Aligning Nanodiscs at the Air–Water Interface, a Neutron Reflectivity

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Aligning Nanodiscs at the Air
Water Interface, a Neutron Reflectivity Study Maria Wads€ater,*,†,r Jens B. Simonsen,*,‡,r Torsten Lauridsen,§ Erlend Grytli Tveten,§,|| Peter Naur,^ Thomas Bjørnholm,† Hanna Wacklin,# Kell Mortensen,‡ Lise Arleth,‡ Robert Feidenhans’l,§ and Marite Cardenas*,† †

Nano-Science Center and Institute of Chemistry, ‡Department of Basic Sciences and Environment, Faculty of Life Sciences, and Nano-Science Center and Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark Department of Physics, Norwegian University of Science and Technology, N-7491 Trondheim, Norway ^ Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark # Institut Laue Langevin, 6 rue Jules Horowitz
BP 156, 38042 Grenoble Cedex 9, France, and European Spallation Source ESS AB, P.O. Box 176, 22100 Lund, Sweden

)

§

bS Supporting Information ABSTRACT:

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air
water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ( 2.6 Å with a surface coverage of 66 ( 4%. This layer is located about 15 Å below a cationic surfactant layer at the air
water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (∼4.5 Å) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.

’ INTRODUCTION Nanodiscs are stable self-assembled nanostructures of phospholipids and an engineered amphipathic helical membrane scaffold protein (MSP).1 Nanodiscs have a finite size and shape, in which the MSP protein wraps around a phospholipid bilayer patch in a belt-like manner as revealed by small-angle scattering and other techniques.1 They offer a potential new platform for manipulating and handling of membrane proteins,1
3 because the latter can be incorporated in the nanodiscs lipid core (for a comprehensive review, see Borch and Hamann4 and Ritchie et al.5). This is of special interest given that membrane proteins r 2011 American Chemical Society

are extremely difficult to handle and crystallize. Indeed, only 1.2% of the known crystal protein structures by the end of 2010 corresponded to membrane proteins.6 Also, among the human membrane proteins, only 12 out of the total number of ∼7.000 have been structurally solved.7 In 2004, it was suggested that membrane proteins reconstituted in nanodiscs could be crystallized,8 and their high-resolution structure was thereby Received: August 9, 2011 Revised: October 28, 2011 Published: November 02, 2011 15065

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Figure 1. Schematic representation of (A) molecular structures for the phospholipids and surfactants used in this work, (B) the nanodisc layer below the air/surfactant
water interface, and (C) the geometrical model used to represent a single nanodisc in the fits for neutron reflectivity data.

mapped by X-ray crystallography. However, this approach has turned out to be more difficult than expected, and, so far, no reports on the successful crystallization of proteins in nanodiscs have been published to the author’s knowledge. Solution studies by electron microscopy of negatively stained samples have been successfully applied to study the voltage-dependent anion channel into a DMPC nanodisc giving a low-resolution 3D structure of the membrane protein-containing nanodiscs.9 An alternative method for obtaining structural information from noncrystalline proteins in a biologically friendly environment is the use of oriented membranes. Membrane proteins reconstituted in a 2D lipid matrix are free from participating in interlayer protein
protein interactions. Thus, the functional properties of isolated membrane proteins cannot be probed using continuous lipid bilayers. Therefore, we suggest that a 2D matrix of nanodiscs is a more appropriate choice of oriented membrane model because in such a model the function and structure of individual functional membrane proteins can be probed. We show in this Article how we can, by careful design of an insoluble surfactant at the air
water interface, align nanodiscs just below the interface, thus enabling future studies of aligned membrane proteins. We propose that the use of nanodiscs at interfaces may result in new ways of probing the structure and also the functionality of membrane proteins. Aligning nanodiscs at a planar interface will result in increased structural sensitivity in the direction perpendicular to the interface. Neutron reflectivity is an excellent tool to gain detailed structural information at the angstrom level in the direction perpendicular to the interface, especially when using appropriate hydrogen/deuterium contrast variation.10 Nanodiscs with and without reconstituted membrane proteins have previously been investigated at the mica
water interface by atomic force microscope (AFM) to probe their overall size and shape in the presence of divalent cations.1,11,12 Surface plasmon resonance (SPR) was used to measure the kinetics of the interaction between a ligand and its membrane bound receptors contained within a solid-supported nanodisc layer, showing that functional studies are possible using nanodiscs layers on surfaces.13 Koppaka et al.14 used total reflection Fourier transform infrared spectroscopy on a system in which app A1 based lipoprotein A-I complexes were adsorbed on top of an immobilized monolayer of partial negatively charged lipids aided by divalent ions, proving that the belt protein was aligned parallel to the solid surface. However, in this type of system, the membrane proteins

