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Colloids and Surfaces B: Biointerfaces 89 (2012) 283– 288

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

hort communication

novel method of AquaporinZ incorporation via binary-lipid Langmuironolayers

uofei Suna, Hu Zhoub, Yi Lia, Kandiah Jeyaseelanc, Arunmozhiarasi Armugamc, Tai-Shung Chunga,∗

Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, SingaporeDepartment of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, SingaporeDepartment of Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore

r t i c l e i n f o

rticle history:eceived 28 May 2011eceived in revised form 3 September 2011ccepted 4 September 2011vailable online 10 September 2011

a b s t r a c t

In this work, a new approach of incorporating transmembrane protein AquaporinZ (AqpZ) into a lipidbilayer has been developed with the aid of the Langmuir–Blodgett technique. The binary-lipid mono-layer for AqpZ incorporation is composed of (1) gel-phase lipids resistant to detergent dissolution and(2) nickel-chelating lipids that can attach the histidine-tagged AqpZ from the subphase. Upon removal ofsubphase detergent with BioBeads, the incorporation is achieved by transferring the AqpZ-associated

eywords:quaporinrotein incorporationetergentipid monolayerangmuir–Blodgett trough

binary-lipid monolayer onto a preformed pure binary-lipid monolayer using the Langmuir–Schaeferdeposition method. AFM images show an indication of AqpZ incorporation in the bilayer. Furthermore,it is also shown that BioBeads can remove a significant amount of detergent in the subphase and lipidfilm integrity is restored after detergent removal. The detergent removal rate is correlated to BioBeadsamount and subphase circulation. The new approach of AqpZ reconstitution revealed in this work couldpotentially be applied in biomimetic membrane formation for water purification applications.

. Introduction

AquaporinZ (AqpZ) is a transmembrane protein that promoteshe transport of water across biological membranes throughn osmotic pressure differential [1,2]. In recent years, success-ul reconstitution of AqpZ in lipid and block-copolymer vesicles

embranes has been demonstrated by significant increment ofembrane permeability in stopped-flow tests [3–5]. The character-

stics of high selectivity and high permeability to water moleculesf AqpZ contribute to its potential application in water purifica-ion technology, for example, forward osmosis. Langmuir–BlodgettLB) technology is known to be a powerful method to studyipid–protein interaction at the gas–liquid interface and form pla-ar lipid bilayers. Membrane insertion behavior of water solubleroteins is commonly investigated on the LB surface to mimiciological membranes. However, hydrophobic membrane proteins

ike AqpZ [6] which have to be solubilized in a detergent solu-ion are poorly studied with the LB technique. This is mainlyue to the fact that the detergent can easily disrupt the surface

onolayer by either adsorbing onto the monolayer or solubiliz-

ng the monolayer [7–10]. As a result, the process of membranerotein insertion is hindered by the detergent–monolayer inter-

∗ Corresponding author. Tel.: +65 6516 6645; fax: +65 6779 1936.E-mail address: chencts@nus.edu.sg (T.-S. Chung).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.09.004

© 2011 Elsevier B.V. All rights reserved.

action. Moreover, the incorporated protein has the tendency todenature and lose its activity when it reaches the air–liquidinterface [11]. All of these reasons make the incorporation of mem-brane proteins with the LB method a challenging and intriguingwork.

In this study, we attempt for the first time to reconstitute AqpZinto the lipid bilayer with the LB technique. As detergent removalis critical for the membrane protein incorporation as well as main-taining the membrane stability and integrity, the first goal of thiswork aims to prove that the detergent adsorption on the lipidmonolayer can be suppressed through the addition of BioBeads inthe subphase, and the detergent removal rate is correlated to theamount of BioBeads and circulation in the subphase. For the sec-ond part of this work, we demonstrate a new AqpZ incorporationapproach with a binary-lipid Langmuir monolayer and propose athree-step mechanism for protein incorporation. As atomic forcemicroscopy (AFM) is the unique method to characterize LB film inthe nano-scale [12–15], it was adopted for the membrane morphol-ogy study in this work.

