Probing the Local Order of Single Phospholipid Membranes Using Grazing Incidence X-Ray Diffraction

We report the first grazing incidence x-ray diffraction measurements of a single phospholipid bilayer at the solid-liquid interface. Our grazing incidence x-ray diffraction and reflectivity measurements reveal that the lateral ordering in a supported DPPE (1, 2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine) bilayer is significantly less than that of an equivalent monolayer at the air-liquid interface. Our findings also indicate that the leaflets of the bilayer are uncoupled in contrast to the scattering from free standing phosphatidylcholine bilayers. The methodology presented can be readily implemented to study more complicated biomembranes and their interaction with proteins.

Figure 1

(a) Reflectivity profile for a DPPE bilayer deposited at $45\mathrm{mN}/\mathrm{m}$ at the quartz-${\mathrm{H}}_2\mathrm{O}$ interface and a DPPE monolayer at the ${\mathrm{H}}_2\mathrm{O}$-air interface ($45\mathrm{mN}/\mathrm{m}$). The vertical momentum transfer vector $q_z$ is $4\pi \sin \theta /\lambda$, where $\theta$ is the angle of incidence to the interface and $\lambda$ is the wavelength of x-ray beam. Symbols represent measured data and solid lines are the fits corresponding to the scattering length density profiles in (b). (Inset) Schematic of the experimental cell. (b) Scattering length density and electron density profiles corresponding to the fits in (a) and obtained by box model fitting procedures. The electron density profile for the DPPE monolayer has been oriented for comparison with the outer leaflet of the bilayer.

Figure 2

Diffracted intensity distribution from a single DPPE bilayer membrane on a quartz substrate deposited by L-B/S deposition at $45\mathrm{mN}/\mathrm{m}$. To enhance the visibility of the diffraction peak the image was produced by subtracting the intensity distribution of a bare quartz substrate in contact with water. The projection of the quartz substrate is defined as $q_z=0{\AA }^{-1}$. The scattered intensity along the line slightly above $q_z=0{\AA }^{-1}$ is the Vineyard-Yoneda peak [22]. The Bragg rod at $q_{xy}=1.5{\AA }^{-1}$ extends from the quartz interface up to $q_z\approx 0.25{\AA }^{-1}$ and is due to the lateral ordering of the alkyl chains of DPPE.

Figure 3

Bragg peaks (integrated over $q_z$) from a DPPE bilayer membrane in comparison to the Bragg peaks from a DPPE monolayer at 20, 30 and $45\mathrm{mN}/\mathrm{m}$ at the air-water interface. Monolayers have been offset vertically for clarity. Analysis of the bilayer peaks reveal a hexagonal unit cell of $a_H=4.84\pm 0.01\AA$. Solid lines are fits to the Bragg peaks using Voight functions. The dashed line marks the center of the Bragg peak of the DPPE bilayer.

Figure 4

Bragg rods (integrated over $q_{xy}$) from a DPPE bilayer in comparison to the Bragg rods from a DPPE monolayer at 20, 30 and $45\mathrm{mN}/\mathrm{m}$ at the air-water interface. The sharp peak at $q_z\sim 0{\AA }^{-1}$ is the Vineyard-Yoneda peak. Lines are fits to the models described in the text. Visual inspection of the DPPE bilayer rod reveals similar FWHM as in the monolayer case at $45\mathrm{mN}/\mathrm{m}$. For the coupled model, small changes in the tilt angle ($\pm 1°$) dramatically impact the fit and the calculated Bragg rod FWHM (as shown by the light gray dotted curves).