Dimers of light-harvesting complex 2 from
Rhodobacter sphaeroides characterized in reconstituted
2D crystals with atomic force microscopy
Lu-Ning Liu
1,2
, Thijs J. Aartsma
1
and Raoul N. Frese
1
1 Huygens Laboratory, Department of Biophysics, Leiden University, The Netherlands
2 State Key Lab of Microbial Technology, Shandong University, Jinan, China
Photosynthetic bacteria use a large part of their internal
volume for functionalized invaginations of the intracyto-
plasmic membrane containing the photosynthetic
machinery. The most abundant protein complexes in
the intracytoplasmic membrane are light-harvesting
(LH) complexes, responsible for the absorption of sun-
light and subsequent excited state energy transfer, and
reaction centers (RCs), to which the energy is directed
to initiate the primary charge transfer reactions [1,2].
There are various photosynthetic purple bacterial spe-
cies that display high similarity between the molecular
structures of the individual photosynthetic protein
complexes [3–9]. Nevertheless, differences exist: purple
bacteria assemble a variety of photosynthetic unit
architectures, the simplest being an array of RC–LH
complex 1 (LH1) core complexes, as seen in Blasto-
chloris viridis and Rhodospirillum rubrum [10], whereas
other species, e.g. Rhodobacter sphaeroides, synthesize
Keywords
2D crystallization; atomic force microscopy;

within the zig-zag lattice and that observed within the dimers corroborate
their similar packing motifs. The type 2 dimer configuration exhibits a tilt
that, in the absence of up-down packing, could bend the lipid bilayer,
leading to the strong curvature of the LH2 domains as observed in
Rhodobacter sphaeroides photosynthetic membranes in vivo.
Abbreviations
AFM, atomic force microscopy; DDM, dodecyl-b-
D-maltoside; DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine;
DOTM, dodecyl-b-
D-thiomaltoside; DPPC, dipalmitoyl-phosphatidylcholine; LDAO, N,N-dimethyldodecylamine-N-oxide; LH, light-harvesting;
LH1, light-harvesting complex 1; LH2, light-harvesting complex 2; LPR, lipid ⁄ protein ratio; OG, octyl-b-glucopyranoside; OTG, n-octyl-b-
D-
thioglucopyranoside; PC, phosphatidylcholine; RC, reaction center.
FEBS Journal 275 (2008) 3157–3166 ª 2008 The Authors Journal compilation ª 2008 FEBS 3157
peripheral LH complexes, LH complexes 2 (LH2’s), or
configure RC–LH1 complexes into dimeric supercom-
plexes [11]. Moreover, the structures of specific photo-
synthetic protein complexes may be variable. This is
exemplified by the 3D crystallographic structures of
LH2’s from the species Rhodopseudomonas acidophila
and Rhodospirillum molischianum [6,7]. Both species
synthesize LH2’s from repeating a-helical protein units
that are cylindrically arranged in a ring-like structure,
but Rhodop. acidophila complexes display nine-fold
symmetry and Rhodos. molischianum complexes display
eight-fold symmetry. Also, the exact arrangement of
the light-interacting chromophores within the protein
scaffold differs between these species. Finally, the
shape of the photosynthetic membranes is highly spe-
cies-dependent: B. viridis membranes are large, flat

mixed with lipids by gradually removing the detergent.
There are essentially two main methods for removing
the detergent for 2D crystallization: flow dialysis and
bio-beads. Other variables involve the types of lipids
or mixtures of lipids used and the lipid ⁄ protein ratio
(LPR). Two-dimensional crystals of the photosynthetic
bacterial LH2 have been extensively studied by means
of AFM [13,14,21–24]. Table 1 summarizes the method
used and the crystal lattices from different species
observed with AFM. It was found that the morphol-
ogy of the 2D crystals and the LH2 arrangement are
highly dependent on the crystallization conditions,
including the lipid, detergent scavenger, protein con-
centration, LPR, and species used. Creating 2D crys-
tals of LH2 by flow dialysis in the presence of dioleoyl
phosphatidylcholine (DOPC) as lipid has been shown
to produce highly structured tubular crystals, although
these may contain different packing lattices [13]. With
Table 1. Summary of AFM data from 2D crystals of LH2. DMPC, dimyristoyl phosphatidylcholine; OG, octyl-b-glucopyranoside; DPPC, dipal-
mitoyl-phosphatidylcholine; DDM, dodecyl-b-
D-maltoside; DOTM, dodecyl-b-D-thiomaltoside.
Species Lipid(s) Detergent Method Lattice type
Rubrivivax gelatinosus [21] Egg PC OTG Bio-beads Square
Rhodobacter sphaeroides [14] DOPC OTG Bio-beads Square 1 (90°)
Rhodopseudomonas acidophila [22] Egg PC LDAO Dialysis Disordered
Rhodobacter sphaeroides [13] DOPC OG Dialysis Square 1 (90°)
Zig-zag 2 (90°)
Disordered
Rhodopseudomonas acidophila [23] DMPC OTG Bio-beads Square 1 (90°)
Zig-zag 2 (90°)

