Accepted Manuscript

A concise method for quantitative analysis of interactions between lipids and

membrane proteins

Masataka Inada, Masanao Kinoshita, Ayumi Sumino, Shigetoshi Oiki, Nobuaki

Matsumori

PII: S0003-2670(19)30127-8
DOI: https://doi.org/10.1016/j.aca.2019.01.042
Reference: ACA 236544

To appear in: Analytica Chimica Acta

Received Date: 5 October 2018

Revised Date: 17 January 2019
Accepted Date: 18 January 2019

Please cite this article as: M. Inada, M. Kinoshita, A. Sumino, S. Oiki, N. Matsumori, A concise method
for quantitative analysis of interactions between lipids and membrane proteins, Analytica Chimica Acta,
https://doi.org/10.1016/j.aca.2019.01.042.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

Graphical abstract

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A concise method for quantitative analysis of interactions between lipids and

membrane proteins

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Masataka Inada a, Masanao Kinoshita a, Ayumi Sumino b,c,d,e, Shigetoshi Oiki b, Nobuaki Matsumori a*

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a
Department of Chemistry, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka,

819-0395, Japan.
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b
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of
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Fukui, Fukui, 910-1193, Japan.

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c
PRESTO, Japan Science and Technology Agency (JST), Saitama, 332-0012, Japan.
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d
High-speed AFM for Biological Application Unit, Institute for Frontier Science Initiative, Kanazawa
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University, Kanazawa, 920-1192, Japan.

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Bio-AFM frontier Research Center, Kanazawa University, Kanazawa, 920-1192, Japan
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*
Corresponding author at: Department of Chemistry, Faculty of Science, Kyushu University, 744

Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.

E-mail address: matsmori@chem.kyushu-univ.jp (N. Matsumori)

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Abstract

Although interactions between lipids and membrane proteins (MPs) have been considered crucially

important for understanding the functions of lipids, lack of useful and convincing experimental methods

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has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance

(SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip

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surface with self-assembled monolayer (SAM) with C6-chain. To develop this method, we used

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bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits

of using C6-SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better

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immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM
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provides immobilized MPs with a partial membranous environment, which is important for the

stabilization of MPs; and (3) a thinner C6-SAM layer (1 nm) compared with MP size forces the MP to
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bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the
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hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C6-SAM is 10 times

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higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules

are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had
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the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported
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co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we
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not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation,

demonstrating the validity and utility of this method.

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Abbreviations: DLPC, 1,2-dilauroylphosphatidylcholine; DMPC, 1,2-dimyristoylphosphatidylcholine;

DPPC, 1,2-dipalmitoylphosphatidylcholine; DOPC, 1,2-dioleoylphosphatidylcholine; DMPG,

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1,2-dimyristoylphosphatidylcholine; DPPS, 1,2-dipalmitoylphosphatidylserine; phytanoylPC,

1,2-diphytanoylphosphatidylcholine; phytanylPC, 1,2-di-O-phytanylphosphatidylcholine; PG,

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phosphatidylglycerol; PGP-MEphosphatidylglycerophosphate methyl ester; PGS, archeal

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phosphatidylglycerosulfate; S-TGA-1, 3-HSO3-Galp-β1,6-Manp-α1,2-Glcp-α

1,1-2,3-diphytanylglycerol; PM, purple membrane; bR, bacteriorhodopsin; OG, n-octyl-β

-D-glycopyranoside.
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Keywords: Lipid, Membrane protein, Quantitative interaction analysis, Surface plasmon resonance,
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Self-assembled monolayer, Bacteriorhodopsin

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1. Introduction

Membrane proteins (MPs) cannot function without membrane environments, and the folding, structure,

and function of MPs were shown to be affected by their lipid environments [1-15]. Therefore, to ensure

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their structural and functional integrity, MPs require support by the surrounding and/or specific lipid

molecules. These effects can be understood in terms of specific interactions between lipids and protein

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residues. In effect, some MP-lipid interactions are sufficiently tight, so that these lipids are retained

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during the purification of MPs and are found in their crystal structures [16-23]. However, quantitative

experimental measurements of lipid-MP interactions have been rarely performed, due to the difficulty in

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the observation of these interactions using traditional structural methods in lipid membrane
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environments. Therefore, the molecular mechanisms underlying the lipid-associated modulation of the
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structure and functions of MPs have not been completely understood. To improve our understanding of

the roles of lipids, the development of a method for quantitative evaluation of the lipid-MP interactions
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is required.
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As described, since MPs require annular lipids, some types of media, such as the supported lipid
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bilayer [24], proteoliposomes [25], nanodisks [26], and Amphipol [27] have been developed to confer

the membranous environment to MPs and to prevent them from denaturation. These substrates cover the
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hydrophobic surface of the MPs, maintaining their natural membranous states and allowing the
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application of nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC),

and surface plasmon resonance (SPR) [28-33] for the analysis of their interaction with extraneously

added drugs [34,35]. However, these media cannot be applied for the analysis of lipid-MP interactions,

as they prevent the extraneously added lipid molecules from accessing and interacting with the MPs.
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Additionally, lipids are generally water-insoluble and aggregated, which further prevents the analysis of

the interactions.