2. Materials and methods

2.1. Materials

A 10-histidine residual tagged (His-tagged) AqpZ used inthis work was produced according to Borgnia et al.’s methods

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84 G. Sun et al. / Colloids and Surfac

4]. Nickel-chelating lipids, 1,2-di-(9Z-octadecenoyl)-sn-glycero--[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]ith Nickle (DOGSNTA) and 1,2-dipalmitoyl-sn-glycero-3-hosphocholine (DPPC) were purchased from Avanti Polar Lipids,irmingham, United States. n-Dodecyl beta-d-maltoside (DDM)ith purity >99.5% was a product of Acros Organics, Geel, Belgium.

0× Phosphate buffered saline (PBS) in ultra high purity (1st BASE,ingapore) was diluted 10 times with Milli-Q water (Millipore)efore use (pH = 7.4). The lipids solution was prepared in chloro-orm (Tedia, HPLC grade) at a concentration of 1 mg/mL. Bio-BeadsM-2 Absorbents from BIO-RAD was used to remove DDM in theubphase. Grade V1 mica was purchased from SPI Supplies.

.2. Methods

.2.1. Surface pressure measurementThe surface pressure measurement was carried out on a cus-

omized LB trough (KSV-NIMA, Finland) with a trough area of6 cm × 5 cm. Before each experiment, the trough was wiped thor-ughly with chloroform twice and subsequently rinsed with Milli-Qater. The subphase in the trough was either Milli-Q water or

BS buffer solution, and all the experiments were performed atontrolled room temperature (23 ± 1 ◦C). Surface pressure waseasured by a pressure sensor based on the Wilhelmy plateethod. The effective surface area was controlled by a pair of Del-

in barriers. Lipid solutions were spread on the aqueous subphasehen the barriers were opened to the maximum and 60 min was

llowed for chloroform evaporation from the surface. The total vol-me of the subphase is 65 ± 2 mL.

For injection of DDM or AqpZ solution into the subphase, barri-rs were held statically after the surface pressure reached 30 mN/m.n injection port located at the center of the trough was utilized

o reduce the disturbance on surface monolayer during injection.fter injection, the subphase was circulated at 3 mL/min to pro-ote equilibration for 2 h and the change in surface pressureas recorded. In the case of detergent removal from the sub-hase, BioBeads were added into the dipping well before spreadingf the monolayer and stirred gently with a mini Teflon stir bar.ubphase circulation was performed throughout the entire deter-ent removal process. The air above the subphase was maintainedt saturation level to limit evaporation of water. To obtain theressure-area isotherms of lipids monolayer, the surface was com-ressed or expanded at the rate of 10 cm2/min. All the experimentsere repeated for at least three times to ensure the reproducibility

f the results.

.2.2. Surface tension measurementA surface pressure sensor was used to determine the surface

ension of detergent/protein solutions. A platinum Whilhelmy plateonnected to the pressure sensor was partially immersed in a 20 mLBS solution. The surface pressure change (�) of PBS buffer withhanging DDM concentration in the buffer was measured by theressure sensor. The mixture was gently stirred until � was stabi-

ized. Subsequently, surface tension (ST) can be calculated based onhe equation

T = STbuffer − �

hile STbuffer is the surface tension of the pure PBS buffer solution

hich is about 72 mN/m. The surface tensions of mixtures were

alculated and plotted against DDM concentration. Three timesepetition was done for each data point and the error for all pointsre within 6%.