but was caused by specific interactions induced by
packing. In contrast, tilted LH2’s and RC–LH1’s have
been observed in native membranes of Rhodob.
sphaeroides [17]. Moreover, in the aforementioned
model of the photosynthetic membrane of this particu-
lar species, we showed the importance of protein pack-
ing for the full appearance of the membrane [18].
More specifically, we showed that the formation of
LH2 domains has a strong influence on the curvature
of the membrane. In the absence of a 3D crystallo-
graphic structure, we assigned an intrinsic curvature to
the LH2 of Rhodob. sphaeroides that could originate
from a slight conical shape or specific binding of a
curved lipid. In any case, a tilted configuration in
packed conditions could ultimately lead to membrane
curvature.
Here we further investigate in detail the packing
effects in artificially created 2D crystals of the LH2
from Rhodob. sphaeroides . Our results show that with
egg phosphatidylcholine (PC) as lipid and bio-beads as
detergent scavenger, a multitude of different packing
configurations can be obtained within one preparation.
AFM images allow the differences in protein packing
and interaction within the membrane to be visualized.
Within the packed lattices, we find a new conforma-
tion of LH2’s, namely a dimeric configuration.
Detailed investigations of the observed LH2 packing
patterns reveal that the different packing lattices con-
sist of similarly interacting LH2’s. The dimeric LH2
configuration is very likely to exist as an intermediate

AFM was shown to produce the highest-resolution
images of naturally curved membranes [17], and
was applied here to obtain detailed information on
possible tilts of the protein complexes above the
membranes.
Figure 1 shows typical 2D crystals of densely packed
LH2’s as observed with AFM. The protruding mass
appears most bright; the mica surface is dark. Even at
this low magnification, the LH2’s were visible as rings,
about 7 nm wide. Crystals had a diameter of up to 2 lm
and a height of 6.5 ± 1.0 nm (n = 20). Areas of empty
lipid bilayer without incorporated LH2’s had an average
height of 4.0 ± 0.7 nm (n = 20). Already at the low
magnification of Fig. 1, the regular arrangement of
LH2’s and the varieties thereof could be observed. It is
noteworthy that several different LH2 arrangements
could coexist in the same membrane fragment. We
found no less than seven different packing arrangements
in terms of the different lateral arrangements and tilts
of LH2’s. There were two rectangular arrays, two
zig-zag lattices, two types of dimeric organization, and
a disordered arrangement. The majority of crystalline
lattices were occupied by so-called ‘zig-zag’ rows of
LH2’s ( 80%). The other 20% contained crystallized
arrangements of LH2’s in a square pattern and with a
L N. Liu et al. AFM study on LH2 2D crystal
FEBS Journal 275 (2008) 3157–3166 ª 2008 The Authors Journal compilation ª 2008 FEBS 3159
disordered distribution. Most surprisingly, a dimeric
LH2 configuration could be observed, as shown in
Fig. 1 (areas 4 and 6). Regardless of the protein-packing

has been well described earlier in Rhodob. sphaeroides
[14], whereas the latter array is observed for the first
time. The 60° square lattice consists of four strongly
protruding LH2’s arranged in rhombic periodicity. The
spacing within this domain could fit for two weakly
protruding LH2’s, which could be occasionally recog-
nized (Fig. 3B, arrows). These down-LH2’s could form
a hexagonal motif surrounding a central up-LH-2.
These available results enabled the arrangements of
up-LH2’s and down-LH2’s in these lattices to be sche-
matically depicted in Fig. 3.
We also found a novel arrangement of LH2 crystal
packing, termed a dimer lattice. Figure 4 shows high-
magnification images of the areas containing dimers;
the larger lattice of origin is indicated in Fig. 1
(area 4). LH2’s were found to be aligned, forming
rows along the long axis of the dimers within a period-
ical lattice. Figure 4A shows an area where two LH2’s
contact closely, separated from neighboring LH2–LH2
dimers. Adjacent rows of dimers are separated such
that the dimeric LH2 in the neighboring row faces the
empty central space. The distance between adjacent
rows of dimers was found to be 5.3 ± 0.4 nm
(n = 20), less than the size of the LH2. Figure 4B,C
presents two different dimer lattices (type 1 and type 2)
with opposing lines of progress. The directions of the
progressing lines were related to that of the surround-
ing zig-zag lattices. This will be discussed below. The
inset of Fig. 4A shows a scheme of the packing config-
uration of dimers. Such a specific arrangement is