are in direct contact with the underlying solid surface that may affect both their structure15
17 and their function. We believe that the 2D arrangement of nanodiscs in a fully aqueous environment at the air
water is expected to be a much more ideal setup for studying functional membrane proteins, given that unfolding of the disks as well as undesired changes in protein conformation upon interaction with the solid substrate15
17 can be avoided. We have therefore developed a system in which nanodiscs are aligned at the air
water interface while always being fully immersed in the aqueous solution. In this way, a fully biologically compatible environment is achieved. The Langmuir trough offers an ideal tool to handle samples at the air
water interface because the film density and surface pressure can be measured and fine-tuned by simple changes in the trough area. We have taken advantage of electrostatic interactions to drive the adsorption of nanodiscs to an insoluble cationic surfactant film at the air
water interface to attempt to form a uniform, welldefined nanodisc monolayer. Similar approaches have previously been used to study various biomolecules adsorbed to insoluble monolayers at the air
water interface. For instance, the electrostatic balance due to the in-plane repulsion between negatively charged DNA molecules and the attraction between DNA and cationic surfactant monolayers led to the formation of a DNA layer below the surfactant populated air
water interface.18,19 The presence of divalent ions has also proved efficient to adsorb DNA below zwitterionic lipid monolayers at the air
water interface.20 Similarly, the mechanism of interaction of soluble proteins with an insoluble lipid monolayer can be determined by neutron and X-ray reflectivity.21,22 The method we present can be briefly described as follows: First, a cationic surfactant monolayer is spread on the water surface and compressed to a certain pressure using a Langmuir film balance. Next, nanodiscs in an aqueous solution are injected below the surface into the subphase. The electrostatic attraction between the insoluble cationic film and the nanodiscs is enough to lead to the formation of a nanodisc layer just below the insoluble cationic film at the air
water interface. We hypothesized that the geometry and charge distribution of the nanodiscs, with the highest charge at the flat bilayer surfaces, should facilitate the alignment of nanodiscs to a parallel orientation to the air
water interface as schematically represented in Figure 1b. We used neutron reflection to elucidate the structure of the nanodiscs and surfactant films to determine the orientation and degree of alignment of the discs. We show that the surfactant
nanodiscs 15066

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Langmuir ensemble at the air
water interface is stable for at least 45 min under reservoir conditions (ionic strength and pH) similar to physiological ones. As a model system, we chose the cationic surfactant dioctadecyldimethylammonium bromide (DODAB) spread on Tris buffer pH 7.4 enriched with 100 mM NaCl and nanodiscs composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) at a 3:1 molar ratio. It should be emphasized that the nanodisc monolayer setup presented herein is directly applicable to techniques that explore the in-plane packing of molecules such as grazing incidence small-angle X-ray and neutron scattering, and to many other techniques that can probe the function of lipids and proteins at the air
water interface, for example, fluorescent techniques.23

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Table 1. Scattering Length Densities, G, of the Components and the Layers in the Nanodisc Film of Both the Deuterated and the Undeuterated Samplesa volume

F

(Å3)

(10
6 Å
2)

DMPC head (C10H18NO8P)

31930

1.9

DMPG head (in D2O) (C8H10D2O10PNa)

