Adsorption Of Lipid Vesicles Research Articles

Interfacial approach to fabricate covalently and noncovalently attached inverse-phosphocholine supported lipid bilayers on TiO2 and SiO2 surfaces

Inverse-phosphocholine lipids such as DOCP are intriguing biomolecules for surface functionalization because they can form supported lipid bilayers (SLBs) on titania surfaces but adsorb weakly on silica surfaces. Interestingly, these trends are nearly opposite to those of conventional phosphocholine lipids, motivating deeper investigation of how environmental parameters affect DOCP lipid vesicle adsorption phenomena. Herein, we systematically investigated how solution pH (4, 6, 8, 10) and ionic strength (50, 150, 250 mM NaCl) influence DOCP lipid vesicle adsorption behavior on titania and silica surfaces. On titania, DOCP lipid vesicles either adsorbed and ruptured to form a covalently attached SLB (acidic pH), adsorbed but did not rupture (basic pH with high salt), or did not adsorb (basic pH with low salt). Conversely, on silica, a narrow range of acidic pH, high-salt conditions triggered DOCP lipid vesicle adsorption and rupture to form a noncovalently attached SLB whereas negligible adsorption occurred in other conditions. The corresponding energetics of the vesicle-surface interaction and lipid attachment properties were analyzed and clarify how the interplay of solution pH and ionic strength modulates DOCP lipid vesicle adsorption behavior. Based on these insights, we identified suitable strategies to fabricate covalently and noncovalently stabilized inverse-phosphocholine lipid bilayers on oxide surfaces.

Modulating noncovalent and covalent forces to control inverse phosphocholine lipid self-assembly on inorganic surfaces: Nanoarchitectonic design principles

While noncovalent forces typically drive lipid vesicle adsorption and rupture to form supported lipid bilayer (SLB) coatings on inorganic surfaces for various material science applications, this strategy only works on a few materials with suitable energetics such as SiO2. The use of coordination chemistry between inverse phosphocholine (PC) lipid headgroups and surfaces has emerged as a promising strategy to enable SLB formation on other materials such as TiO2 based on covalent forces. However, until now, a cohesive picture of how noncovalent and covalent forces jointly contribute to the SLB formation process on TiO2 has been lacking and addressing this gap is important to design functional lipid biointerfaces with tailored properties such as antifouling capabilities. Herein, we investigated inverse PC lipid vesicle adsorption onto TiO2 and SiO2 surfaces and discovered how adsorption pathways can be controlled by tuning the balance of noncovalent and covalent forces, which enabled us to establish molecular design rules to fabricate physically robust SLB coatings on macroscopically flat TiO2 surfaces. On TiO2, SLB formation depended on two key factors: (1) favorable noncovalent forces to facilitate initial vesicle adsorption; and (2) a critical density of lipid-TiO2 covalent bonds to enable sufficient vesicle deformation triggering fusion and rupture. In other cases, either no adsorption or intact vesicle adsorption without rupture occurred even when covalent bonds were present. Conversely, on SiO2, conditions were identified to support inverse PC lipid adsorption whereas vesicles were repelled otherwise. The experimental results were supported by interfacial force modeling and our findings demonstrate how a subtle interplay of noncovalent and covalent forces plays a deterministic role in modulating lipid self-assembly pathways. Specific conditions in which the physically stable, fabricated SLBs on TiO2 surfaces exhibit antifouling properties were also identified based on optimizing the lipid composition to enhance vesicle-surface interactions while preventing other nonspecific interaction events.

Mechanical and Electrical Interaction of Biological Membranes with Nanoparticles and Nanostructured Surfaces.