To address these problems, we developed an SPR-based method for the lipid-MP interaction analysis,

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in which the surface of sensor chip is coated with self-assembled monolayer (SAM) [36] with relatively

short alkyl chains. The alkyl chains of SAM were expected to cover the hydrophobic surface of

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immobilized MP molecules partially and stabilize the MP, while allowing the remaining MP

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hydrophobic surface to be exposed to aqueous phase and interact with extraneously added lipid

molecules. In the development of the methodology as well as the confirmation of its validity, we used

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bacteriorhodopsin (bR) as an MP [37,38], and examined its interaction with various types of lipids,
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including commercially available and halobacterium-derived natural lipids.
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2. Experimental section
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2.1. Materials
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DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPC

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(dioleoylphosphatidylcholine), DMPG (dimyristoylphosphatidylcholine), DPPS

(dipalmitoylphosphatidylserine), phytanoylPC (diphytanoylphosphatidylcholine), and phytanylPC

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(diphytanylphosphatidylcholine) were purchased from Avanti Polar Lipids (Alabaster, AL, USA), while
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DLPC (dilauroylphosphatidylcholine) was purchased from Wako (Osaka, Japan). The sensor chips and

materials for the SPR measurements were purchased from GE Healthcare (Little Chalfont,

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Buckinghamshire, UK). All other reagents and materials were purchased from Wako, Tokyo Chemical

Inc. (Tokyo, Japan), and Sigma-Aldrich (St. Louis, MO USA).

2.2. Preparation of solubilized bR

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Purple membranes (PMs) were obtained from H. salinarum (strain R1M1) culture, according to the

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standard method [39,40] and stored at 4°C in dark. A PM suspension containing 15-20 mg bR was

sonicated for 10 min and incubated for 40 h under constant magnetic stirring with 100 mM OG in 10

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mM HEPES buffer (pH 7.0). The solubilized bR solution was centrifuged (10 min at 8,000×g, Hybrid

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High Speed Refrigerated Centrifuge Model 6200, Kubota, Japan) to removed insoluble residues, and the
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supernatant was dialyzed overnight using Biodesign dialysis tubing 8000MWCO (#D102, BioDesign

Inc. NY, USA) in 10 mM HEPES buffer (pH 7.0) including 25 mM OG, in dark at 4°C. The
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concentrations of solubilized bR samples were estimated using the absorption coefficient at 554 nm (ε =

47,000 M−1 cm−1) [41]. The solubilized bR samples were stored in dark at 4°C until further use.
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2.3. Extraction and isolation of lipids from PM

Lipids in the PMs were extracted using Bligh & Dyer method [42] with CHCl3/MeOH/H2O = 1/1/1
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solution. Extracted lipids were subjected to preparative HPTLC (silica gel 60, 20 cm × 20 cm × 0.2 mm,

Aluminum back, Merck) with acidic solvent (CHCl3/MeOH/90% AcOH, 65/4/35, v/v) [43]. Lipids on
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each TLC plate were detected by spraying sulfuric acid/ethanol solution (5:95, v/v) over a part of the
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TLC plate and charring it at 180°C. Crude lipids were recovered from the silica gel with CHCl3/MeOH

mixed solvent. Further purification was performed using preparative TLC (silica gel 60, 20 cm × 10 cm

× 0.5 mm, thick layer, Glass plate, Merck) with neutral solvent (CHCl3/MeOH/H2O, 65/25/4, v/v),

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followed by lipid recovery from the silica gel with CHCl3/MeOH mixed solvent. The purified lipids

were dried with N2 gas and in vacuo, and stored at 4°C. The lipids were identified with

MALDI-TOF/MS (Autoflex Ⅲ, Bruker, USA) using 9-aminoacridine as a matrix [44].

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2.4. Preparation of the SAM-modified sensor chip

SIA Kit Au was purchased from the GE Healthcare, and bare Au sensor chips were used. Before using,

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the surface of sensor chip was washed with H2SO4/H2O2 (3/1) solution and dried with N2 gas. The

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sensor chip was immersed in an ethanol solution of mercaptocarboxylic acid/mercaptoalcohol (total

concentration was 10 mM) for 3 days at room temperature, to coat the Au surface with SAM. Afterward,

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the sensor chips were washed with ethanol and Milli-Q water and dried using N2 gas. Thus prepared
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SAM-coated sensor chips were stored in dark at 4 °C and used within a day.
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2.5. Immobilization of bR onto the sensor chip

HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05 % (v/v) Tween 20) was
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used as the running buffer. Solubilized bR was immobilized onto the sensor chips CM5, or the
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SAM-modified sensor chips, according to a standard amino-coupling protocol. Briefly, carboxyl groups
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on the surface were activated by injecting a mixture of 390 mM

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 100 mM N-hydrosuccinimide for 7 min, followed

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by the immobilization of bR by injecting the solubilized bR solution (500 µg mL−1) for 20 min, and the
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deactivation of unreacted carboxyl group by injecting 1 M ethanolamine-HCl (pH 8.5) for 7 min, at a

flow rate of 5 µL min−1.

