylation
of LHC II. Furthermore, our solvent perturbation measurements show that
LHC II has a higher number of aromatic amino acid residues associated
with the hydrophobic membrane domain than LHC II(P). Residue 16 is Trp
and residue 17 is Tyr. It is therefore plausible to assume that these
aromatic amino acid residues are the ones that are shielded by the
lipids in the non-phosphorylated LHC II, whereas upon phosphorylation of the N terminus, they are exposed to the surrounding medium. Thus, a
good candidate for the
-turn site is the same region as that at
which the phosphatidylglycerol interaction has been shown to take
place. There are two prolines located at positions 15 and 19. Prolines
are able to cis-trans-isomerize, and a total isomerization
of these two prolines would induce a complete turn of the protein
backbone. Proline groups are also known to induce hinges in different
proteins (49-51). Such relocation of the negatively charged phosphate
group can move it closer to the highly positively charged region around
the helix-membrane interface (see Fig. 7). This model
would then also explain why LHC II(P) is not found as trimers and
hence does not crystallize. The interaction between phosphatidylglycerol and the region around residue 16 was shown to be
of importance for trimerization and thereby crystal formation. In LHC
II(P) this lipid-protein interaction is broken by structural and
interactional changes.
The Functions of LHC II and LHC II(P)
As discussed above, our results imply a quaternary and tertiary structural change upon phosphorylation of LHC II. We propose a model, shown in Fig. 7, describing the events of phosphorylation and protein migration. The tight complex between LHC II and PS II is based on specific protein-protein/protein-lipid interactions, which include the phosphorylation site. The regulatory effects of phosphorylation of LHC II are decoupling of adjacent thylakoids, destacking of the membranes, and migration and docking of LHC II(P) with PS I (5). Dephosphorylation of LHC II(P) has the opposite effects. These effects have previously been proposed to depend mainly on simple electrostatic repulsion and/or attraction (52), where the addition of the negatively charged PO4 group induces a repulsive force between opposing, stacked thylakoids and, to reduce that force, LHC II(P) migrates laterally into unappressed regions of membrane. However, earlier it has been shown that photosystem segregation and membrane stacking are separate events (53). Furthermore, it has been shown that importing LHC II into stacked and unstacked thylakoid membranes causes the LHC II in the unstacked regions to migrate toward the PS II-rich stacked regions and not in the opposite direction toward regions with high density of PS I complexes (54). This result is in agreement with LHC II and LHC II(P) having different affinities for the two photosystems. The findings of dimer formation by the non-phosphorylated peptide and dissociation of the dimer by phosphorylation may help to explain the role of phosphorylation in destacking of the thylakoid membranes.
Our results are more in favor of an explanation of control of LHC II function based on structural changes (5, 55, 56), reducing the distance of electrostatic effects to short range, intramolecular interactions. The tertiary structure of LHC II may have a higher affinity for PS II than that of LHC II(P) and a lower affinity for PS I, and migration of LHC II between PS II and PS I will then be simply a result of normal lateral diffusion (57). Structures have now been obtained for some of the primary components of photosynthetic light harvesting (8, 58-60), reaction centers (10, 61-63), and secondary electron transfer and CO2 assimilation (64, 65). Further work can now logically be directed at an atomic resolution description of the structural changes involved in regulation of light harvesting in photosynthesis, where the complexes involved are intrinsic to photosynthetic membranes.
ConclusionsThe results presented here imply that regulation of light-harvesting by means of phosphorylation of chloroplast LHC II can be understood in terms of effects of the phosphate group on protein structure and on molecular recognition. This conclusion removes a conceptual barrier between regulation of ligand binding in soluble proteins and regulation of the function of membrane proteins, where, in photosynthesis at least, emphasis has been placed on the effect of protein phosphorylation on net membrane surface charge. Our results indicate that understanding regulation of photosynthesis will likewise depend on a full three-dimensional structural description of effects of post-translational, covalent modification.
We thank Drs. H. Franzén and I. Blaha for the synthetic peptides used, Dr. L. Cheng for the help with preparation of LHC II and LHC II(P), and Dr. P.-O Arvidsson for drawing Fig. 7.