In this review paper, we theoretically explain the origin of electrostatic interactions between lipid bilayers and charged solid surfaces using a statistical mechanics approach, where the orientational degree of freedom of lipid head groups and the orientational ordering of the water dipoles are considered. Within the modified Langevin Poisson–Boltzmann model of an electric double layer, we derived an analytical expression for the osmotic pressure between the planar zwitterionic lipid bilayer and charged solid planar surface. We also show that the electrostatic interaction between the zwitterionic lipid head groups of the proximal leaflet and the negatively charged solid surface is accompanied with a more perpendicular average orientation of the lipid head-groups. We further highlight the important role of the surfaces’ nanostructured topography in their interactions with biological material. As an example of nanostructured surfaces, we describe the synthesis of TiO2 nanotubular and octahedral surfaces by using the electrochemical anodization method and hydrothermal method, respectively. The physical and chemical properties of these nanostructured surfaces are described in order to elucidate the influence of the surface topography and other physical properties on the behavior of human cells adhered to TiO2 nanostructured surfaces. In the last part of the paper, we theoretically explain the interplay of elastic and adhesive contributions to the adsorption of lipid vesicles on the solid surfaces. We show the numerically predicted shapes of adhered lipid vesicles corresponding to the minimum of the membrane free energy to describe the influence of the vesicle size, bending modulus, and adhesion strength on the adhesion of lipid vesicles on solid charged surfaces.

Changes in membrane elasticity caused by the hydrophobic surfactant proteins correlate poorly with adsorption of lipid vesicles.

To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used X-ray diffuse scattering (XDS) to determine an order parameter of the lipid chains (Sxray) and the bending modulus of the lipid bilayers (KC). Samples contained different amounts of the proteins with two sets of lipids. Dioleoylphosphatidylcholine (DOPC) provided a simple, well characterized model system. The nonpolar and phospholipids (N&PL) from extracted calf surfactant provided the biological mix of lipids. For both systems, the proteins produced changes in Sxray that correlated well with KC. The dose-response to the proteins, however, differed. Small amounts of protein generated large decreases in Sxray and KC for DOPC that progressed monotonically. The changes for the surfactant lipids were erratic. Our studies then tested whether the proteins produced correlated effects on adsorption. Experiments measured the initial fall in surface tension during adsorption to a constant surface area, and then expansion of the interface during adsorption at a constant surface tension of 40 mN m-1. The proteins produced a sigmoidal increase in the rate of adsorption at 40 mN m-1 for both lipids. The results correlated poorly with the changes in Sxray and KC in both cases. Disordering of the lipid chains produced by the proteins, and the softening of the bilayers, fail to explain how the proteins promote adsorption of lipid vesicles.

Stimulations inducing tethered lipid bilayer formation on protein‐modified substrate

AbstractSupported lipid bilayer (SLB) is one artificial biomembrane system used to study transportation reactions through biomembranes using solid devices and surface scientific methods. A key step in the SLB formation is the transformation of spherical lipid vesicles to a planar membrane. We investigated stimulation methods for adsorbed lipid vesicles on a streptavidin‐modified SiO2/Si substrate to induce tethered‐type SLB. We describe the effects of osmotic stress, Ca2+ addition, and a freeze–thaw cycle on the adsorbed vesicles, and on the condition of t‐SLB.

Interfacial water at synthetic and natural lipid bilayers probed by vibrational sum-frequency generation spectroscopy

Background: At lipid interfaces, water plays a crucial role in carrying biological processes, so that there is a huge interest in unravelling the behaviour of water close to membranes. At charged bio-interfaces, water dipoles form an organized layer. Probing such an interfacial thin layer buried between macroscopic bulk environments is a real challenge. Vibrational sum frequency generation (SFG) spectroscopy is intrinsically specific to interfaces, and has already proven to be an ideal tool to investigate model membranes and their surrounding water. Objectives: The goal of this work is to measure the vibrational SFG response of interfacial water around different model membranes — from easiest synthetic lipids to more complex natural lipids, — in order to use it as diagnostic signal able to distinguish the lipid bilayer interface by its charge properties. Materials and methods: Lipid bilayers made either of synthetic or natural lipids (Avanti Polar Lipids) were physisorbed on CaF2 prisms (Crystran), by using the method of the spontaneous fusion of lipid vesicles, to form so called solid-supported lipid bilayers (SSLBs). The model membranes were investigated by SFG spectroscopy at the solid/water interface. Results: The SFG response was measured between 3600 cm-1 and 2800 cm-1, where OH stretching vibrations of water molecules show-up. The SFG intensity of the OH peak maximum at 3125 cm-1 was recorded during the adsorption of lipid vesicles on the surface, and provided knowledge of the changes of the charge properties of the interface due to the adsorption of the model membranes. The SFG signal indicated that the organization of water was larger at negatively charged than at positively lipid interfaces, and reached the highest value with natural E. сoli сardiolipin layers. Moreover, when the full composition of natural lipids was unknown, the behaviour of the SFG response enabled establishing the charge characteristics of the corresponding lipid interfaces. Conclusion: The SFG response of water enabled estimating average charge behaviour of synthetic and natural lipid bilayers in pure water, thus paving the way to use the SFG signal of water as new diagnostic tool to identify lipid interfaces.