2.6. SPR analysis

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SPR measurements were performed at 25°C using Biacore T100 system (GE Healthcare, Chicago, IL,

USA). In all protocols, HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05%

(v/v) Tween 20), higher salinity HBS-EP buffer (10 mM HEPES [pH 7.4], 450 mM NaCl, 3 mM EDTA,

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0.05% (v/v) Tween 20), and HBS-N buffer (10 mM HEPES [pH 7.4], 150 mM NaCl) containing 3 mM

EDTA and 25 mM OG were used as running buffers. Evaluation of bR-lipid interactions was made

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within 2 days after bR immobilization. Analyte samples were prepared by dissolving dried lipids into the

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buffer at the concentrations ranging from 10 to 50 µg mL−1, and the samples were sonicated prior to use.

All procedures were automated, using repetitive cycles of sample injection and regeneration. The

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binding assays were performed after three cycles of start-up injections to normalize the two flow cells,
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and just before the injection of lipid solutions, the buffer alone was injected in the first two cycles in
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order to obtain baseline value. Afterward, five solutions with increasing lipid concentrations were

injected for 180 s at a flow rate of 20 µL min−1, followed by 180 s of dissociation at the same flow rate.
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The surface of the chip was regenerated by one sequential injection of 10 mM Gly-HCl solution (pH
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2.5) for 30 s at a flow rate of 20 µL min−1. Association and dissociation of lipid molecules were
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expressed as sensorgrams, representing the time-dependent changes. To remove the contribution from

non-specific binding between SAM and lipids, a blank channel without bR immobilization was used as a
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reference, and the response of the reference channel (SAM-lipid interactions) was subtracted from that
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of the sample channel. The evaluation of interactions was performed using BiaEvaluation software (GE

Healthcare), and kinetic parameters were extracted by a global fit of the corrected sensorgrams using a

1:1 interaction (Langmuir interaction) and heterogeneous ligand model [45]. The correlations of the

fitting were evaluated using χ2 analyses and residual plots.

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2.7. Isothermal titration calorimetry (ITC) analysis

ITC measurements were performed using VP-ITC calorimeter (Microcal Inc., Northampton, MA, USA).

Dried lipid powder and bR were separately dissolved in HBS-EP buffer (10 mM HEPES [pH 7.4], 150

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mM NaCl, 3 mM EDTA, 0.05% (v/v) Tween 20) or in HBS-N buffer (10 mM HEPES [pH 7.4], 150

mM NaCl) containing 3 mM EDTA and 25 mM OG. The final concentrations of lipids and bR were

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1.96 mM and 0.02 mM, respectively. All samples and buffer were well sonicated and degassed at 25°C

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prior to their use. Fifty aliquots of lipid solution (4 µL) were injected sequentially into 1412 µL titration

cell containing bR samples at 3-min intervals. The solution in the titration cell was stirred at a speed of

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130 rpm, and maintained at 25°C. Prior to the analyses of the interactions, the measurements were
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corrected for the heat from the dilution of lipid or bR. The obtained data were analyzed using Microcal
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Origin. During the evaluation, each parameter was calculated by the fitting with the sequential binding

sites model [46], and therefore, bR/lipid complexes stoichiometry changed from 1/1 to 1/15. The
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reaction enthalpy ∆H and KD were calculated from the obtained heat changes, and the entropy term –
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T∆S was obtained as ∆H–∆G.

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2.8. Dynamic light scattering (DLS) measurement

DLS was measured using LB-500 (HORIBA, Ltd., Kyoto, Japan). The lipid powders were dispersed
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with 10 mM HEPES buffer (pH 7.4, 150 mM NaCl, 3 mM EDTA), HBS-EP buffer (10 mM HEPES [pH
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7.4], 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Tween 20), or HBS-N buffer (10 mM HEPES [pH 7.4],

150 mM NaCl) containing 3 mM EDTA and 25 mM OG, and the mixture was vortexed. The final

concentration of lipid was 200 µg mL−1. Measurements were performed at 25°C, and the cumulative

number was 50.

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2.9. Atomic Force Microscopy (AFM) imaging of the SPR sensor chip surface

AFM imaging was performed using Cypher (Oxford Instruments Asylum Research, Inc., Santa Barbara,

CA, USA) with the AC mode. The cantilever used was BL-AC40TS with a tip radius of ~7 nm, the

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resonant frequency was 25 kHz, and the spring constant was 0.1 N m−1. All images were obtained in

HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Tween 20).

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bR-immobilized sensor chip was prepared using the Biacore system, and 132 pg mm−2 bR molecules

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were immobilized onto each flow cell.

3. Results and Discussion

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3.1. SAM-modified SPR sensor chip for the enhanced immobilization of bR
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To directly observe the interactions between MPs and lipids using the SPR method, two experimental

patterns are possible. Lipids can be immobilized as ligands, while MPs are injected as analytes, or vice
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versa. The immobilization of lipids can increase the sensitivity of the SPR observations, since the
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molecular weights of MPs are generally much higher than that of lipids. However, the method is not
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suitable for examining the interactions of a particular MP with different lipid types, because, although

lipids can be immobilized using a commercially available L1 sensor chip, many different types of lipids
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have to be immobilized in respective channels on the L1 chips, and all of the channels have to be tested
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for the interaction with the MP. This requires more time and labor in comparison with the case of

immobilizing the MP. Furthermore, since MPs are generally more precious and expensive than lipids, it

costs more to use MPs as analytes. Additionally, the interactions may include not only simple lipid-MP

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interactions, but membrane insertion and penetration processes of MP as well, which would further

complicate the analysis.

