Differential Stiffness and Lipid Mobility in the Leaflets of Purple Membranes

Abstract

Purple membranes (PM) are two-dimensional crystals formed by bacteriorhodopsin and a variety of lipids. The lipid composition and density in the cytoplasmic (CP) leaflet differ from those of the extracellular (EC) leaflet. A new way of differentiating the two sides of such asymmetric membranes using the phase signal in alternate contact atomic force microscopy is presented. This method does not require molecular resolution and is applied to study the stiffness and intertrimer lipid mobility in both leaflets of the PM independently over a broad range of pH and salt concentrations. PM stiffens with increasing salt concentration according to two different regimes. At low salt concentration, the membrane Young's normal modulus grows quickly but differentially for the EC and CP leaflets. At higher salt concentration, both leaflets behave similarly and their stiffness converges toward the native environment value. Changes in pH do not affect PM stiffness; however, the crystal assembly is less pronounced at pH > or = 10. Lipid mobility is high in the CP leaflet, especially at low salt concentration, but negligible in the EC leaflet regardless of pH or salt concentration. An independent lipid mobility study by solid-state NMR confirms and quantifies the atomic force microscopy qualitative observations.

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

“…Figure 1B shows a high magnification image of the inner region of the cytoplasmic leaflet captured at 1 frame/sec. The distinction of PM cytoplasmic and extracellular sides was made using the high-resolution AFM image as previously reported (Müller et al, 1996;Voitchovsky et al, 2006). The hexagonal lattice of bR trimers is clearly visible, demonstrating the ability of our HS-AFM apparatus to visualize individual bR molecules even at high imaging rate.…”

Origins of phase contrast in the atomic force microscope in liquids

“…Again, mica appears bright in the phase-lag image, confirming that energy propagation between eigenmodes is the primary source of phase contrast. Furthermore, although it has been demonstrated in high salt-concentration buffers that the stiffnesses of the CP face and the EC face are indistinguishable by force-distance "jump-mode" imaging methods (18), the difference in stiffnesses between the CP and EC faces is clearly resolved in the phase-lag image. Moreover, the phase-lag image suggests that the CP face is slightly softer than the EC face as expected (18).…”

“…Here we focus on the common scenario, where a nonfunctionalized tip interacts with a soft biological sample supported on a stiff substrate in a high-ionic-concentration buffer where the DLVO forces are highly screened (18,19) and adhesion hysteresis is negligible. Accordingly, interactions between the tip and sample can be modeled by a rate-dependent (viscous) force F tsnc (d,ḋ) combined with a conservative contact force F tsc (d).…”

Compositional Contrast of Biological Materials in Liquids Using the Momentary Excitation of Higher Eigenmodes in Dynamic Atomic Force Microscopy

“…We chose purple membrane (PM) for this study because it is a well-studied protein membrane that is easy to deposit with submonolayer coverage [19][20][21][22][23]. The sample preparation details are given in [18].…”