A Comparative Study of Phosphatidylcholine versus Phosphatidylserine-Based Solid Supported Membranes for the Preparation of Liposome-Rich Interfaces.

Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.

Time-Resolved Neutron Reflectivity during Supported Membrane Formation by Vesicle Fusion

The formation of supported lipid bilayers (SLB) on hydrophilic substrates through the method of unilamelar vesicle fusion is used routinely in a wide range of biophysical studies. In an effort to control and better understand the fusion process on the substrate, many experimental studies employing different techniques have been devoted to the elucidation of the fusion mechanism. In the present work, we follow the kinetics of membrane formation using time-resolved (TR) neutron reflectivity, focusing on the structural changes near the solid/liquid interface. A clear indication of stacked bilayer structure is observed during the intermediate phase of SLB formation. Adsorbed lipid mass decrease is also measured in the final stage of the process. We have found that it is essential for the analysis of the experimental results to treat the shape of adsorbed lipid vesicles on an attractive substrate theoretically. The overall findings are discussed in relation to proposed fusion mechanisms from the literature, and we argue that our observations favor a model involving enhanced adhesion of incoming vesicles on the edges of already-formed bilayer patches.

Influence of Divalent Cations on Deformation and Rupture of Adsorbed Lipid Vesicles.

The fate of adsorbed lipid vesicles on solid supports depends on numerous experimental parameters and typically results in the formation of a supported lipid bilayer (SLB) or an adsorbed vesicle layer. One of the poorly understood questions relates to how divalent cations appear to promote SLB formation in some cases. The complexity arises from the multiple ways in which divalent cations affect vesicle-substrate and vesicle-vesicle interactions as well as vesicle properties. These interactions are reflected, e.g., in the degree of deformation of adsorbed vesicles (if they do not rupture). It is, however, experimentally challenging to measure the extent of vesicle deformation in real-time. Herein, we investigated the effect of divalent cations (Mg(2+), Ca(2+), Sr(2+)) on the adsorption of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles onto silicon oxide- and titanium oxide-coated substrates. The vesicle adsorption process was tracked using the quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) measurement techniques. On silicon oxide, vesicle adsorption led to SLB formation in all cases, while vesicles adsorbed but did not rupture on titanium oxide. It was identified that divalent cations promote increased deformation of adsorbed vesicles on both substrates and enhanced rupture on silicon oxide in the order Ca(2+) > Mg(2+) > Sr(2+). The influence of divalent cations on different factors in these systems is discussed, clarifying experimental observations on both substrates. Taken together, the findings in this work offer insight into how divalent cations modulate the interfacial science of supported membrane systems.

Mechanism of rupture of single adsorbed vesicles

Rupture of single adsorbed lipid vesicles is believed to occur via pore formation in the membrane. The latter process is related to the support-induced membrane strain. It includes membrane bending primarily near the rim of the vesicle-substrate contact area and tension distributed over the whole membrane. Herein, the relative role of these two factors is scrutinized from the perspectives of general theory and available experiments. The former factor is concluded to dominate.

Nanoplasmonic biosensing for soft matter adsorption: kinetics of lipid vesicle attachment and shape deformation.

