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Tiêu đề Dimers of light-harvesting complex 2 from Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy
Tác giả Lu-Ning Liu, Thijs J. Aartsma, Raoul N. Frese
Trường học Leiden University
Chuyên ngành Biophysics
Thể loại Research article
Năm xuất bản 2008
Thành phố Leiden
Định dạng
Số trang 10
Dung lượng 893,02 KB

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Nội dung

To further investigate in detail the packing effects of like-protein photosyn-thetic complexes, we report an atomic force microscopy investigation on artificially created 2D crystals of t

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Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy

Lu-Ning Liu1,2, Thijs J Aartsma1and Raoul N Frese1

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;

light-harvesting complex 2; polymorphism;

Rhodobacter sphaeroides

Correspondence

R N Frese, Huygens Laboratory,

Biophysics Department, Leiden University,

2333CA Leiden, The Netherlands

Fax: +31 (0)71 527 5936

Tel: +31 (0)71 527 5970

E-mail: frese@physics.leidenuniv.nl

(Received 26 February 2008, revised 28

March 2008, accepted 16 April 2008)

doi:10.1111/j.1742-4658.2008.06469.x

Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the pho-tosynthetic protein complexes to be arranged in a densely packed energy-transducing network Protein packing may play a determining role in the formation of functional photosynthetic domains and membrane curvature

To further investigate in detail the packing effects of like-protein photosyn-thetic complexes, we report an atomic force microscopy investigation on artificially created 2D crystals of the peripheral photosynthetic light-har-vesting complexes 2 (LH2’s) from the bacterium Rhodobacter sphaeroides Instead of the usually observed one or two different crystallization lattices for one specific preparation protocol, we find seven different packing lat-tices The most abundant crystal types all show a tilting of LH2 Most sur-prisingly, although LH2 is a monomeric protein complex in vivo, we find

an LH2 dimer packing motif We further characterize two different dimer configurations: in type 1, the LH2’s are tilted inwards, and in type 2, they are titlted outwards Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2’s that constitute a lattice of zig-zagging LH2’s In addition, analyses of the tilt of the LH2’s 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 sphaeroidesphotosynthetic 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.

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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

sheets, Rhodos molischianum membranes are stacked

thylakoids, and Rhodob sphaeroides membranes

con-tain bud-like chromatophores [12]

The photosynthetic bacterium Rhodob sphaeroides is

one of the few purple bacterial species that is amenable

to genetic manipulation, which facilitates a study of the

interdependence of membrane organization, membrane

shape, protein structure and protein composition

Dif-ferent types of LH antenna complexes and RCs from

Rhodob sphaeroideshave been structurally analyzed by

X-ray crystallography, cryoelectron microscopy, and

atomic force microscopy (AFM) [3,13–16] Recent

advances in AFM imaging and polarized spectroscopy,

utilizing these structural models, has revealed the

molecular architecture of native Rhodob sphaeroides

membranes [17,18] In all cases, images revealed close

proximity of the photosynthetic components, which thus comprise a densely packed energy-transferring net-work Polarized light-spectroscopic measurements on intact membranes revealed remarkable homology in supramolecular organization in Rhodob sphaeroides membranes [19] Monte Carlo simulations, assessing the effect of the differences in size and shape of the protein complexes, showed the importance of protein-packing effects on the formation of like-protein domains, membrane curvature, and the creation of dif-fusive pathways within crowded membranes [18] Packing effects of like proteins can be most clearly observed in artificially created 2D crystals [20] Such crystals are formed from detergent-solubilized proteins 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.