progressing along the \-side of the V (or zig-side),
whereas in Fig. 4C, the dimers progress along the
⁄ -side of the V (or zag-side). It thus seems that the
LH-2s associated with either the zig-side or the zag-side
of zig-zag lines actually originate from LH2 dimers.
More evidence for the dimeric origin of zig-zag
lattices can be obtained from high-resolution AFM
images of dimers and zig-zag LH2’s, as shown in
Fig. 5. In dimeric LH2’s, there were two tilted types
found with different protruding heights, type 1 dimers
(Fig. 5A) and type 2 dimers (Fig. 5B). Type 1 dimers
have high contacting sides and low peripheral sides
(Fig. 4A), whereas type 2 dimers show an opposite tilt-
ing orientation. The tilt of LH2 dimers was further
studied by measuring the protruding profiles of each
LH2 within the dimer on the basis of the height
differences of both sides and the size of LH2’s
(Fig. 5C,D). This allowed the tilting angles to be calcu-
lated as 5.0 ± 0.5° and 3.5 ± 0.3° (n = 20), respec-
tively. These two tilting angles bear a striking
resemblance to that of the zig and zag LH2’s as shown
in Fig. 5F,G. Depending on the directions along which
LH2’s were measured, we found the same angles as for
A
B
Fig. 2. Raw AFM images of the two different zig-zag lattice types.
(A) Zig-zag lattice (type 1) with 120° angle: zoomed-in image of the
areas indicated by the dashed line in Fig. 1A (area 1). Inset: 3D
enhanced close view showing weakly protruding LH2’s (black
arrows) in between the strongly protruding zig-zag lines (white

two observations strongly suggest that the zig-zag
lattice is composed of LH2 dimers.
Why LH2 is dimerized within this particular prepa-
ration, whereas it is absent in native membranes, is
unknown. The presence of dimers in these crystals may
reflect a more dense packing condition. In mutant
Rhodob. sphaeroides membranes where RC–LH1
dimerization was inhibited, we also observed a particu-
lar RC–LH1 dimer effect [18]. There, we found the
RC–LH1 monomers to be rotationally locked within
one unique orientation in half of the cases. Rotational
locking had been observed before, but only for
RC–LH1 dimers, and not for monomers [33]. We
could relate this effect to an increased packing strain
within the mutant membranes induced by dense pack-
ing. As the protein helices constituting LH2 and LH1
are similar, the dimerization that we observed here
might just reflect another packing effect acting on
these similar LH proteins.
Packing lattices
By means of AFM, we observed a multitude of differ-
ent packing arrangements within one 2D crystal prepa-
ration. In Table 1, we summarize our findings and
those of previous published studies. Within the five
separate AFM studies on 2D crystals of LH2’s
published to date (two of which were on Rhodob.
sphaeroides LH2), only one type of square (square 1
with 90° angle) and one type of zig-zag lattice
(zig-zag 2 with 90° angle) have been reported. Here we
find two types of square lattices, two types of zig-zag

different patterns of LH2 packing coexist within the
same crystal preparation suggests that this is not the
case for the Rhodob. sphaeroides LH2.
On the basis of the observations shown in Figs 1–4,
we represent five schematic models of LH2 arrange-
ments in either up or down (and in one case unknown)
configuration, without considering the tilt of LH2 in
Fig. 6. The strongly protruding LH2’s, or up-LH2’s,
protrude about 1.0 nm, in good agreement with the
previous data obtained on Rhodob. sphaeroides
and Ru. gelatinosus 2D crystals [13,14,21]. In the latter
AB
C
Fig. 4. High-resolution AFM images of the dimer areas surrounded
by zig-zag lattices. Images are 3D-enhanced for clarity. (A) Zoomed-
in image of Fig. 1A (area 4). Inset: closer view of dimeric LH2’s.
Strongly and weakly protruding LH2’s are schematically illustrated
as green and turquoise circles, respectively. (B) Type 1 dimer con-
figurations. Inset: zoomed-in images show strongly and weakly (indi-
cated by white arrows) protruding LH2’s. ‘Dimer’ and ‘zig-zag’ LH2
lattices are indicated by white lines. (C) Type 2 dimer configurations.
‘Dimer’ and ‘zig-zag’ LH2 lattices are again indicated by white lines.
Note the opposing lines of progress as compared to type 1. See
text for details. Scale bars: (A) 30 nm; (B) 50 nm; (C) 30 nm.
AFM study on LH2 2D crystal L N. Liu et al.
3162 FEBS Journal 275 (2008) 3157–3166 ª 2008 The Authors Journal compilation ª 2008 FEBS
study, it was shown that the strongly protruding side
was the periplasmic side of the complex by means of
thermolysin digestion that only affected this face [21].
Also in Rhodob. sphaeroides native membranes, which