An indirect nanoplasmonic sensing platform is reported for investigating the kinetics of attachment and shape deformation associated with lipid vesicle adsorption onto a titanium oxide-coated substrate. The localized surface plasmon resonance (LSPR) originates from embedded gold nanodisks and is highly sensitive to the local lipid environment. To interpret the corresponding results, we have extended treatments of diffusion-limited adsorption kinetics and adsorbate-related LSPR physics, identified the expected scaling laws for the LSPR-tracked kinetics measured at different lipid concentrations and/or nanometer-scale vesicle sizes in the case when vesicle deformation is negligible, and scrutinized experimental deviations accordingly. After adsorption, the smallest 58 nm diameter vesicles were found to maintain shape on the time scale of adsorption at high lipid concentrations in solution, and shape deformation became more appreciable at lower lipid concentrations. Higher saturation coverage was observed with increasing lipid concentration, which is attributed to the difference in relative time scales of vesicle attachment and deformation. For larger vesicles between 80 and 160 nm diameter, deviations associated with their shape deformation and correlations with the location of gold nanodisks became more apparent at moderate and high coverages. Taken together, the results obtained support that the quantitative measurement capabilities of nanoplasmonic biosensing should be considered for applications demanding highly surface-sensitive characterization of soft matter adsorption and related phenomena at liquid-solid interfaces.

Lipid Nanodomains on Modified Gold Surfaces - A Biomimetic Platform to Study Electroactive Biomolecule-Membrane Interactions

Biomimetic platforms based on supported lipid bilayers (SLB) on gold are becoming prominent due to their wide applications in biosensor developing and redox biology studies. A recent innovation was the use of multicomponent lipid bilayers with phase coexistence (Marques et al. 2013). Here, the formation of ternary (DOPC/DPPC/Cholesterol 2:2:1) SLBs exhibiting lipid rafts in modified gold will be described. Surface modifications are required to confer hydrophilicity to gold facilitating the formation of a planar and uniform bilayer. Two surface modifications, 11-mercaptoundecanoic acid (MUA) which generates a long and compact self-assembled monolayer (SAM) and L-cysteine (Cys) which forms a short and non-compact SAM, were used. Similar kinetics was observed for lipid vesicle adsorption on both SAM by surface plasmon resonance and quartz crystal microbalance. On both modified electrodes a continuous lipid bilayer with topographical details assigned to lipid rafts was imaged by atomic force microscopy. Moreover the thickness estimated by ellipsometry lies in the range expected for a bilayer.Since the compact MUA monolayers hinder the electronic exchange between electroactive molecules and the gold electrode, Cysteine emerges as a suitable solution to develop lipid-based interfaces to study electrochemical phenomena. Cys-modified electrodes with raft-containing SLB were employed to study membrane-interactions of a redox active biomolecule, epinephrine. Cyclic voltammetric results revealed an influence of lipid composition on the membrane-adsorbed epinephrine. Interestingly, fluorescence spectroscopy revealed a rather weak interaction between epinephrine and lipid bilayers in suspension, highlighting the applicability of the platform cys-SLB to investigate and detect membrane-associated processes with high sensitivity.Marques, JT, de Almeida, RFM, Viana, AS, 2013, Electrochim. Acta (in press).This work was financed by Portuguese national funds through FCT: Ph.D. fellowship: SFRH/BD/64442/2009, grant PEst-OE/QUI/UI0612/2013, and IF2012 initiative (POPH, Fundo Social Europeu).

Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous Silicon Using Reflectance Interferometric Fourier Transform Spectroscopy

The ability to observe interactions of drugs with cell membranes is an important area in pharmaceutical research. However, these processes are often difficult to understand due to the dynamic nature of cell membranes. Therefore, artificial systems composed of lipids have been used to study membrane properties and their interaction with drugs. Here, lipid vesicle adsorption, rupture, and formation of planar lipid bilayers induced by various antibiotics (surfactin, azithromycin, gramicidin, melittin and ciprofloxacin) and the detergent dodecyl-b-D-thiomaltoside (DOTM) was studied using reflective interferometric Fourier transform spectroscopy (RIFTS) on an oxidized porous silicon (pSi) surface as a transducer. The pSi transducer surfaces are prepared as thin films of 3 μm thickness with pore dimensions of a few nanometers in diameter by electrochemical etching of crystalline silicon followed by passivation with a thermal oxide layer. Furthermore, the sensitivity of RIFTS was investigated using three different concentrations of surfactin. Complementary techniques including atomic force microscopy, fluorescence recovery after photobleaching, and fluorescence microscopy were used to validate the RIFTS-based method and confirm adsorption and consequent rupture of vesicles to form a phospholipid bilayer upon the addition of antibiotics. The method provides a sensitive and real-time approach to monitor the antibiotic-induced transition of lipid vesicles to phospholipid bilayers.