Zig-zag 2 (90) Disordered

Zig-zag 2 (90) Disordered

Square 2 (60) Zig-zag 1 (120) Zig-zag 2 (90) Dimer type 1 Dimer type 2 Disordered

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the use of bio-beads and DOPC, the more common

membrane vesicles are obtained, which have been

reported to contain only one type of packing motif

[14] Chami et al [25] found that

n-octyl-b-d-thioglucopyranoside (OTG) can greatly increase the

crystal size Detergent removal using bio-beads can be

achieved at a much higher rate than with dialysis [26]

On the basis of AFM imaging of both the periplasmic

and cytoplasmic sides of LH2’s after reconstitution,

ellipticity and tilt of the LH2’s have been reported

[14,21,23] A comparison between the different packing

configurations found in 2D crystals of LH2’s from

Rhodop acidophila showed for one specific LPR a

square-packing lattice consisting of LH2’s that were

tilted relative to the lipid-membrane plane, and for

another LPR a lattice of zig-zagging nontilted LH2’s

[23] In that study, it was concluded that the observed

tilt of LH2’s within the square lattice was not due to

an intrinsic property of LH2’s of Rhodop acidophila,

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

packing configuration Furthermore, we find that

LH-2, in all cases, show a tilted conformation One of

the two types of LH2 dimer configurations is shown to

possess a tilt that could bend the lipid bilayer, leading

to the bud-like membrane curvature as observed for Rhodob sphaeroides in vivo Finally, on the basis of these images, schematic models of possible LH2 arrangements and protein–protein contacts in the reconstituted 2D crystal membranes are proposed

Results

Two-dimensional crystals of Rhodob sphaeroides LH-2s were prepared according to the protocol of Rigaud et al [26], involving the addition of OTG for LH2 solubilization and detergent removal by bio-beads This method has been applied successfully for the 2D crystallization of LH2’s from a range of photosynthetic bacterial species, as shown in Table 1

In contrast to other studies, here we utilized egg PC (Sigma, St Louis, MO, USA) as lipid in combination with Rhodob sphaeroides LH2’s A range of prepara-tions were examined with different LPRs (0.35, 0.4, 0.45, 0.5, 0.55, 0.6) All preparations were shown to form large vesicles as determined by electron microscopy (data not shown) For the AFM imaging, all samples were prepared with LPR = 0.5 Our imaging method has been described before [13,17] In essence, ultrasoft tapping-mode AFM was applied in combination with the choice of appropriate buffer for electrostatic balancing of the AFM tip and the substrate Electrostatically balanced tapping-mode 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

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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

arrangement, the LH-2s were clearly resolved in all these

types of periodicities, with outer and inner diameters of

6.5 ± 0.5 nm and 3.2 ± 0.3 nm (n = 50), respectively,

in agreement with previous descriptions [7,13,14,21]

High-resolution AFM topographs showing the

‘zig-zag’ lattices in close detail are shown in Fig 2

Differ-ent angles between adjacDiffer-ent strongly protruding LH2

rings, approximately 120 (Fig 2A) and 90 (Fig 2B),

indicated two types of zig-zag lattice The inset of

Fig 2A shows a high-magnification image of LH2

rings embedded in the 2D crystals Within this zig-zag

lattice, we were able to visualize the weakly protruding

LH2’s These lower LH2’s had an average height of

 4.5 A˚ (n = 20) above the lipid bilayer, whereas the

strongly protruding LH2’s had an average height of

 9.9 A˚ (n = 30) On the basis of the spacing between

LH2 rows measured in this work and previous

estima-tions [27], the arrangements of up-LH2’s and

down-LH2’s in the zig-zag lattices are schematically

illustrated as green and turquoise circles

Twenty per cent of crystal lattices in the preparation

were found to occupy the square arrangement of

LH-2s, as shown in Fig 3 We recorded two different

square lattices with a variation of angles between the

progressing lines of protruding LH2’s: approximately

90 (Fig 3A) and 60 (Fig 3B) The former pattern

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 exemplified in the inset of Fig 4B, in which the up and down configuration of LH2’s can be viewed Here, the less protruding LH2’s are visualized, showing their location to be precisely within the gap between two adjacent dimers

Discussion

Dimeric LH2 Surprisingly, we observed dimeric configurations of LH-2 Native membranes from four different LH2-containing photosynthetic bacteria, including Rhodob sphaeroides, have been imaged by AFM before [17,28– 32] In all cases, no sign of LH2 dimers has been reported, although LH1–RC complexes are mainly arranged in rows of dimers in Rhodob sphaeroides An

Fig 1 Overview of typical crystals imaged with AFM (raw images) Areas of different crystal lattices are indicated by dashed lines (A) Two types of zig-zag (areas 1 and 2), disordered (area 3) and dimer (area 4) lattices (B) Zig-zag lattice (area 2), square lattice (area 5) and dimers (area 6) (C) Zig-zag lattice (area 1) and dimers (area 6) Scale bars: (A) 100 nm; (B) 50 nm; (C) 200 nm.

