To understand how interactions at the lipid-water interface can influence the function of biological membranes, we study the physical properties of lipid bilayers. In particular, we measure the mechanical stress associated with lipid packing and interactions with biologically active perturbants such as sterols and antimicrobial peptides.

Because ions vary widely in their effects on biological materials, ion specificity beyond mere charge properties is a major issue in biology. One overlooked property of ions is their polarizability, the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces.  A surprising consequence of ion polarizability is the ability to stick to charged bilayers to an extent beyond what is expected from charge-charge attraction. This stickiness changes the way membranes interact, it also introduces strains that can alter the way proteins are accommodated and are able to change conformation as in the opening and closing of trans-membrane ionic channels.

Topics:

		Ions at the lipid-water interface and interbilayer forces
		Lipids, membrane additives (channels, sterols) and the balance of forces within bilayers
		Lipid-DNA complexes

Methods and Collaborators:

		X-ray (NIH, CMU, CHESS). A special sample chamber constructed in collaboration with the laboratory of  Prof. John F. Nagle (CMU) is used for oriented lipids.
		Density measurements.
		Osmometry, refractometry
		Electrophoretic mobility (Dr. Thomas Zemb, Saclay, France)
		Surface tension measurements (Langmuir trough)
		Solid state NMR (Dr. Klaus Gawrisch, NIAAA, NIH. Prof. Michael Brown, University of Arizona)
		Molecular dynamics simulations (Dr. Richard Pastor, CBER/FDA, Prof. Tomas B. Woolf, Johns Hopkins University)

Lipids and Ions

Neutral lipids and monovalent salt

Biological membranes work in salt solutions. A major component of membrane phospholipids are electrically neutral but show, significant and puzzling interactions with ions, as documented in the biological and physical literature.

Immersed in salt solutions, lipid bilayers swell though salt-driven swelling cannot be explained by simple electrostatics.  Measured by X-ray diffraction, the effect of salt added to neutral multilayers increases swelling in the Hofmeister order KF < KCl < KBr. An almost 100% increase in the water separation is measured in the presence of 1M KBr, to a distance that is 10 times the Debye length (3.6 Å) at this concentration. At close distances, ~10 Å, neutral lipid bilayers interact mainly through attractive van der Waals and repulsive hydration forces. At greater distances, an additional repulsion arises from the restriction of bilayer undulations within the multilayer structure.  Through computation and measurements we are investigating the extent to which the measured swelling curves can be explained by a combination of weakening of the van der Waals attraction, enhancement of repulsive terms and ion binding to the lipid headgroup.

Reflecting the competition between ions and lipids for the available interlamellar water, salt ions are excluded from the interface. There is salt deficit next to lipid headgroups, measurable by mass density gradients.

References

Charged lipids and salt

Suddenly we have become aware of the organizing power of small ions changing membrane protein behavior. Guided by the unexpectedly strong attraction between charged phospholipids and simple ions, we have begun to examine Li + ions with phosphatidylserine bilayers.  Why?  Earlier work by others suggests that Li⁺ acts like protons.  We are now asking whether Li + can stress lipids assembly and consequently whether low levels of Li + modify channel gating.

Charged bilayers repel as expected when interlamellar forces are measured by osmotic stress and X-ray diffraction. Why does this same repulsion not occur between charged groups on the same bilayer?  Why are the charged lipids stiffer against bending? One likely culprit is strong ion binding. Indeed we found a few years ago that protons bind so strongly to these same lipids that at 10 -4 Molar concentrations (pH 4), the membrane is wrenched from the lamellar form into an inverted hexagonal structure.  More intriguing, when these same lipids are used as a scaffold for protein channels, exposure to protons shifts channel gating.   

We further probe interactions at the lipid-water interface by 31 P and 2 H NMR. A significantly larger  hydration shell is measured around the PS headgroup, altough orientational fluctuations are reduced compared to the zwitterionic PC  headgroups. 

References

Forces within Bilayers, Strains

Pure lipids

The structure of lipid bilayers is dictated by conflicting forces between hydrophobic chains and hydrophilic headgroups. These forces are non uniform as a function of depth: monolayers tend to bend.

By modifying lipid structures under osmotic stress, we can measure the net effect of lateral forces. For lipids with strong bending tendencies, such as PE in combination with unsaturated chains, or PC with polyunsaturated chains, we measure the energy needed to induce a transition from lamellar geometry to inverted hexagonal. For lipids such as disaturated PCs that prefer the lamellar geometry we measure the energies needed to compress lipids laterally (reduction of area per headgroup).

To determine details about the distribution of lateral forces (i.e. higher moments of the distribution function) requires more local investigations. By deuterium NMR we and our collaborators can measure order parameter profiles along the lipid chains. These order parameters  are in essence a measure of chain disorder that yield information on lateral forces.

Using a basic mean-torque model, we and our collaborators have established a universal chain-packing curve for saturated chains of phospholipids. This curve is simply shifted by membrane additives or by the presence of polyunsaturated (PUFA) chains. For the latter, the order profile shifts towards more disordered states at the bilayer center, implying a redistribution of forces within the bilayer with consequent influence on bending moments and spontaneous curvature.

References

Lipids and Guests

Channels

Modifications of lateral stress and monolayer bending moments have been implicated in the formation and function of channels such as alamethicin and syringomycin. Working with the Section on Molecular Transport, we are manipulating the lateral stress in charged DOPS bilayers by controlling the pH and the chemical composition of the bathing ionic solution.

Sterols

As might be expected from their dramatic biological effects, sterols significantly shift the balance of forces within lipid bilayers. We measure these shifts by osmotic stress and x-ray diffraction, to compute energies relevant to the function of biological membranes. When caused by biosynthesis defects, cholesterol deficiency can lead to developmental disorders and malformations, with possible implication of lipid membrane properties (NICHD, Laboratory of Developmental Neurobiology, Yoke Loh, Chief). . Compared to its metabolic precursors, cholesterol is most efficient in stiffening membranes, a possible clue to why depletion or replacement with other sterols can affect cellular processes.

Oils, detergents, and alcohols

The absorption of oils, detergents, and alcohols into lipid membranes give us a detailed view on the physics of lipid packing. The dual stress method, by which the water and oil (e.g. tetradecane) fractions are regulated by sample preparation, permits the determination of bending rigidities and intrinsic curvature values for lipids of general interest.

References