In contrast, the immobilization of MP molecules would enable us to study the interactions between an

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MP of interest and different types of lipids. However, this approach has two main issues: a low level of

the MP immobilization on the sensor chip and a low water solubility of lipids. The former is an

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especially serious problem, because the molecular weights of lipids are generally much lower than those

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of the immobilized MP, and therefore, higher levels of MP immobilization are required in order to

obtain sufficient sensitivity and reliability of the SPR measurements. To address these problems, we

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devised a novel sensor chip modified with SAM to enhance the immobilization of MPs, and utilized
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mixed micelles to solubilize lipid molecules in water.
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For the development of the modified sensor chip, we used bR monomer as an MP, which was

obtained from the culture of Halobacterium salinarum and solubilized with n-octyl-β-D-glucopyranoside
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(OG) (Fig. S1). Initially, we immobilized the solubilized bR onto a commercial CM5 chip (GE
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Healthcare), coated with hydrophilic carboxymethylated dextran, by using the standard amino-coupling
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method at pH 7. In the amino-coupling method, the lysyl or terminal amino groups of bR were expected

to react with activated carboxyl groups on the CM5 chip. However, the immobilization levels of bR
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were not high enough to allow the evaluation of MP-lipid interactions (Fig. S2 and Table 1). As the pI of
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bR is approximately 5-6 [47], we further attempted to use a buffer at pH 4 and 5, for a more efficient

preconcentration of bR on the sensor chip surface. However, the immobilization level of bR on the CM5

chip remained insufficient for our purpose (182 ± 5 RU for pH 5 and 296 ± 23 RU for pH 4, Table 1).

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This may be because the hydrophilicity of the CM5 surface hampers the preconcentration of bR, which

has a large hydrophobic membrane-penetrating region.

Therefore, to increase the hydrophobicity of the sensor chip, we modified the surface of a bare Au

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sensor chip with a SAM composed of mercaptocarboxylic acid with 3, 6, or 11 carbons (C3, C6, C11),

and bR molecules were immobilized on the sensor chips using the conventional amino-coupling method

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at pH 7 (Fig. 1). This led to the immobilization of a considerably larger quantity of bR on the

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SAM-modified sensor chips (Table 1), enabling a clearer observation of their interactions with lipid

molecules.

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Of the three investigated SAMs, C6- and C11-SAMs were shown to allow a better immobilization of
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bR than C3-SAM (Table 1), suggesting that bR can be more efficiently preconcentrated on the chip
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surface via hydrophobic interactions with longer carbon chains. However, when we observed the SPR

sensorgrams showing the interactions between the SAM-immobilized bR and DPPC, the sensorgram
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obtained using C11-SAM had a higher noise level in comparison with that obtained using C6-SAM (Fig.
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S3). This is most likely due to the more prominent hydrophobic interactions between lipid molecules
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and longer SAM chains, which led to the generation of increased background noise. Taking into

consideration both the amount of bR immobilization and the noise levels, we selected C6-SAM for the
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use in the following interaction analyses.

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Since the electrostatic repulsion due to the negative charge formed by deprotonation of

mercaptocarboxylic acid can induce defects on the SAM surface, upon SAM coating we mixed

mercaptocarboxylic acid with non-charged mercaptoalcohol to remove these defects, and examined its

effect on the immobilization of bR (Table 1). Although the addition of 40 mol% mercaptoalcohol
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decreased the bR immobilization, 20 and 0 mol% of mercaptoalcohol allowed sufficient immobilization

levels. Therefore, to avoid a possible defect formation on the SAM surface, we selected C6-SAM

composed of 80% mercaptohexanoic acid and 20% mercaptohexanol.

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3.2. Evaluation of the bR-lipid interactions

Using C6-SAM sensor chip, we analyzed interactions between immobilized bR and different

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commercially available lipids (Fig. 2), which were dissolved in the running buffer containing 0.05%

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(v/v) Tween 20 as a detergent. Using DLS measurements, we confirmed that the lipids were completely

dissolved in the running buffer by forming mixed micelles with Tween 20 at the concentration used for

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the SPR analysis (lipid/Tween 20 = 1/5.57) (Fig. S4 and Table S1). In this study, to remove the
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contribution from non-specific binding between SAM and lipids, a blank channel without bR
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immobilization was used as a reference, and the response of the reference channel (SAM-lipid

interactions) was subtracted from the sensorgram.

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For fitting the sensorgrams, we compared a 1:1 Langmuir binding model and a heterogeneous ligand
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model, the latter of which assumes that bR has two binding sites for the lipid, and therefore provides two
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kinds of dissociation constants KD1 and KD2. Although the heterogeneous model seems more reasonable

because MPs can interact with plural annular lipids in biological membranes, both models fitted very
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well as judged by residual plots and χ2 values, and the obtained KD values have no major differences
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between the two models (Fig. S5). In particular, KD2 values obtained from the heterogeneous model are

much larger than KD1 values, indicating that the second binding hardly occurs in this system. In addition,

the rise in the RU values upon the binding of lipid molecules is not so large as expected for the multiple

binding of lipid molecules. Taken together, we concluded that the contribution of the second lipid
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binding is much smaller than that of the first binding at the concentrations tested, and therefore the 1:1

Langmuir binding model can be safely applied for this system. Of course, an appropriate fitting model

should be changed according to the system, and the heterogeneous model may be better when a MP has

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two or more lipid-binding sites with similar affinity.