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important clue about the origin of the dimers can be

found upon close inspection of the crystal lattices LH2

dimers are always surrounded by zig-zag lattices

(Fig 4) With respect to these surrounding zig-zag

lattices, we found two different dimer morphologies

(Fig 5A,B) When we represented a zig-zag line as

progressing Vs (or VVVV), where the corners of the

V are occupied by LH2’s, we found dimers progressing

along both sides of the V For instance, in Fig 4B, the

V-shaped zig-zag lines appear to correlate with dimers

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

arrows) (B) Zig-zag lattice (type 2) with 90 angle: zoomed-in image

of the area indicated by the dashed line in Fig 1B (area 2) Strongly

and weakly protruding LH2’s are schematically illustrated as green

and turquoise circles, respectively Scale bars: (A) 10 nm;

(B) 20 nm.

A

B

Fig 3 Raw AFM images of the two types of square-packing lat-tices observed Strongly and weakly protruding LH2’s are schemati-cally illustrated as green and turquoise circles, respectively (A) Square lattice (type 1) with 90 angle Note that the schematic arrangement of LH2’s is according to earlier descriptions [14,27] (B) Square lattice (type 2) with 60 angle (see text) Arrows indicate weakly protruding complexes The rhombic domain formed by up-LH2’s and the potential hexagonal motif of down-LH2’s are shown Scale bars: (A) 30 nm; (B) 20 nm.

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the two dimer types In the absence of up-down

pack-ing, such as in native membranes, the observed tilt

could bend the lipid bilayer Remarkably, the direction

and degree of tilt of the type 2 dimer were consistent

with the parameters that were used in modeling the

packing-induced membrane curvature of native

Rhod-ob sphaeroidesmembrane buds [18]

To conclude, we found that the dimers within the

two dimer lattices tilt similarly to the zig-zag LH2’s

Furthermore, the alignment of the dimers along their

long axis coincides with their zig or zag LH2

counter-parts within the surrounding zig-zag lattices These

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 arrangemdiffer-ents 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 lattices and two dimer lattices, including the commonly found disordered ‘lattice’; no less than seven different packing configurations for LH2 (see Table 1) In any AFM study, a certain amount of selectivity cannot be avoided, as only those areas that reveal a protruding mass can be discussed In this respect, possible rear-rangements due to the adhesion to the mica surface represent an intrinsic variability that is largely beyond the control of an experimenter Nevertheless, unlike other studies on Rhodob sphaeroides LH2, this study combined egg PC as lipid and bio-beads as detergent scavenger Egg PC is a mixture of very similar lipids containing all different fatty acid side-chains In contrast, the commonly used DOPC contains only one type of lipid Here we speculate that the differences between the lipids in egg PC, although small, may induce different LH2–LH2 interactions This effect may be further enhanced by the use of bio-beads, instead of the dialysis method, which does not uniformly remove detergent throughout a preparation

On the other hand, 2D crystals of LH2’s from Rubrivivax gelatinosus have been prepared using bio-beads and egg PC as well, and there only one type

of crystal lattice was found [21] The differences and variations in packing lattices might therefore possibly originate from the structural differences between the LH2’s from different species Similarly, it has been documented that the LPR is a critical factor in packing LH2 from Rhodop acidophila in either a square or a zig-zag lattice [23] Our observation that 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

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.