iointerfaces 89 (2012) 283– 288

2.2.3. LB film deposition and AFM scanningLB film deposition was performed to transfer the monolayer

from the air–liquid interface to the mica surface. The surfacepressure for deposition was always at 35 mN/m. To study the mor-phology of the DPPC layer with DDM disruption and BioBeadsintervention, the DPPC monolayer was deposited onto a freshcleaved mica surface first by the vertical Langmuir–Blodgett (LB)method and followed with the horizontal Langmuir–Schaefer (LS)method. The transfer speed for the LB method is 2 mm/min.The samples were immersed in Milli-Q water before and dur-ing AFM scanning. In the case of protein-associated monolayerdeposition, a first monolayer of pure DPPC–DOGSNTA (5:1) wasprepared by LB deposition on the mica surface using another trough(KSV-NIMA, Inverted Microscopy Trough). The protein-associatedDPPC–DOGSNTA monolayer was subsequently deposited onto thefirst layer by the LS method. The samples were incubated in PBSbuffer for 1 h, and then imaged by AFM in PBS buffer environ-ment.

The films were imaged by a PicoSPM atomic force microscope(Agilent) in the Acoustic alternating current mode. The sampleswere scanned with ULTRASHARP NSC15/AIBS cantilevers (res-onance frequency between 265 and 400 kHz and typical forceconstant of 46 N/m) in aqueous solutions (Milli-Q water or PBSbuffer) at room temperature (23 ± 1 ◦C). The scanning speed is lessthan 0.5 ln/s.

3. Results and discussion

3.1. DDM effects on DPPC monolayer

A well-characterized lipid, DPPC, was selected in this exper-iment to demonstrate that detergent penetration into the lipidmonolayer could be eliminated with the addition of BioBeads.At room temperature (23 ± 1 ◦C), DPPC at the air–water interfaceexhibits a liquid-condensed phase upon compression to above7 mN/m; therefore, detergent has the poor accessibility to such acompact monolayer [8]. To avoid a heavy loss of surface lipids bydetergent solubilization, the DDM concentration in the subphasewas controlled at 4 �M (2 mg/L), which was much lower than itscritical micelle concentration (CMC), 0.18 mM [16]. As shown in thedashed lines of Fig. 1, the isotherm cycle of the monolayer after theaddition of DDM displays a significant counterclockwise hystere-sis, while it is known that the pure DPPC monolayer does not showany hysteresis [17]. The hysteresis is caused by the DDM adsorp-tion onto the monolayer at low surface pressures and incompletedesorption from the monolayer at high surface pressures [7]. Dif-ferent amounts of BioBeads (50 mg, 150 mg and 300 mg, in wetweight) were introduced into the trough to study the influence ofBioBeads addition on the detergent removal rate. Since BioBeadsis only confined in the dipping well (1.5 cm in diameter) locatedat the center of the trough, effective detergent removal cannot beachieved without subphase circulation.

The isotherm cycle of DPPC monolayer was obtained after 6 hof detergent removal. The relative amount of remaining DDM inthe subphase can be reflected by the intensity of isotherm-cyclehysteresis as well as the lowest surface pressure in the cycle. When50 mg BioBeads was used (Fig. 1(a)), the isotherm cycle shows simi-lar intensity of hysteresis with the one that has no BioBeads, but thelowest surface pressure has been reduced to 0 mN/m from 5 mN/m,indicating that only a small fraction of the detergent in the subphasehas been removed. A significant improvement can be observed from

the isotherm cycle as the BioBeads amount increased to 150 mg(Fig. 1(b)), with milder hysteresis and better proximity to the pureDPPC isotherm. With 300 mg BioBeads (Fig. 1(c)), though the mono-layer displays a gentle hysteresis, the shape of the compression

G. Sun et al. / Colloids and Surfaces B: B

Fig. 1. Pressure-area isotherm cycles of DPPC at 23 ◦C after 6 h detergent removalwith (a) 50 mg, (b) 150 mg, and (c) 300 mg BioBeads in the trough (dotted line)and subphase volume of 65 ± 2 mL. The initial DDM concentration in the subphaseis 2 mg/L. The isotherm cycles after detergent removal are compared with pureDPPC isotherm (solid line) and isotherm cycle without using BioBeads (dashedline).

iDtb

stfgwcwatltsftr

[20], the His-tagged membrane protein with strong hydrophobic-ity could bind to the DOGSNTA monolayer within 1 h after injection

sotherm has reverted to that of a pure DPPC isotherm. Therefore,DM can be eliminated from the interface at a faster rate when

here is a higher BioBeads to DDM ratio, which is reflected by aetter recovery of DPPC isotherms.