Table 2 lists dissociation constants KD and free energy values ∆G between bR and commercial lipids,

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obtained from the analysis of the sensorgrams using the conventional Langmuir binding model. The data

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suggests that the interactions with bR depend on the structural characteristics of lipids, longer- or

bulky-chain lipids (DPPC and phytanoylPC) have higher affinity toward bR, compared with those with

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short- or unsaturated-chain lipids (DLPC and DOPC), and that bR interacts more strongly with anionic
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lipids, DMPG and DPPS, than with neutral PCs. To determine if the electrostatic interactions are
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responsible for the higher affinity of the anionic lipids, we performed the SPR analyses under a higher

ionic strength condition (Table 2 and Fig. S6). Consequently, the interaction between bR and anionic
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DMPG or DPPS decreased due to the electrostatic shielding, while the interaction between bR and
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DMPC was less affected at the high salt concentration. This indicates that the electrostatic force
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considerably contributes to the interactions between anionic lipids and bR. Taken together, the

interactions between the commercial lipids and bR are based on the hydrophobic interactions with the
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alkyl chains and on the electrostatic interactions with the head groups.
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We further analyzed the interactions between bR and halobacterium-derived lipids, PG, PGS,

PGP-ME, and S-TGA-1 (Fig. 3) [48-50], which share the same phytanyl chains that are linked to the

head groups via ether bonds, instead of ester bonds. These lipids were extracted from the purple

membrane of H. salinarum, and purified using HPTLC (Fig. S7). For reference, phytanylPC, purchased
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from a commercial source, was also examined, because it is a PC congener sharing the same alkyl

chains with the halobacterium-derived lipids. The obtained sensorgrams are presented in Fig. 3, and the

affinity data are listed in Table 3, which shows that these lipids have a higher affinity toward bR in

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comparison with the commercial lipids listed in Table 2. Table 3 also shows that dianionic PGS and

PGP-ME have stronger interactions with bR than neutral phytanylPC and monoanionic PG, supporting

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the hypothesis that electrostatic interactions at the lipid head groups play functional roles in the bR-lipid

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interactions.

Of a particular note is the strongest interaction between bR and glycolipid S-TGA-1, at nanomolar

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order of KD. A lower RU value for S-TGA-1 (Fig. 3) suggests that bR contains a specific binding site for
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S-TGA-1, while the interactions between bR and other lipids are less specific, as their larger RU values
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indicate. Based on the aforementioned electrostatic hypothesis, monoanionic S-TGA-1 was expected to

show smaller affinity toward bR than dianionic PGS and PGP-ME. However, the reported co-crystal
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structure of bR and S-TGA-1 [51] suggests that several hydrogen bonds are established between bR and
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the triglycoside moiety of S-TGA-1, conferring a specific and strong molecular interaction between
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S-TGA-1 and bR.

3.3. Comparison with the ITC results

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To examine the validity of the SPR data obtained using newly developed SAM-modified sensor chip,
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we performed ITC measurements under the same condition, and compared two datasets. The ITC

thermograms were best fitted using sequential binding sites model (Fig. S8), which assumes that bR has

plural (n) binding sites for lipids. Although this model is reasonable because MPs can interact with

plural annular lipids in biological membranes, it is not consistent with the treatment of the SPR data with
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the 1:1 Langmuir binding model. The reason for this inconsistency is probably because the ITC

experiments were performed under the condition of higher lipid/bR stoichiometry (15/1) compared with

the SPR experiments, which promoted the multiple non-specific binding of lipid molecules.

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Table 4 lists the thermodynamic parameters representing the strongest affinity between bR and lipids

among n sets of affinity parameters (full lists of the thermodynamic parameters are provided in Table

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S2). Except for halobacterium-derived S-TGA-1 and PGP-ME, the observed affinity data were shown to

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be well consistent between two types of experiments (Fig. 4, red circles). Large binding site n and KD

values indicate relatively nonspecific interactions between these lipids and bR [52]. Additionally, the

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ITC analysis demonstrates that enthalpy term ∆H contributes more to the higher affinity of anionic lipids,
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DMPG and DPPS, toward bR, which is consistent with the SPR results showing the importance of the
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electrostatic interaction for the high affinity of these anionic lipids. Taken together, with the exception

of the results obtained for PGP-ME and S-TGA-1, the results obtained using ITC analysis agree with
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those obtained using the SPR method (Fig. 4), supporting the validity of the newly devised
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SAM-modified SPR method.

