Characterization of Lipid Bilayers Adsorbed to Functionalized 1 Air/Water Interfaces 2

18 Lipid bilayers immobilized in planar geometries, such as solid-supported or 19 "floating" bilayers, have enabled detailed studies of biological membranes with 20 numerous experimental techniques, notably x-ray and neutron reflectometry. 21 However, the presence of a solid support also has disadvantages as it 22 complicates the use of spectroscopic techniques as well as surface rheological 23 measurements that would require surface deformations. Here, in order to 24 overcome these limitations, we investigate lipid bilayers adsorbed to inherently 25 soft and experimentally well accessible air/water interfaces that are functionalized 26 with Langmuir monolayers of amphiphiles. The bilayers are characterized with 27 ellipsometry, X-ray scattering, and X-ray fluorescence. Grazing-incidence X-ray 28 diffraction reveals that lipid bilayers in a chain-ordered state can have significantly 29 different structural features than regular Langmuir monolayers of the same 30 composition. Our results suggest that bilayers at air/water interfaces may be well 31 suited for fundamental studies in the field of membrane biophysics. 32


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On a quantitative level, is proportional to the spatial integral over the product 254 of ( ) and the known intensity profile Φ( ) introduced above,  The coverage of a DMPC-DMTAP bilayer adsorbed to a PFOL-PFOA monolayer 299 was determined in the same way, but with a suitably adapted slab model which 300 considers that the perfluorinated monolayer is -free. As a -containing 301 calibration reference, the DPPE-DSTAP monolayer was again used.  The associated best-matching electron density profile is shown in Fig. 3B. The 414 first layer has a comparatively low electron density and represents the tails of the 415 monolayer ("tm"), while the second layer has a higher electron density and 416 represents the headgroups of the monolayer ("hm"). The best-matching 417 parameters are summarized in Table 1 and are similar to those reported earlier  adsorbed underneath the monolayer. In addition to the "tm" and "hm" slabs 446 introduced before, slabs for the headgroups, tails, and the central methyl dip of 447 the bilayer ("hb", "tb", and "mb", respectively) as well as a water layer "w" between 448 monolayer and bilayer were considered. Note that the "hb" and "tb" layers appear 449 twice in the bilayer for reason of symmetry (see Fig. 4 B). In order to minimize the  Table 2. However, in order 455 to realistically model an imperfect, fluctuating bilayer adsorbed to a monolayer, 456 we further considered a global bilayer roughness, achieved by convolution of the 457 bilayer profile with a Gaussian function of width conv (see Table 3, 1 st column).

458
This was achieved analytically by correcting all bilayer-related slab roughness 459 parameters as corr = √ 2 + conv 2 . Moreover, we allowed for scenarios of     Tables 1 and 2 54 . Importantly, the SW intensity is 531 higher at the monolayer surface than at the bilayer surfaces, which is why the 532 bilayer contributes less to the fluorescence intensity than the monolayer.
In other words, no full bilayer coverage was achieved after 6h, which is consistent 537 with the non-saturated intensity in Fig. 5A and with the ellipsometry results 538 which show that full coverage can take more than 8h. By analyzing the 539 ellipsometry results (Fig. 2), it can even be seen that the apparent bilayer the presence of salt appears to correlate also with an increase in the bilayer 573 roughness (see Table 3), as was previously discussed for solid-supported floating 574 bilayers 15 . 575 576 with and without NaCl. a Known from TXRF measurement and therefore fixed.

579
Error estimates include systematic uncertainties.  the best-matching values for the two remaining parameters were obtained as 615 ≈ 5 Å and conv < 2 Å (see also Table 4). As can be seen, the interstitial water 616 layer is significantly thinner than that of the DPPE-DSTAP-DMPC-DMPG system, 617 which can be explained due to a much weaker hydration repulsion. In fact, it has 618 been already reported that hydration repulsion is of much longer range for 619 zwitterionic phospholipid surfaces than for surfaces bearing OH-groups 61 .

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Besides, a stronger charge attraction can be attributed to a higher charge density 621 in the monolayer. Regarding conv we can assume that its remarkably low value 622 is likely a consequence of the stronger adhesion and the overall higher rigidity of 623 the perfluorinated monolayer.    The GIXD pattern recorded 6 hours after the injection of DMPC/DMTAP SUVs 662 features additional diffraction intensity around = 1.49 A -1 , which can safely 663 be attributed to the formed bilayer (Fig. 7B). The first impression is that this 664 additional intensity is from a single diffraction peak centered at ≈ 0, which 665 would indicate a fully upright hexagonal packing of the lipid tails in the bilayer.

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However, the width of the peak in -direction (FWHM = 0.   were carried out at PETRA III and we would like to thank René Kirchhof (Chen 748 Shen) and Milena Lippmann for assistance in using P08 and chemistry lab,