The effect of DDM on the DPPC monolayer was also demon-trated by AFM images in Fig. 2. Without the addition of BioBeads,he DPPC bilayer loses the original compact structure (Fig. 2(a)) andorms a “fluid-like” defective structure (Fig. 2(b)), while with deter-ent removal by BioBeads, the compact bilayer structure (Fig. 2(c))as restored. Therefore, it can be concluded that the detergent

ontent at the air–liquid interface can be substantially reducedith a decrease in its subphase concentration. BioBeads reduce the

dsorbed DDM in the DPPC monolayer by removing the DDM inhe bulk either before or after the DDM association with the mono-ayer. In the former case, DDM adsorbs to BioBeads faster thano the monolayer; thus less detergent is available to disrupt theurface monolayer. In the latter case, DDM quickly reaches the sur-ace monolayer, but the elimination of DDM in the subphase leads

o DDM desorption from the monolayer. Both hypotheses may beeasonable but require further verification.

iointerfaces 89 (2012) 283– 288 285

3.2. AqpZ incorporation via DPPC–DOGSNTA monolayers

Monolayers of DPPC and DOGSNTA mixture were producedby spreading the mixed lipid solution with certain ratio on thePBS buffer surface. Fig. 3 shows the pressure-area isotherms ofpure DPPC, pure DOGSNTA and their mixtures of different molarratios (5:1 and 10:1). The pure DOGSNTA monolayer has no liquid-expanded to liquid-condensed transition plateau in the isothermshowing that DOGSNTA exist in the fluid-phase at the room temper-ature. In contrast, DPPC has a transition temperature of 40 ◦C thus isin the gel-phase at the room temperature. Therefore, their mixturesdisplay characteristics of both. To preserve the compact structureof the monolayer at high surface pressures, DPPC and DOGSNTAmixed at a ratio of 5–1 was adopted for protein insertion. As men-tioned previously, DPPC is able to form a closely packed monolayerat high surface pressures and resist the invasion of detergents. Inthe meantime, DOGSNTA provides binding sites for His-tagged pro-teins [18–20], which ensures the monolayer to have a good affinityto AqpZ. Upon addition of DOGSNTA in the DPPC monolayer, thesurface pressure at liquid expanded-liquid condensed phase tran-sition increases and the transition plateau becomes less prominentcompared with the pure DPPC monolayer. These suggest that themonolayer formed with the DPPC–DOGSNTA mixture possessesmore fluidity than that formed with the pure DPPC.

The AqpZ-DDM solution was injected at a surface pressure of30 mN/m. This surface pressure is selected for two reasons. The firstis to minimize the detergent penetration into the monolayer. Asdescribed above, DDM can be partially desorbed (i.e. squeezed out)from the tightly packed LC phase monolayer, which causes the hys-teretic feature in the isotherm cycle of DPPC/DDM. Thus, at a highsurface pressure of 30 mN/m, less disruption of the monolayer bydetergent is expected [21]. Secondly, the transmembrane proteinAqpZ has a great tendency to aggregate and be denatured whenexposed to air. Therefore, the densely packed monolayer wouldlimit the exposure of proteins to air at the air–liquid interface.