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In contrast, the ITC results obtained for PGP-ME and S-TGA-1 interactions demonstrated a low

affinity toward bR in the ITC analysis (Table 4), which is not consistent with the above SPR data or with
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the co-crystal structure that indicated the specific interaction between bR and S-TGA-1[51]. This
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inconsistency is most likely due to trimer formation of bR during the ITC measurements, where high

lipid/bR molar ratios were used to obtain better ITC thermograms. At such high lipid/bR molar ratios,

PGP-ME was shown to promote the trimerization of solubilized monomeric bR [53], and recently, we

demonstrated that S-TGA-1 promotes the trimer formation more efficiently than PGP-ME (to be
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published in due course). In contrast, the trimer formation of bR during the SPR analysis, following the

interaction with these lipids, is unlikely, because the protein is immobilized on the sensor chip.

Therefore, the heat emission due to the trimer formation can lead to the disagreement between the SPR-

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and ITC-obtained data for PGP-ME and S-TGA-1. Furthermore, the ITC analyses require larger sample

amounts of both lipids and MPs, which is not feasible or easily obtainable in many cases. Hence, our

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newly developed SPR analysis offers advantages over the ITC analysis.

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3.4. Effects of the detergent on the interaction analysis results

Detergent is thought to be important for this method, not only for the dissolving of lipids and MPs,

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but also to prevent MPs from denaturing. Because cationic or anionic detergents can interact with MPs
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via electrostatic interactions, nonionic detergents would be more appropriate. Here, we analyzed the
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effects of nonionic detergents on the SPR and ITC results using OG instead of Tween 20, because they

have extremely different critical micelle concentrations (CMCs); the CMC of OG is 20 mM, while that
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of Tween 20 is 0.06 mM [54,55]. Similar to the above experiments with Tween 20 at 0.05% v/v (0.4
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mM), DLS measurements confirmed that lipids form mixed micelles with 25 mM OG (Fig. S9 and
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Table S3). As in the case of using the buffer containing Tween 20, SPR and ITC data were also

correlated in the buffer containing OG (Table 5 and Fig. 4, blue circles). Comparison of the affinity data
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obtained using 25 mM OG (Table 5, Figs. S10 and S11) and 0.05% v/v (0.4 mM) Tween 20 (Tables 2-4)
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further demonstrated that when using OG, the interactions became weaker both in SPR and ITC analyses

(Fig. 4, red and blue circles), most likely because OG, which has to be used at a higher concentration

due to its higher CMC, can compete with lipids for the interactions with bR. If this is so, Tween 20,

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which can be used at a lower concentration, would be a more appropriate detergent for the analysis of

MP-lipid interactions.

3.4. Implication of this method for the analysis of MP-lipid interactions

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Although SPR has been frequently used for the analyses of protein-small molecule interactions, such

as protein-drug interactions, it has been rarely applied in the evaluation of MP-lipid interactions. This

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can be attributed to the following reasons; difficulties in immobilizing a large amount of MPs on the

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sensor chip without denaturation and difficulties in reproducing membranous environments for the

interaction analysis. Here, we achieved a much higher level of bR immobilization on the SAM-modified

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sensor chip in comparison with that on the conventional CM5 sensor chip. We have also confirmed that
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a larger amount of potassium channel can be immobilized on the SAM-modified sensor chip than on the
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CM5 (to be published in due course), and its general applicability in the analysis of other MPs is

currently investigated.
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Considering the reproduction of membranous environments, which are thought to be important for the
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stability and functioning of MPs, it is possible to reconstitute MPs into membranous media, such as
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liposomes, nanodisks, or amphipathic polymers, and to immobilize them on the SPR sensor chip [30-33].

When using those methods, however, hydrophobic interfaces of MPs, which contribute to the interaction
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with lipid molecules, are buried into or covered with the media, preventing the access of extraneously
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added lipids to the MPs. SAM is considered to provide membranous environments to MPs as well, but

C6-SAM is not thick enough to completely cover and accommodate the transmembrane regions of MPs.

For example, bR dimensions are 4 × 6 × 11 nm (11 nm in the transmembrane direction) [56], exceeding

the thickness of C6-SAM, which is ca 1 nm. Therefore, the hydrophobic membrane spanning regions are
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assumed to be partially inserted in the SAM and stabilized, but a considerable part of the MP molecule

remains exposed out of the SAM surface, allowing the contact between the hydrophobic regions and the

extraneously added lipid molecules. To support this assumption, we observed, using atomic force

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microscopy (AFM), the SAM-modified sensor chips with and without immobilized bR molecules, and

confirmed that bR molecules are protruding from the SAM surface (Fig. 5).

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Therefore, the hydrophobic nature of SAM-modified sensor chip is expected not only to allow a high

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level of MP immobilization due to the efficient preconcentration, but also to provide a partially

membranous environment for the immobilized MP molecules. Furthermore, C6-SAM thickness, lower

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than the MP dimensions, allows the MP’s hydrophobic regions to protrude from the SAM surface,
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making it possible to interact efficiently with extraneous lipid molecules.
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4. Conclusions
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Measuring the affinity of lipids for MPs is an important unmet need in analytical chemistry, albeit
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challenging. In this study, to evaluate lipid-MP interactions quantitatively, we developed a concise

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SPR-based methodology by using C6-SAM-coated sensor chip, and applied this novel method to the

analysis of the interactions between bR and various lipids. The merits of using C6-SAM-modified sensor
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chip are that (1) the alkyl chains of SAM allow an improved immobilization of MPs through the
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efficient preconcentration due to hydrophobic interaction, (2) SAM provides immobilized MP molecules

with a partial membranous environment, important for the stabilization and functioning of the MP, and

(3) lower thickness of C6-SAM (ca. 1 nm), compared with the MP dimensions, allows the exposure of

the hydrophobic regions of MPs out of the SAM surface and the contact with extraneously introduced
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lipid molecules solubilized by Tween 20. In fact, the amount of bR immobilized on C6-SAM was shown

to be 10 times larger than that on the CM5 sensor chip, and AFM observation further confirmed that bR

molecules protrude from the SAM surface.