30431

3.2

1:3 PG:PC head

315.3

component

lipid heads and D2O in nanodisc cylinder in Lheads DMPG or DMPC tailsh/d

721.430
0.4h/7.4d 30 95432

lipid tail core (1:3 DMPG:DMPC) and

’ EXPERIMENTAL SECTION work was MSP1D1, which was expressed and purified according to Bayburt et al.1 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (h-DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (h-DMPG), as well as chain deuterated 1,2-dimyristoyl (d54)-sn-glycero-3-phosphocholine (d-DMPC), were purchased from AVANTI and used as received. 20 mM tris buffer pH 7.4 (Sigma Aldrich) and 100 mM NaCl (Sigma Aldrich) were prepared in D2O provided by the neutron scattering facilities ILL and SNS prior to use. Dioctadecyldimethylammonium bromide (DODAB) from Sigma Aldrich was dissolved in chloroform prior to use. Nanodiscs were prepared as described in detail elsewhere.1,2 In this text, h-ND refers to nanodiscs made by mixing h-DMPG and h-DMPC to a molar ratio of 1:3, while d-ND refers to nanodiscs made of h-DMPG and d-DMPC to the same molar ratio. The chemical structures of lipids and surfactants used are schematically represented in Figure 1A. Methods. Specular neutron reflection is measured as a function of the scattering vector, Q, perpendicular to the reflecting surface (Q = (4π)/(λ) sin θ, where θ is the reflecting angle and λ is the neutron wavelength). The reflectivity (R) is related to the scattering length density (F) of a material (F = ∑inibi, where n is the number of nuclei i in a given volume and b is their coherent scattering length) via an inverse Fourier transformation.24 The neutron interacts with the nucleus of an atom, and therefore the scattering lengths from different isotopes of the same element can differ significantly. This allows for contrast variation by isotopic substitution, particularly by deuterium substitution for hydrogen, isotopes that have a large difference in their coherent scattering. Neutron reflectivity profiles are typically analyzed by optical matrix formalisms.25 All neutron reflectivity profiles obtained in this study were analyzed by fitting simulated reflectivity profiles for an optical model structure of the system to the experimental data via the software Motofit,26 which uses the Abeles optical matrix method to calculate the reflectivity of thin layers and enables simultaneous fitting of data sets of different isotopic compositions.27 The fitting parameters used for each layer were the thickness (d), the interfacial roughness (δ), and the solvent volume fraction (ϕ). The uncertainty of these parameters was estimated by varying each parameter separately. The maximum accepted deviation for each parameter, for which the model still fit to both of the two contrasts, was assigned as the uncertainties28 given in Tables 2 and 3. In the Supporting Information, we show the effects on the calculated reflectivities of the variation of each fitted parameters to values larger than those accounted for by the given uncertainty. From the fitted parameters ϕ and d as well as the known molecular volumes (Vm) of the cationic surfactant,29 phospholipid heads/tails,30,31 and belt protein,32 the average wet area per molecule at each fitted layer can be calculated: Awet = (Vm)/(d(1
ϕ)).