A
B
E
6.3
9.8
10.0
7.9
6.2
6.1
7.6
8.5
10.6
6.7
10.0
11.0
6.4
8.8
6.4
C
D
F
G
Fig. 5. Comparison between two types of dimer configuration (A, B) and the zig-zag lattice (E). (A) LH2 dimer type 1 isolated from the image
shown in Fig. 4B. (B) LH2 dimer type 2 originating from Fig. 4C. (C, D) The two distinct height profiles of LH2 dimers, indicating the tilt
(n = 20). (E) Zig-zag LH2 lattice isolated from Fig. 2B. (F, G) The height profiles of zig-zag LH2 with respect to two vertical directions
(n = 20), indicated as E1 (\) and E2 ( ⁄ ), respectively in (E) (height in A
˚
). Scale bars: (A, B) 5 nm; (E) 10 nm.
L N. Liu et al. AFM study on LH2 2D crystal
FEBS Journal 275 (2008) 3157–3166 ª 2008 The Authors Journal compilation ª 2008 FEBS 3163

induced interactions among neighboring LH2’s are the
predominant factors that drive the arrangements of
LH2. In addition, it has been observed that tilting of
LH2’s shows some dependency upon the packing den-
sities of different lattices. The least packed, disordered
organization presents the smallest tilt 1.3 ± 0.2°
(n = 20), whereas the largest tilt, 6.5 ± 0.5° (n = 20),
is observed in the zig-zag lattice, which also represents
the most densely packed configuration.
In conclusion, crystalline packing in a high number
of configurations of LH2’s in 2D crystals has been
resolved by AFM. We characterized no less than seven
different LH2 lattices in only one specific preparation.
All individual LH2’s of Rhodob. sphaeroides are tilted,
depending upon the packing densities of LH2’s in the
crystal lattices. We found a novel dimeric organization
of LH2, and showed the close resemblance to the
LH2’s that form the zig-zag lattice. Such a lattice has
also been observed in LH2-only domains of adhered
Rhodob. sphaeroides membrane patches [27], which in
native conditions are spherically shaped [34]. One type
of the dimers observed here displays a tilt capable of
curving the membrane in such a manner, leading to
the spherical domains as observed in intact LH2-con-
taining Rhodob. sphaeroides membranes [17,18,34].
Although long-range curvature is strongly reduced in
2D crystals, the similarity in configuration of this
dimer and that of the LH2 complexes forming the
11.4
10.8

measured distances in nanometers and
angles (n = 20). (A, B) Square-packing
lattices corresponding to the images from
Fig. 3A,B. (C, D) Zig-zag lattices correspond-
ing to Fig. 2A,B. (E) The dimer lattices from
Fig. 4 (note: packing configurations for both
lattices in Fig. 4 are the same). (F) Total
LH-2 densities of the different lattices.
AFM study on LH2 2D crystal L N. Liu et al.
3164 FEBS Journal 275 (2008) 3157–3166 ª 2008 The Authors Journal compilation ª 2008 FEBS
curved domains in native Rhodob. sphaeroides mem-
branes indicates the existence of specific, packing-
induced interactions between LH2 and the lipid mem-
brane of this species in vivo.
Experimental procedures
LH2 purification
After 4 days of growth, Rhodob. sphaeroides wild-type cells
were harvested and disrupted by sonication. Unbroken
cells and cell debris were removed by centrifugation
(10 000 g for 20 min). The supernatant containing
chromatophores was pelleted by centrifugation at
265 000 g for 90 min at 4 °C, and then resuspended in
10 mm Tris buffer (pH 7.5) to A
850 nm
= 200Æcm
)1
. Mem-
branes were solubilized with 1% (w ⁄ v) N,N-dimethyldode-
cylamine-N-oxide (LDAO) (Fluka, Buchs, Switzerland) for
40 min. Insoluble material was removed by centrifugation

instrument (NanoscopeIII; Digital Instruments, Santa Bar-
bara, CA, USA) and standard silicon nitride cantilevers with
a length of 85 lm, a force constant of 0.5 NÆm
)1
and operat-
ing frequencies of 25–35 kHz (in liquid) (Veeco NanoProbe
Tips, Santa Barbara, CA, USA) were used. High-resolution
AFM images were obtained using tapping mode in liquid
and with amplitude setpoint adjusted to minimal forces.
Acknowledgements
The authors thank Dre
´
de Wit for growing the Rhodob.
sphaeroides cells and purifying the LH2. This research
was sponsored by the Dutch Science Foundation
[Netherlands Organization for Scientific Research
(NWO)]. This project is part of the research
programme ‘From Molecule to Cell’, funded by the
NWO and the Foundation for Earth and Life Sciences
(ALW). R. N. Frese gratefully acknowledges the
NWO for a veni-grant. Lu-Ning Liu acknowledges
financial support from a PhD Study-Abroad Scholar-
ship of Shandong University (China).
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