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The affinities of different lipid molecules toward bR can be explained by hydrophobic interactions

with the lipid tails and electrostatic interactions with the head group. However, an exceptionally high

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affinity of S-TGA-1 toward bR can be explained by the formation of several hydrogen bonds between

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the carbohydrate parts of S-TGA-1 and bR, which is consistent with their reported cocrystal structure

[51]. Although the ITC measurement failed to reproduce the specific interaction between S-TGA-1 and

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bR, our method not only accurately reproduced the specific interaction, but succeeded in the quantitative
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evaluation of the interaction, demonstrating the validity and usefulness of our method. Additionally, it
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can be easily extended to encompass the quantitative analysis of interactions between MPs and

hydrophobic small molecules, instead of lipids. Further investigations of this method using other MPs,
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including potassium channel [57], are currently underway.

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Conflicts of interest

The authors declare that they have no competing interests.

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Acknowledgements

We thank Professor Michio Murata (Osaka University) for his help in halobacterium culture, and

Associate Professor Ayami Matsushima (Kyushu University) for her help in SPR measurements. M.

Kinoshita acknowledges JSPS KAKENHI (JP17K15107) for funding. A. Sumino acknowledges JST
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PRESTO, the Shiseido Female Researcher Science Grant, JSPS KAKENHI (JP17H05058 and

JP16K15178) for funding. S. Oiki acknowledges JSPS KAKENHI (JP17H04017 and JP16K15179) and

MEXT KAKENHI (JP16H00759). N. Matsumori acknowledges JSPS KAKENHI (JP15H03121),

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MEXT KAKENHI (JP16H00773), and JST ERATO (Lipid Active Structure Project).

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Tables
Table 1. Immobilization levels of bR on CM5 and SAM-modified sensor chips a
Sensor chip b Immobilization level of bR (103 RU)
CM5 pH 7 0.158 ± 0.009

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pH 5 0.182 ± 0.005
pH 4 0.296 ± 0.023
c
C3-SAM (100:0) 1.85 ± 0.33

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c
(80:20) 1.90 ± 0.06
c
(60:40) 1.33 ± 0.13

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C6-SAM (100:0) c 2.67 ± 0.44
c
(80:20) 2.61 ± 0.32

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(60:40) c 1.06 ± 0.23
C11-SAM (100:0) c 2.42 ± 0.50
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(80:20) c 2.27 ± 0.20
(60:40) c 1.46 ± 0.08
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a
Data are presented as mean ± SD (N = 3). b CM5 and SAM-modified chip surfaces were covered with
hydrophilic carboxymethyldextran and SAM, respectively. c Molar ratios of mercaptocarboxylic
acid:mercaptoalcohol.
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Table 2. Affinities of commercial lipids toward bR, as determined using SPR a

Lipid KD (µM) ∆G (kJ mol−1)
DLPC 359 ± 32 −19.6 ± 1.7
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DMPC 31.5 ± 2.2 −25.7 ± 1.8

(20.0 ± 2.5) b (−26.8 ± 3.3) b
DPPC 15.9 ± 1.3 −27.4 ± 2.3
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(2.53 ± 0.20) ×
DOPC −15.2 ± 1.2
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103
PhytanoylPC 5.52 ± 0.37 −30.0 ± 2.0
DMPG 0.453 ± 0.044 −36.2 ± 3.5
(25.5 ± 4.9 ) b (−26.2 ± 5.0) b
DPPS 0.687 ± 0.068 −35.1 ± 0.35
(33.7 ± 1.8) b (−25.5 ± 0.9) b

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a
Data are presented as mean ± SD (N = 3). b Data in parentheses were obtained using high-salinity HBS-EP (10
mM HEPES [pH 7.4], 450 mM NaCl, 3 mM EDTA, 0.05% (v/v) Tween 20) as a running buffer. Other data were
collected using HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05%(v/v) Tween 20).

Table 3. Affinities of halobacteirum-derived lipids toward bR, as determined using SPR a

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Lipid KD (nM) ∆G (kJ mol−1)
PhytanylPC (3.72 ± 0.90)×103 −31.0 ± 7.5
PG 187 ± 47 −38.4 ± 95

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PGS 99.2 ± 5.1 −39.9 ± 2.1
PGP-ME 78.3 ± 1.3 −40.5 ± 0.7

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S-TGA-1 9.44 ± 0.19 −45.8 ± 0.9
a
Data were collected using HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% (v/v)