2.2 3.7

(C26H54/C26D54) MSP1D1

Materials. The amphiphatic scaffold protein used in the present

38 747

177 332

3.532 0.96h‑ND/4.8d‑ND

MSP1D1 in Lcore cationic surfactant C38H80N+

102029


0.4

a

The scattering length densities of the different components were calculated as: (∑ibini)/(V), where b is the scattering length of atom i and n is the number of atom i in the component, and V is the volume of the component. A single layer interfacial model was used to fit neutron reflectivity profiles of the cationic film on D2O prior to nanodisc injection. The cationic surfactant film is termed as Lsurfactant. Between the Lsurfactant and the nanodisc monolayer, the presence of a water layer is required in the model to best fit the neutron reflectivity profiles (discussion about other models is given in regard to Figure 5). This solvent layer is termed Lsolvent. The nanodiscs are modeled as flattened cylinders, for which the area is equal to the area of the belt and lipid core in Lcore (Acore), while its height is equal to the thickness of the lipid bilayer as shown in Figure 1b, c. In this model, it is assumed that the belt protein has the same height as the lipid tails. Thus, the nanodisc can be modeled via a symmetrical three-layer interfacial model (Figure 1c) described as follows: lipid heads (Lheads), lipid tails plus belt protein (Lcore), and lipid heads (Lheads). By exploiting that, in flat bilayers, the cross-sectional area of the lipid tails equals that of the lipid head groups, the volume of the water in the phospholipid head groups (Vsolvent) can be calculated as the difference between the total volume of the headgroup region per lipid (Vlipidhead) and the partial specific molecular volume of the phospholipid head (Vsolvent = AheaddLheads
Vlipidhead). The area per nanodisc in Lcore, assuming no solvent is present within this layer, is thus Acore = (2Vbelt + Vlipidtailcore)/(dLcore), while Ahead = (Vlipidheadcore + Vsolvent)/(dLheads), where Vlipidtailcore = VlipidtailNlipids, Vlipidheadcore = Vlipidhead(Nlipids)/(2), and Nlipids is the number of lipid molecules within the nanodiscs and taken to be 160 as measured by other techniques.30,31 Vlipidhead is a weighted average (3:1) of the volumes of the PC and PG heads. On the other hand, Vsolvent in the lipid headgroup layer is calculated from AcoredLheads
Vlipidheadcore.32 Using these geometrical relationships and knowing that for a two-component system, Flayer = F1X + F2(1
X), where X refers to volume fractions of one of the materials, we can calculate FLcore and FLheads. The fits now need to satisfy that the number of nanodiscs (or the area per ND) at each layer (Lheads and Lcore as shown in Figure 1b and c) is constant, that is, Aheads,wet = Acore,wet. Table 1 gives all F and Vm values used in this work. Two contrast situations, both in D2O, were used with nanodiscs made out of hydrogenated DMPG and either deuterated (d-ND) or hydrogenated DMPC (h-ND). All measurements were made on D2O containing 20 mM tris buffer pH 7.4 and 100 mM NaCl at 20 C. In this work, we take advantage of buoyancy to increase dramatically the kinetics of nanodisc layer formation by injecting a H2O-based nanodisc stock solution into the D2O subphase. Reflectivity measurements were performed 15067

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Table 2. Parameters Used in the Fit for h/d-ND under the Insoluble Cationic Film in D2O at Steady-State Conditions, As Shown in Figure 4a F (10
6 Å
2)

layer


0.4 ( 0.1h‑ND

Lsurfactant Lsolvent

1.8 ( 0.7 6.36

Lheads

2.2

ϕ (v/v%)

d (Å) 18 ( 2

Awet (Å2)

0
10c

δ (Å)b
d

d‑ND

15.0 ( 1.5 6.2 ( 1.5

8(1

100 57 ( 2.5h‑ND

9428 ( 608h‑ND

4.5 ( 3.5

77 ( 4d‑ND 28.5 ( 1.5

h‑ND

Lcore

0.96 4.8

Lheads

34 ( 4h‑ND 65 ( 7d‑ND

d‑ND

6.2 ( 1.5

2.2

57 ( 2.5h‑ND

4.5 ( 3.5 16 344 ( 4086d‑ND 4.5 ( 3.5

77 ( 4d‑ND

The corresponding surface area coverage and surface mass are 35 ( 7% and 148 ( 30 ng/cm2 for d-ND and 66 ( 4% and 279 ( 17 ng/cm2 for h-ND. The total thickness of the nanodisc layer is 40.9 ( 2.6 Å. b The cationic surfactant
air interface had a roughness of 8 ( 3 Å, using the d-ND contrast. c The solvent penetration for the cationic film was assumed to be negligible given the high surface pressure achieved. d The fit is highly insensitive to the roughness of this layer. a

Table 3. Parameters Allowed To Fluctuate in the Best Fits Shown in Figure 4 for the Various Kinetic Neutron Reflectivity Measurements in Which h-ND Was Injected under the Insoluble Cationic Film in D2Oa ϕ (v/v %)

Lheads Lcore Lheads

Awet (Å2) δLsurfactant (Å)

15 min

30 min

60 min

65 ( 3.5 46 ( 5

62 ( 3.5 42 ( 5

58 ( 3 36 ( 4

65 ( 3.5 11 553 ( 1176 5(2

62 ( 3.5 10 728 ( 1012 7(2

58 ( 3 9722 ( 1012 8(2

The corresponding surface area coverage is 54 ( 5%, 58 ( 5%, and 64 ( 4% for h-ND after 15, 30, and 60 min, respectively. The scattering length density of Lsurfactant was kept constant at
0.4  10
6 Å
2. a