As the protein–detergent mixture is injected into theDPPC–DOGSNTA subphase, one critical issue is that the lipid mono-layer may be dissolved when the detergent concentration is aboveCMC [8,22]. This is especially true for DOGSNTA because it is inthe fluid phase at room temperature and therefore has no liq-uid condensed phase on the air–liquid interface. However, it isknown that AqpZ needs to be stored at a detergent concentrationhigher than CMC to prevent aggregation. The lowest associationconcentration, the critical aggregation concentration (CAC), of DDMin the presence of AqpZ in the system, was therefore studied bythe surface tension method [23,24]. With the presence of proteinsor polyelectrolyte in the solution, complexation of the detergentwith protein/polymer molecules is thermodynamically favored atthe CAC rather than the formation of regular micelles [24–28].The hydrophobic segment of proteins associates with hydrocarbonchains of detergents and forms a protein–surfactant complex. Asshown in Fig. 4, at an AqpZ concentration of 0.5 mg/L (0.02 �M), theCAC of the mixed system is at 4 �M (2 mg/L) DDM, which is wellbelow the CMC value of pure DDM solution 0.18 mM (85 mg/L) [16].The existence of CAC proves that nonspecific hydrophobic interac-tions between DDM and AqpZ are strong enough to induce complexassociation of the two at a DDM concentration lower than CMC.The protein–detergent complex formed at CAC has been named asa “necklace” model with detergent micelles as beads on the proteinchain [28–32].

The CAC value was selected as the DDM concentration in theprotein association experiments. According to previous research

into the subphase. Due to the hydrophobic nature of transmem-brane proteins, AqpZ stays preferentially either in the detergent

286 G. Sun et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 283– 288

Fig. 2. AFM topograph of (a) pure DPPC bilayer, (b) disrupted DPPC bilayer by 2 mg/L DDM in the subphase, and (c) DPPC bilayer with removal of subphase DDM. All thei o the

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tion. After combining the protein associated monolayer with asecond lipid monolayer, AqpZ could possibly refold to its nativestate within the bilayer film. Many previous studies have reportedthat the unfolded membrane protein could be refolded to its native

mages were scanned in ultrapure water environment. The dark area corresponds tnd (c). However with DDM disruption, the DPPC thickness is reduced below 4 nm at the dashed lines are shown at the bottom of respective images. The scale bars (in

icelles or at the air–water interface; therefore, a reduction inetergent concentration could promote association of proteinsith the surface monolayer. In order to achieve a better detergent

emoving rate, 300 mg BioBeads was added for detergent removalor 3, 6 or 12 h. With the LS technique, the final protein-associated

onolayer was deposited onto a second DPPC–DOGSNTA mono-ayer prepared on the Mica surface. The bilayer was imaged by AFMfter incubation in the PBS buffer for 1 h. Only the images from 6 hetergent removal condition show hints of incorporated AqpZ (seeig. 5). AFM imaging shows small protrusions on top of the bilayerlm with an average height of 4 A above the lipid bilayer. A simi-

ar protein protrusion height was reported by Scheuring et al. whosed AFM to study AqpZ 2D crystal structures in 1999 [33]. There-ore, Fig. 5 might give an indirect indication of incorporated AqpZ.owever, more research should be done in the future to validate

he hypothesis. Detergent removal for a longer time span may causehe protein aggregation in the low detergent environment, whileor a shorter time span, DDM would destabilize lipid monolayer

hich can be rapidly collapsed after deposition. Furthermore, as theDM adsorption and removal from the surface monolayer is a com-licated process, it is very unlikely to interpret AqpZ-monolayerssociation from the surface pressure evolution profile.

ig. 3. Pressure-area isotherms of DPPC, DOGSNTA and their mixtures of differentolar ratios (5:1 and 10:1). The temperature of experiments is 23 ± 1 ◦C.

mica surface. The typical DPPC thickness should be around 4–5 nm as shown in (a)e “fluid-like” defective structure is formed in the bilayer. The cross-section profiles

line) are 1 �m in all the three images.