Formation and Diffusivity Characterization of Supported Lipid Bilayers with Complex Lipid Compositions

The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO(2). By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.

Lipid-polymer hybrid nanoparticles with rhamnolipid-triggered release capabilities as anti-biofilm drug delivery vehicles

In lung biofilm infection therapies, the use of lipid-polymer hybrid nanoparticles to encapsulate drugs has emerged as a promising alternative to using liposomes because they have superior physicochemical stability and still possess the biofilm affinity and sputum-penetrating ability of liposomes. To be deemed equally efficacious as liposomes against bacterial biofilms, however, the capability of hybrid nanoparticles to target-release encapsulated drugs at biofilm colonies must be demonstrated. This communication details our investigations into the trigger-release characteristics of hybrid nanoparticles in response to encountering rhamnolipids, which are ubiquitously present in biofilm colonies of Pseudomonas aeruginosa, a major respiratory pathogen. Poly(lactic-co-glycolic acid) and phosphatidylcholine were used as the polymer nanoparticle core and lipid coat, respectively. These investigations were performed using compounds from various biopharmaceutical classification systems (BCS) that differ in their lipid-membrane permeabilities. The release of BCS Class III compounds, which have poor lipid-membrane permeabilities, was successfully triggered by rhamnolipids at a concentration approximately equal to their clinically observed value, and this release was attributed to the disruption of lipid coats by rhamnolipid micelles. Not unexpectedly, BCS Class I compounds, which have high lipid-membrane permeabilities, were released freely whether or not rhamnolipids were present. The rate of the triggered release can be controlled by incorporating an additional lipid layer on the hybrid nanoparticles via the electrostatically driven adsorption of lipid vesicles.

Lipid vesicle adsorption on micropore arrays prepared by colloidal lithography-based deposition approaches

We present a new method for obtaining patches of fluid lipid membranes by patterning zinc oxide nanorods in 2D–3D micropore arrays via colloidal lithography combined with metal organic chemical vapour deposition and chemical bath deposition.

A universal method for planar lipid bilayer formation by freeze and thaw

A procedure based on freezing and thawing was developed to induce the rupture of adsorbed lipid vesicles on solid surfaces into supported lipid bilayers (SLBs). The SLB assembly exploits the phase transition of both lipids and water during freezing. It enables SLB formation independent of the type of substrates and lipids as long as the vesicles spontaneously adsorb onto the surface. The created SLB is a single bilayer, and has a diffusion coefficient of (0.6–4) × 10−8 cm2 s−1 on TiO2, which is in the same range as the SLBs formed by conventional techniques. The presented approach has the advantages of both the Langmuir–Blodgett method (the versatility in the selection of lipids and substrates) and vesicle fusion (self-assembly) at the same time.

Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.

Pore Spanning Lipid Bilayers on Mesoporous Silica Having Varying Pore Size

Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.

Effect of Hydrophobic Surfactant Proteins on the Structure of Oriented Lipid Bilayers

The hydrophobic surfactant proteins, SP-B and SP-C (SP), enhance the adsorption of phospholipids to an air-water interface. Previous evidence suggests that the SP-B forms amphipathic helices that are oriented parallel to the surface of phospholipid bilayers, whereas SP-C forms transmembrane helices. To determine the mechanism by which the SP induce adsorption of lipid vesicles, we measured small-angle X-ray scattering from dioleoyl phosphatidylcholine (DOPC) bilayers aligned on solid supports with the proteins in their physiological ratio. Increasing amounts of protein (0-5%) decreased the difference between maximum and minimum electron density, flattening the electron density profiles (EDP) across the bilayer. This flattening of the EDP with increasing protein concentration has been attributed previously to lateral variation of bilayer thickness. Larger amounts of protein also increased the lamellar d-spacing in the supported films. The EDP explained this increase by a greater head-head distance (dHH) of the membrane. Prior studies have found that amphipathic peptides cause thinning of the dHH in contrast to the thickening of dHH observed with the SP. Our results therefore suggest that the increased dHH observed here is unrelated to the amphipathic SP-B, and instead reflects hydrophobic matching with the SP-C transmembrane helix, the length of which exceeds the thickness of the DOPC bilayer.Acknowledgements: Stanford Synchrotron Radiation Lightsource (SSRL); NIH.