on the FIGARO time-of-flight neutron reflectometer at the ILL Facility, Grenoble, France. Measurements were carried out at two incident angles (0.624 and 3.78) and a wavelength range of 2 Å < λ < 30 Å, which covers a Q range from 0.005 to 0.42 Å
1. Reflectivities down to 10
6 relative to the incident beam could be measured in 30 min up to q ≈ 0.25 Å
1 due to the incoherent background from the bulk liquid subphase. The beam collimation slits were set at both angles so as to ensure constant illuminated length of the sample and constant resolution taking into account the wavelength resolution used (9.8% dλ/λ). These parameters ensured that about 35 cm2 of the water surface area was illuminated; thus, there is no ambiguity about representative sampling of the interface. Measurement times required 10
30 min for all of the Q-range probed. Some additional measurements were performed on the liquids reflectometer at the Spallation Neutron Source at the Oak Ridge National Laboratory, over a similar Q range using a sequence of incident angles (0.47
4) and wavelength bands (2.5
12 Å).

’ RESULTS AND DISCUSSION We planned to form a monolayer of nanodiscs just below the air
water interface taking advantage of electrostatic forces between negatively charged nanodiscs and a positively charged insoluble surfactant film. In the absence of the cationic surfactant layer, addition of nanodiscs only leads to the formation of a phospholipid monolayer at the air
water interface as indicated by the neutron reflectivity profile shown in Figure 2. When nanodiscs containing only the neutral phospholipid DMPC are injected below the insoluble cationic surfactant film (compressed

Figure 2. Neutron reflectivity for nanodiscs made of pure DMPC in the absence (green) and presence of a cationic surfactant layer after 30 (red) and 60 min (blue) of nanodisc injection. The black data correspond to the cationic surfactant monolayer on the aqueous solution in the absence of ND. The inset shows the scattering length density, F, profile for a monolayer fit to the cationic surfactant monolayer at the air
water interface. Data were recorded in the Figaro beamline, ILL.

to the surface pressure Π = 25 mN/m, see a typical isotherm in Figure 3), no major changes in the neutron reflectivity profile for the cationic film are observed even 1 h after nanodisc injection. This profile can be fitted to a single surfactant layer model, and the F(z) profile is given in the inset of Figure 2. We conclude that no nanodisc film is formed when using only zwitterionic lipids. Thus, the observed increase in surface pressure from 25 to 31 mN/m upon nanodisc injection (see inset Figure 3) is not related to adsorption of nanodiscs below the surfactant monolayer (as the reflectivity does not change) but, instead, should be due to transfer of phospholipids from the nanodisc sample into the surfactant monolayer at the surface. Indeed, the incorporation of lipids at the surface is confirmed when deuterated lipids are used in the nanodiscs as discussed below regarding Figure 4a. The lipid incorporation is initially energetically favorable, because the electrostatic repulsion within the cationic film is decreased by incorporation of the zwitterionic DMPC that is also accompanied by an entropic gain due to mixing. The initially fast increase in surface pressure slows about 15 min after nanodisc injection as the monolayer becomes saturated with molecules (inset in Figure 3). Assuming that the incoming phospholipids occupy the same mean molecular area as the DODAB molecule at the 15068

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Figure 3. Surface pressure
molecular area isotherm for the cationic surfactant film on 20 mM tris pH 7.4, 100 mM NaCl in D2O. Inset: The cationic surfactant film is compressed to 25 mN/m and the Π evolution upon injection of nanodiscs made of DMPC (blue) and DMPC/DMPG to a molar ratio 3:1 (red) below the interface.