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Tween 20), and are presented as mean ± SD (N = 3).
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Table 4. Affinities of the artificial and halobium-derived lipids toward bR, as determied using ITC a
∆G (kJ mol−1) ∆H (kJ mol−1) T∆S (kJ mol−1)
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Lipid n KD (µM)
DLPC 7 18.9 ± 1.0 −26.9 ± 1.4 1.03 ± 0.51 37.2 ± 1.5
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DMPC 10 11.8 ± 0.2 −28.1 ± 0.4 20.6 ± 3.1 48.7 ± 3.1

DPPC 10 5.32 ± 0.14 −30.1 ± 0.8 126 ± 13 156 ± 14
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PhytanoylPC 8 2.60 ± 0.12 −31.8 ± 1.5 204 ± 25 236 ± 25

DPPS 10 0.648 ± 0.031 −35.3 ± 2.5 −31.1 ± 6.8 4.18 ± 7.00
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DMPG 8 0.391 ± 0.059 −36.5 ± 5.5 −48.3 ± 1.3 11.8 ± 5.7

S-TGA-1 4 8.20 ± 0.04 −29.0 ± 1.3 −26.6 ± 0.55 2.37 ± 1.44
PGP-ME 4 3.45 ± 0.13 −31.1 ± 1.1 −53.2 ± 0.6 −22.0 ± 1.2
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a
Data were collected using HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% (v/v)
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Tween 20), and are presented as mean ± SD (N = 3). Binding isotherms were fitted to a model in which
interaction involves the participation of “n” lipid molecules (sequential binding site model), and the listed
thermodynamic parameters represent the strongest binding among n sets of the parameters. Full lists of the
thermodynamic parameters are provided in Table S2.

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Table 5. Affinities of lipids toward bR, analyzed using SPR and ITC, with 25 mM OG as a detergent a
SPR ITC

Lipid KD (µM) ∆G (kJ mol−1) KD (µM) ∆G (kJ mol−1)

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DMPC 40.2 ± 2.7 −25.1 ± 1.7 12.1 ± 0.8 −28.0 ± 2.0
DPPC 19.8 ± 3.3 −26.8 ± 4.4 9.26 ± 0.43 −28.7 ± 1.3
PhytanoylPC 13.2 ± 0.6 −27.8 ± 1.2 2.53 ± 0.29 −31.9 ± 3.7

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DMPG 7.17 ± 0.41 −35.0 ± 2.0 2.31 ± 0.15 −32.1 ± 2.1
a
Data were collected using HBS-N buffer (10 mM HEPES [pH 7.4], 150 mM NaCl) containing 3 mM EDTA

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and 25 mM OG, and are presented as mean ± SD (N = 3). Binding isotherms were fitted to a model in which
interaction involves the participation of “n” lipid molecules. Full lists of ITC data are provided in Table S4.

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Figures

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Fig. 1. Immobilization of MPs on the SAM-modified sensor chip and MP-lipid interaction analysis. (A)
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Bare Au-coated sensor chip was modified to form SAM, on which bR molecules were immobilized
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using the amino-coupling method. Lipids were solubilized by Tween 20. NHS: N-hydrosuccinimide,
EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. (B) To remove the effect of the adsorption of
lipid molecules onto the SAM surface, the SPR response of the reference channel where bR was not
immobilized was subtracted from that of the sample channel.

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Fig. 2. SPR sensorgrams showing the interactions between the commercial lipids and bR immobilized
on the C6-SAM modified sensor chip. Sensorgrams from bottom to top were obtained after injection of
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10, 20, 30, 40, and 50 µg mL−1 of the lipids dissolved in HBS-EP buffer. Black lines indicate
experimentally obtained sensorgrams, while red lines indicate theoretical curves (Langmuir binding
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model).
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Fig. 3. Chemical structure of PhytanylPC and halobacterium-derived lipids, PG, PGS, PGP-ME, and
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S-TGA-1 (left) and SPR sensorgrams between those lipids and bR immobilized on the C6-SAM
modified sensor chip. PhytanylPC was purchased from a commercial source, while other lipids were
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isolated from H. salinarum. Sensorgrams from bottom to top were obtained after injection of 10, 20, 30,
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40, and 50 µg mL−1 of the lipids dissolved in HBS-EP buffer. Black lines indicate experimentally
obtained sensorgrams, while red lines indicate theoretical curves (Langmuir binding model).
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Fig. 4. Correlation of MP-lipid dissociation constants between SPR and ITC analysis. Red circles and
blue triangles indicate the data obtained in HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3
mM EDTA, 0.05% (v/v) Tween 20) and in HBS-N buffer (10 mM HEPES [pH 7.4], 150 mM NaCl)
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containing 3 mM EDTA, 25 mM OG, respectively.

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Fig 5. AFM images of the SPR sensor chip surfaces modified with C6-SAM without (A) and with (B)
the immobilization of bR. (C) Height profile of an immobilized bR molecule along the dashed line in
panel (B). All images were obtained using HBS-EP buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3
mM EDTA, 0.05% (v/v) Tween 20). Scale bars: 20 nm.

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SPR-based concise method for analyzing the interaction between lipid and membrane
proteins.

SPR sensor chip is coated with self-assembled monolayer.

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Membrane proteins are efficiently immobilized on the sensor chip.

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Lipids are accessible to the hydrophobic regions of the immobilized membrane proteins.

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The sensor detects the specific interaction between bacteriorhodopsin and glycolipid
S-TGA-1.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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