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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

preferentially showed their periplasmic face up, similar

heights of LH2 protrusions above the membrane were

measured [13,14] We thus conclude that the strongly

protruding LH2’s expose their periplasmic face Here,

we occasionally visualized configurations of lower-lying

LH2’s We found that these protruded 4.5 A˚ above the

membrane, in good agreement with the earlier reports

of the protrusions of the down-LH2’s from Rhodob

sphaeroides, which represents the cytoplasmic face

[13,14]

In contrast to the previously reported arrangements

(Fig 6A,D), we visualized a new square lattice

(Fig 6B), a new zig-zag lattice (Fig 6C), and a novel organization, a dimer lattice (Fig 6E) Actually, we observed zig-zag lines in  80% of the lattices, indicat-ing that there is a strong preference for LH2’s to constitute the zig-zag lines The 90 lines of zig-zagging up-LH2’s and down-LH2’s (Fig 6D) have been characterized before for both Rhodob sphaeroides and Rhodop acidophila [13,14,23] The 120 zig-zag lattice could represent a more dense packing motif accom-plished by a translation of alternating rows of LH2’s (Fig 6C) On the other hand, this lattice could also represent a hexagonal lattice of all up-LH2’s, with alternating lines of zig-zagging LH2’s significantly lowered due to adhesion to the mica surface Such a lattice has been reported before to exist in LH2-only Rhodob sphaeroides membranes, which contains all

A

B

E

6.3

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.

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up-LH2’s [27] In that case, the significant lowering

of alternating rows of LH2 implies the existence of

specific interactions between the LH2’s constituting a

zig-zag row This study provides further evidence for

this, as we found similar interactions between the

LH2’s within the zig-zag lattices and those within

the LH2 dimer configuration In addition, we observed

non-zig-zagging LH2’s within two different

square-packing lattices (Fig 6A,B) As seen in Fig 6F, as

compared to the zig-zag, 90 square-packing lattices

represent a less packed configuration, indicating that

dense packing may induce the specific interactions

leading to zig-zagging LH2’s The intermediate density

of the dimer lattice, on the other hand, indicates that

the dimer organization might be a transient

configura-tion between 90 square lattice and others

Tilting of LH2

Our images also allow us to characterize in detail the

heights and tilts of the LH2’s for all different crystal

lattices In contrast to the many differences regarding

packing lattices and configurations discussed in the

previous section, we found all LH2’s to be tilted on the

periplasmic side similarly in all lattices Similar tilts have

been observed for Rhodob sphaeroides LH2 before,

packed within a square lattice [14] In contrast,

Gonc¸al-ves et al [23] reported that no tilt of LH2 from

Rhodop acidophila was observed in type 2 crystals

(zig-zag), but a 4 tilt was observed in type 1 crystals

(square) The similar tilts of Rhodob sphaeroides LH2 in all lattices indicate that the packing density and the 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

6.0

12.9 7.0

7.0

7.1 5.3

10.9

Strongly protruding LH2 (up) Weakly protruding LH2 (down)

12.0 15.0

22 000

F

A

B

C

D

E

20 000

21 000

18 367

A B C D E

21 208 21 224

20 408

19 047

19 000

18 000

–2 )

Fig 6 Schematic models for the different lattice types of LH2 observed by AFM with 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.

Trang 9

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

N,N-dimethyldode-cylamine-N-oxide (LDAO) (Fluka, Buchs, Switzerland) for

40 min Insoluble material was removed by centrifugation

incubated in 10 mm Tris buffer and 0.5% Triton, and

centrifuged for 5 min at 10 000 g The pellet was

resus-pended in 10 mm Tris buffer (pH 7.5) and incubated with

1% LDAO for 20 min at room temperature LH2’s were

purified using a discontinuous sucrose density gradient

of 1.2 m and 0.6 m sucrose in 10 mm Tris buffer, and

centrifuged for 2 h at 300 000 g

Two-dimensional crystallization

removal was performed through three additions of 5 mg

of SM2 Bio-Beads (Bio-Rad, Hercules, CA, USA) [35,36]

After 2.5 h of stirring at room temperature, the

AFM

The AFM sample of LH2 crystals was prepared by adsorbing

2 lL of sample solution onto the surface of freshly cleaved

pH 7.5, 150 mm KCl) in order to remove weakly bound

crys-tal patches Imaging was performed with a commercial AFM

instrument (NanoscopeIII; Digital Instruments, Santa

Bar-bara, CA, USA) and standard silicon nitride cantilevers with

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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