interface, the decrease in mean molecular area corresponding to the increase in surface pressure from 25 to 31 mN/m would imply that only 1% of the phospholipids from the injected nanodisc sample actually incorporates into the surface monolayer. Thus, migration of phospholipids from the nanodiscs does not threaten the stability of the nanodisc complex. Figure 4a gives neutron reflectivity profiles after injection of anionic h-ND below a cationic surfactant film on 20 mM tris buffer pH 7.4 enriched with 100 mM NaCl, in which the surface area is kept constant after the surface pressure reached 25 mN/m. Upon nanodisc injection, the neutron reflectivity profile dramatically differs from that of the pure cationic surfactant already after 15 min, when a clear minimum appears at Q = 0.05 Å
1. With time, the reflectivity features become more pronounced, although the periodicity of the fringes remains constant (Figure 4), until about 60 min after injection after which only minimal changes in reflectivity are observed. The surface pressure Π (inset in Figure 3) stabilizes about 10 min after injection, while it takes 60 min for the reflectivity to stabilize. This indicates that the saturation of the surfactant layer with lipids occurs quicker than the saturation of the ND layer as expected on the basis of diffusion. The slow kinetics regime is probably due to the saturation of the surface sites (and thus a decrease of the free energy of adsorption per nanodisc with time) or perhaps to some reorganization of the surfactant monolayer to optimize the interactions with the underlying nanodiscs layer. Similar issues have been discussed earlier for the adsorption of cholera toxin on a lipid monolayer, although in this case the toxin had an expanding effect on the lipid monolayer.33 The reflectivity profiles shown in Figure 4a were fitted to a five-layer interfacial model, as illustrated in Figure 1b, in which there is a water layer with thickness, dLsolvent, between the cationic surfactant (Lsurfactant) and the nanodisc layer (modeled by three layers Lheads, Lcore, and Lheads). Figure 4b gives the scattering length density F(z) profiles for the best fits shown in Figure 4a. To validate the model used, a second contrast was used in which d-DMPC was used instead of h-DMPC (d-ND), and the NR data for steady-state conditions are also given in Figure 4a. This contrast has less structural detail features in the reflectivity profile, giving less detail about the nanodiscs. However, this contrast is more sensitive to the cationic surfactant layer, especially if this becomes enriched with deuterated phospholipids from the nanodiscs solution. For simultaneous fitting of both contrasts, all

Figure 4. Kinetic data for the formation of nanodisc layer under the cationic film at the air
D2O interface: (a) neutron reflectivity profiles and (b) scattering density profiles after 15 (purple), 30 (black), 45 (pink), and 105 (green) min of h-ND injection. The figure also includes a measurement for d-ND 15 (orange) and 105 min (blue) after d-ND injection. Data were recorded in the Figaro beamline, ILL.

parameters were kept constant except FLsurfactant, ϕLsurfactant, ϕLheads, ϕLcore, and dLsolvent. Given the differences in the isotopic composition of DMPC (deuterium substitution within the alkyl chain only) and assuming deuterated lipid incorporation in Lsurfactant, FLsurfactant and FLcore are significantly different for the two contrasts (see Table 1). The structural parameters obtained from the best fits shown in Figure 4 are listed in Tables 2 and 3. The changes in neutron reflectivity profile and Π seem to indicate that two different processes occur after injection of negatively charged nanodiscs: increase in the total thickness of the interfacial layer due to nanodisc film formation, and, at the same time, changes in the surfactant layer structure at the air
water interface. These results allow us to confirm that the cationic surfactant layer is able to drive the formation of a negatively charged nanodisc film below the air
water interface, in a similar way as it attracts negatively charged polymers.18,29,34 The main characteristics of the scattering length density profiles after nanodisc injection can be summarized as follows: (1) The cationic surfactant layer is enriched with phospholipids from the nanodisc solution. This explains both the increase in Π and the observed increase in scattering length density F and roughness, δ, especially noticeable in the d-ND contrast. The incorporation of phosphatidylcholine molecules into this layer leads to a change of δ in Lsurfactant from 4 ( 1 Å (Figure 2) to 8 ( 1 Å (Figure 4 and Table 2) prior and after nanodiscs injection. This may be due mainly to two reasons: there is a mismatch between the tail lengths of the phospholipid and surfactant, and there is a large difference in F for the lipids chain and tails 15069

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Figure 5. The experimentally observed neutron reflectivity data for a h-ND (A) and d-ND (B) and the simulated reflectivities for a nanodisc layer just below (model 1, blue line) and 15 Å below (model 2, green line) the surfactant layer. The figure also includes a reflectivity profile calculated for a model in which an additional nanodisc layer (model 3, purple line) was allowed in direct contact to the surfactant layer having only 10% surface area coverage, another in which the nanodiscs aligned themselves with their long axis perpendicular to the surfactant layer (model 4, black line), and finally where the nanodiscs are slightly tilted along the long axis (model 5, red line). From the figure, it is obvious that the best fits correspond to the well-aligned nanodiscs layer 15 Å below the interface.

