Grazing Incidence X-ray Diffraction Studies of Lipid-Peptide Mixed Monolayers during Shear Flow

Abstract

Grazing incidence X-ray diffraction (GIXD) studies of monolayers of biomolecules at an air-water interface give quantitative information of in-plane packing, coherence length of crystalline domains, etc. Rheo-GIXD measurements can reveal quantitative changes in the nanocrystalline domains of a monolayer under shear. Here, we report GIXD studies of monolayers of alamethicin peptide, DPPC lipid, and their mixtures at an air-water interface under steady shear stress. The alamethicin monolayer and the mixed monolayer show a flow jamming transition. On the other hand, the pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer under constant stress flows steadily with a notable enhancement of the area/molecule and coherence lengths, suggesting the fusion of nanocrystallites during flow. The DPPC-alamethicin mixed monolayer shows no significant change in the area/DPPC molecule, but the coherence lengths of the individual phases (DPPC and alamethicin) increase, suggesting that the crystallites of individual phases grow bigger by merging of domains. More phase separation occurs in the system during flow. Our results show that rheo-GIXD has the potential to explore in situ molecular structural changes under rheological conditions for a diverse range of confined biomolecules at interfaces.

Figure 1

Schematic of the in situ rheo-GIXD setup, showing the water-filled IRS cell on the rheometer’s Peltier base, the position of the bi-cone on the interface, and the path of the X-ray beam through the Kapton window striking the annular-shaped interface (top). (Bottom left) schematic of the GIXD mechanism: the vertical incidence angle (α_i), the horizontal scattering angle (2θ), and the vertical exit angle (α); in-plane wave vector q_xy ≃ (4π/λ) sin (2θ/2) and out-of-plane wave vector q_z = (2π/λ) (sin α + sin α_i) are shown. (Bottom right) photograph of the experimental setup showing the X-ray source, the rheometer on a z-stage, and the detector assembly attached to the goniometer.

Figure 2

GIXD intensity contours in the (q_xy, q_z) plane and Bragg peaks (I vs q_xy) of the three monolayers, (a) alamethicin, (b) DPPC, and (c) DPPC–alamethicin, are shown under no-shear conditions at 285 K. Solid lines are fits using a Voigt function. In (c), for the bottom Bragg peak, the solid line is the resultant fit with two peaks (blue dotted line and red shaded black dotted line). Color bars represent intensity values in contours.

Figure 3

Flow curve, shear stress (σ) vs shear rate (γ̇), obtained in the controlled shear stress (CSS) mode with a waiting time of 30 s for each data point is shown for the DPPC monolayer and DPPC–alamethicin mixed monolayer at the air–water interface. Solid lines are of slope ∼1. The dotted line is the approximate cutoff of the linear flow region (∼250 μPa·m).

Figure 4

Rheo-GIXD creep data of the alamethicin monolayer (presheared for 200 s followed by a waiting time of 300 s before each measurement; see text): (a) creep curves; shear rate (γ̇) vs time (t) (applied stress σ is mentioned close to the curves), (b) Bragg peaks (I vs q_xy) for different σ values. Solid lines are fits using a Voigt function. The Bragg peak corresponds to the helix pitch of alamethicin. (c) The helix pitch (p) and the coherence length (L_p) are plotted vs σ. Straight horizontal lines represent the average values of p and L_p.

Figure 5

Rheo-GIXD creep data of the DPPC monolayer (presheared for 200 s followed by a waiting time of 300 s before each measurement; see text): (a) creep curves; γ̇ vs t are plotted. Bragg peaks q₀₂ (bottom) and q₁₁ (top) for different σ values of (b) 10 μPa·m, (c) 20 μPa·m, (d) 50 μPa·m, (e) and 100 μPa·m are shown. The peaks are fitted using a Voigt function.

Figure 6

Rheo-GIXD creep data of the DPPC–alamethicin mixed monolayer with the molar ratio P/L = 1:2 (presheared for 200 s followed by a waiting time of 300 s before each measurement; see text): (a) creep curves; γ̇ vs t are plotted. Bragg peaks q₀₂ (blue solid fit) and q₁₁ (black solid fit) and the alamethicin helix peak (red solid fit) for different σ values of (b) 25 μPa·m, (c) 50 μPa·m, (d) 75 μPa·m, and (e) 150 μPa·m are shown. The peaks are fitted using a Voigt function.

Figure 7

q_z-integrated intensity vs q_xy plot for the monolayers during creep flow. The diffraction data from the clean buffer subphase surface are also shown.

Figure 8

(a) Area/molecule of DPPC (A_molecule) and the coherence lengths L₀₂ (b) and L₁₁ (c) corresponding to the Bragg peaks for pure DPPC (open circles) and DPPC–alamethicin mixed (red squares) monolayers are plotted against σ. Dotted curves are guides to the eyes.