A possible mechanism of protein incorporation made up of threephases is proposed. In the first phase, His-tagged AqpZ attachesto the DOGSNTA domain in a short time after injection into thesubphase and the monolayer is meanwhile destabilized by DDM.Proteins are maintained at the CAC and form a “necklace” complexwith detergent molecules. In the second phase, BioBeads furtherdecreases the detergent concentration, which accounts for thepartial insertion of AqpZ into the DOGSNTA monolayer. The trans-membrane protein may be reversibly unfolded in the first twophases due to the low detergent concentration; presumably theprotein–protein aggregation process is slow in the diluted solu-

Fig. 4. Surface tension versus DDM concentration in the AqpZ-free solution andAqpZ solution. The base solution is PBS buffer with pH = 7.4. For pure DDM solutions(N), the transition point of the trend indicates the CMC value of DDM. For DDM–AqpZsolutions (©), the first transition point (from left to right) is the CAC value of theDDM–AqpZ complex system, and the second transition point is the same with theCMC of DDM. At an AqpZ concentration of 0.5 mg/L (0.02 �M), the CAC of the mixedsystem is at about 2 mg/L DDM, which is well below the CMC value of pure DDMsolution, 85 mg/L.

G. Sun et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 283– 288 287

Fig. 5. AFM topograph of AqpZ associated lipid bilayer after 6 h of detergent removal (recorded in PBS buffer solution, pH = 7.4). The dark area corresponds to the micasurface while the lighter area corresponds to the bilayer surface. The thickness of the bilayer is about 4–5 nm. (a) Lower magnification topograph of the bilayer shows hintso h shoa 2, res

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f incorporated AqpZ (arrows). Scale bar is 1 �m. (b) Higher magnification topograpnd (d) are the cross-sectional images of protrusions pointed by arrow 1 and arrow

tate when it is inserted into the lipid bilayer as long as its secondarytructure is still preserved [34–38]. The amphiphilic environmentrovided by the lipid bilayer could spontaneously adsorb, host andefold the membrane proteins, while the detergent is not neces-arily involved in the whole process. This reconstitution of AqpZn the lipid bilayer is the third phase. However, it is known that

embrane protein can only insert into fluid-phase lipid bilayers39]. In this experiment, although the gel-phased DPPC contributeso maintain the monolayer integrity, it becomes an obstacle dur-ng the protein incorporation process. In Fig. 5(a), other than themall round protrusion features (pointed by arrows), there is aarge corrugated domain (upper left corner) which is suspectedo be the incompletely constituted protein clusters. One possiblexplanation is that, in this local region, AqpZ associated DOGSNTAonolayer domain was deposited onto a DPPC monolayer domain;

hus the proteins are permanently denatured and aggregated onhe bilayer surface.

. Conclusion

In summary, as the first attempt to reconstitute hydrophobicembrane proteins with the aid of LB technique, the removal

f detergent from the air–liquid interface was investigated and protein incorporation model was predicted. Since detergentsring many complications in LB studies, we selected the lowestetergent–protein association concentration to prevent dissolu-ion of surface monolayer and to minimize the membrane defects.oth the subphase circulation and the quantity of Biobeads addedan determine the detergent removal rate. Further investigations

ill focus on different lipid–protein ratio and incorporated protein

unctionality tests. This newly developed approach of AqpZ recon-titution may potentially be applied in biomimetic membranes forater purification and biosensor applications.

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ws the inserted proteins are distributed in the lipid bilayer. Scale bar is 200 nm. (c)pectively.

Acknowledgements

This work was financially supported by the Environment andWater Industry Programme Office (EWI) (NUS grant number:R-279-000-293-272) under the Singapore National Research Foun-dation (NRF). The authors appreciated the suggestions from Prof.Wolfgang Meier (University of Basel, Switzerland) and Prof. Si-ShenFeng (NUS, Singapore). The authors would also like to give thanks toDr. Qingsong Lin, Dr. Shirlaine Koh, Ms. Honglei Wang, Ms. BingfangWang, and Ms. Peishan Zhong.

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