(see Table 1). From Figures SI4,5, it is clear that the effects of Fsurfactant and δsurfactant on the reflectivity profiles are opposing each other, so an alternative explanation is that the mixed surfactant
lipid film is rougher than 8 Å and has less deuterated lipids within it. The chosen roughness for the surfactant layer (8 Å) is physically reasonable because the expected length difference between the surfactant and lipid molecules used in this work is about 8
10 Å. (2) There is a solvent (water) layer between the nanodiscs and the cationic surfactant with a thickness of 15.0 ( 1.5 Å. Interestingly, the thickness of this water layer corresponds well with that found for bulk lamellar phases or multilamellar vesicles of DMPG and DMPC, where typical D-spacings of 62 Å for DPPG35 and 48 Å for DMPC 36 are observed at similar ionic conditions and above the lipid melting temperature, Tm. This D-spacing would correspond approximately to a solvent layer of 12 and 10 Å for DPPG and DMPC, respectively. Hydration forces are known to determine the D-spacing in lamellar phases, giving larger repulsive forces for mixed lipid systems than any of the individual systems (for a review, see refs 37,38), which is in agreement with the observed thickness of the water layer between the cationic film and  et al.39 the nanodiscs film. On the other hand, Stahlberg showed that for planar plates bearing surface charge densities with different signs, an equilibrium distance between the plates larger than zero was found if the absolute values of the surface charge density differ. Differences in the overall charge densities of the nanodiscs layer and the surfactant layer could also contribute to the observed total thickness of the water layer. (3) The nanodiscs are aligned in a configuration in which the lipid bilayer is parallel to the cationic surfactant monolayer. As the fitted roughness of this layer, 4.5 ( 3.5 Å, is essentially the same as that of the air
water interface (determined by capillary waves to have a minimum amplitude of 3 Å),40 we conclude that the nanodiscs layer is well organized. Furthermore, the obtained roughness validates the choice of number of layers to model the nanodisc film. In addition, a single nanodiscs layer model cannot satisfactorily represent the reflectivity data unless a

physically unrealistic high value of the scattering length density of the surfactant layer is used. Such value would imply that there are more molecules (lipids and surfactants) than those that could be accommodated at the air
water interface without causing the film to collapse. We have no experimental evidence for film collapse, and thus this is not a possible scenario. For the best fit shown in Figure 4b, the total thickness of the nanodisc layer (40.9 ( 2.6 Å) corresponds well to the height of the DMPC lipid bilayer, which has been reported to be 40 Å below Tm41 and 36 Å above Tm.42 The observed thickness of the lipid headgroups (6.2 ( 1.5 Å) is in accordance with typical dimensions of phospholipid head groups found for various phospholipid bilayers: for DMPC 5 Å at 10 C41 and 8 Å at 29 C43 in pure water or 6 Å for DOPC and POPC at 25 C44 and ionic conditions similar to the ones used in this work. To show that the model described above is unique at describing the reflectivities measured for both deuterated and hydrogenated nanodiscs, we have calculated the reflectivities for alternative models, and the results are summarized in Figure 5. The data in the figure are the same as for Figure 4 at 105 min but represented as R*Q4 versus Q for h-ND (A) and d-ND (B) as to maximize the differences in the models specially for d-ND data. In the alternative models, the nanodiscs align with their long axis parallel to the interface but in direct contact with the surfactant layer (model 1), parallel to the interface but 15 Å below the surfactant layer (model 2), parallel to the interface and 15 Å below to the interface with an additional more diluted layer of nanodiscs in direct contact with the surfactant layer (model 3), perpendicular to the interface (model 4), and slightly tilted along the long axis (model 5). From the figure, it is clear that the best fits correspond to the well-aligned nanodiscs layer located 15 Å below the interface. However, this model could allow discs with a surface area coverage of 2% located in direct contact with the surfactant layer (model 3) without significantly affecting the quality of the fit. A detailed analysis of the time-dependent data for h-ND shows that with time the cationic surfactant layer becomes rougher as compared to the cationic surfactant film prior to nanodisc injection (Figures 2, 4 and Table 3) and more nanodiscs are incorporated into the nanodisc monolayer. The data for d-ND show 15070

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Langmuir

Figure 6. Differential calorimetric scan for the main melting transitions of hydrogenated (red) and deuterated (blue) DMPC-containing nanodiscs.

that the lipids are incorporated into the surfactant monolayer already during the first neutron reflectivity measurement (15 min). Additionally, the nanodisc layer is also formed quickly. The surface area coverage for the nanodiscs layer, that is, the cross-sectional area occupied by the nanodiscs relative to a close-packed arrangement of the nanodiscs in Lcore, is found to be ∼35% and 54% already 15 min after injection of d-ND and h-ND, respectively. However, more h-ND is slowly incorporated into the monolayer until 66 ( 4% of the full surface area coverage is reached. For d-ND, however, there are no major changes in the reflectivity profile after 15 min as the neutron reflection data are practically indistinguishable from the data recorded after 95 min (shown in Figure 4a), giving a surface area coverage of 35 ( 7%. Although both the d-ND and the h-ND show similar initial adsorption kinetics, the maximum coverage observed seems to significantly differ for the two types of nanodiscs. A systematically lower ϕ (and surface area coverage) is found for d-ND as compared to h-ND: the area available/molecule is 42% lower for the h-ND than the d-ND and corresponds to a surface area coverage of 35% and 66% for d-ND and h-ND, respectively. One major difference between deuterated and hydrogenated phospholipids is their main phase transition temperatures, Tm,26 which is about 2 C lower per hydrocarbon tail. DSC measurements on this system (Figure 6) showed that Tm is lower for d-ND (24.6 C) than for h-ND (28.0 C) at identical conditions, and thus a larger part of the lipids in the deuterated nanodiscs might be in the fluid phase at the experimental conditions used in this work (20 C). If the lipids within the d-ND are in the fluid state and thus the nanodiscs layer is thinner (37 Å as for supported lipid bilayers42), then the scattering length density calculated for the nanodiscs layer is not correct, implying that the surface coverage in this layer (Table 2) was underestimated by ∼37%. However, fitting a thinner nanodisc layer implies that both the water layer thickness and the nanodiscs layer interfacial roughness have to increase by 4 Å. We believe that a change in the nanodiscs layer thickness to 37 Å cannot explain the difference in surface coverage found for d-ND as compared to h-ND, given that only a 10% increase in the diameter has been measured by SANS/SAXS.21 Another source of error in the fits is the assumption that there are 160 lipids per nanodisc, which would change FLcore and thus jLcore. A variation of 160 ( 10 translates to a change of (0.03  10
6 Å2 in FLcore and gives only a small difference in the surface coverage of nanodiscs ((0.6% units). It is also possible that the composition of the phospholipids in the disk is not exactly the nominal composition for nanodisc preparation, which could explain some of the difference in surface

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coverage. For example, if the d-ND were composed by 9 mol % DMPG instead of the nominal composition of 25 mol %, then the surface coverage in the d-ND layer would be the same as that for h-ND. Deuteration effects have been observed earlier, for instance, in lipid
protein interactions when using D2O or H2O under otherwise identical conditions that are reflected by dissimilar surface area coverages.22 Nevertheless, the maximum coverage is considerably lower than the theoretical surface area coverage of a closed 2D packing of round or elliptical circles (91% for hexagonal packing). One possible explanation for such lower surface coverage is the electrostatic repulsion between the sides of the nanodiscs given that the belt protein carries a net charge in solution.45,46 Moreover, the MSP1D1 used in this experiment features a poly histidine tag linked to the N-terminal region. This sequence is flanking the nanodiscs and can be described as a flexible chain in Gaussian random coil conformation32 and may also prevent a close-packed arrangement. Interestingly, for the dimensions of the trough used (30 cm, 10 cm, 6 mm) and using the surface mass given in Table 3, the total adsorbed mass of nanodiscs can be calculated for h-ND (∼84 μg) and d-ND (∼44 μg). This is about 14
23% of the total amount of nanodiscs in the system (∼350 and ∼330 μg for h-ND and d-ND, respectively) and considerably larger in proportion to that found in most typical thermodynamically stable adsorption process at area to volume ratios similar to the ones used in this work (