V. Adrian Parsegian, PhD, Head, Section on Molecular Biophysics
Rudi Podgornik, PhD, Visiting Scientist
Daniel Harries, PhD, Visiting Fellow
Jason DeRouchey, PhD, Postdoctoral Fellow
Horia I. Petrache, PhD, Postdoctoral Fellow

With a long-term goal to build a practical physics of biological material, we measure, characterize, and codify the forces that govern the organization of all types of biological molecules. The way we look at molecules sometimes fortunately generates ideas about what goes wrong in molecular disease. Our undertaking is strengthened by its strong connection with physical theory. Through a series of measurements and analyses of diverse forces as revealed in vivo, in vitro, and in computation, we are working with DNA/lipid assemblies for gene therapy; DNA assemblies such as those seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid-crystals. In all these systems, we simultaneously observe the structure of packing and measure intermolecular forces or interaction energies.

Molecular forces in molecular disease

Harries, Parsegian, Petrache, Podgornik; in collaboration with Gondre-Lewis, Loh, Porter

We are directing our efforts to determine how the last step in cholesterol synthesis creates the deadly Smith-Lemli-Opitz syndrome (SLOS) and why removal of a double bond creates a species that frustrates normal vesicular secretion. Using a mouse model of SLOS, Marjorie Gondre-Lewis, Peng Loh, and Forbes Porter investigated the mechanism by which cholesterol affects sterol granule biogenesis in vivo. The absence of one of the last two enzymes in the cholesterol biosynthetic pathway results in an accumulation of precursors. Cholesterol-deficient mice showed a decrease in the number of secretory granules in pancreas, pituitary, and adrenal glands as well as morphologically aberrant granules in exocrine pancreas. Remarkably, exogenous normal cholesterol could restore regulated secretion. The possibility of reversal with the simple addition of normal cholesterol immediately suggested that physical forces are at work that can differ with small differences in sterols. Horia Petrache and Daniel Harries showed that modification of sterol chemical structure significantly alters membrane physical properties. Using X-ray diffraction and osmotic stress, we measured changes in the bending rigidity of bilayers containing either cholesterol or one of its metabolic precursors. We showed that membrane elasticity differed dramatically between slightly different, metabolic-neighbor sterols and increases in the sequence lanosterol<7-dehydrocholesterol <lathosterol<cholesterol. We interpreted the results in terms of sterol location within lipid structures and modification of lateral stress, a structural feature relevant to interactions within biological membranes. We found that cholesterol is most efficient in enhancing membrane rigidity, a possible clue as to why depletion or replacement with other sterols can affect cellular structures. The stiffness of a granule is likely an important factor in the deformations it must endure to undergo secretion. We can thus see a clear physical logic of how physical, mechanical properties conferred by sterols couple with biological action.

van der Waals forces

Parsegian, Podgornik; in collaboration with French, Mkrtchian, Nagle, Tristram-Nagle

As the dominant force that coheres membranes and proteins and the source of the powerful surface tension at membrane interfaces, the van der Waals force has regained its prominence as perhaps the sole attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. The key has been to begin with the elements of physical theory that relate the polarizability of materials to the fluctuations of charges within them. Accordingly, we have been able to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. We have thus furthered our investigations through a tight coupling of modern electromagnetic theory of structured materials with experiments and measurements that reveal electromagnetic properties.

We have teamed with groups that measure absorption spectra in order to formulate and compute charge fluctuation forces involving lipids, water, and ions as well as synthetic structures such as carbon nanotubes. The results have shown how charge fluctuation forces conferred by ions in solution can modify forces between lipid membranes. We have measured those forces and computed van der Waals charge fluctuation forces.

The measured differences between different types of ions clearly show how the identity of different salts influences forces that organize large structures. With regard to the interactions of membranes, ion "specificity" beyond simple charge properties is a major issue in biology, given that ions vary widely in their effects on biological materials. One overlooked property of ions is their polarizability, which is the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces. Another surprising feature of ions is their tendency to stick to charged bilayers to an extent beyond that expected from charge-charge attraction. Such stickiness changes the way membranes interact and introduces strains that can alter the way proteins are accommodated and change conformation, as in the opening and closing of transmembrane ionic channels.

Further, we have seen how the attraction between membranes varies when chloride versus bromide salts are dissolved in the intervening water. Membrane multilayers swell by 50 percent with bromide but not with chloride salts. We have reformulated van der Waals forces between membranes to show how they would respond to changes in solutions so as to have a strategy to control membrane assembly. One unexpected byproduct has been collaboration with engineers using our equations to design production procedures for thin-film resistors in computer chips. We expect the collaboration to work to our benefit by providing us with experimental data for computing van der Waals forces.

We also progressed in extending the Lifshitz theory of van der Waals interactions in stratified media such as lipid multilamellar systems to be able to compute forces between bodies with extended interfaces, with applications ranging from the practical (the composite media of electric insulators) to the biological (the action of extended polymer layers on biological membranes).

Solute control of molecular association

Bezrukov, Kimchi, ¹ Harries, Parsegian, Petrache, Rau; in collaboration with Belloni, Dubois, Nagle, Tristram-Nagle, Zemb

To monitor how small adherent molecules affect molecular association, we measured the changes in binding free energy versus changes in water activity for the specific binding of cyclodextrin to an adamantane derivative. The dependence of the binding constant on osmotic pressure, using different salts and neutral agents, suggests a release of 15 to 25 water molecules from the interacting surfaces upon association, depending on the type of solute used. The observed dependence of binding free energy and enthalpy on added solute indicates that the osmolytes primarily interact enthalpically with the surfaces.

The osmotic action of small solutes controls a remarkable number of cellular processes, including the gating of ionic channels and specific versus non-specific DNA-protein interactions regulating gene expression. Osmotic sensing at the molecular level can probe the forces acting between and within macromolecules. By varying the salt or neutral "osmolyte" concentration in the bathing solution, we controlled osmotic pressure and measured the effect of the varied pressure on the association of carbohydrates with membrane protein channels. We observed a single event of βcyclodextrin (CD) nesting in the lumen of a maltoporin channel as a transient drop in ionic current due to the partial occlusion of the channel pore. The change in equilibrium constant of CD binding to the channel versus solution osmotic pressure translated into the number of water molecules released in the specific binding. Osmotic pressure differently affected the on- and off-rates of CD-ion channel binding. By changing the species of salt used to exert the osmotic stress, we further probed the properties of the hydration water and the interactions of different salts with both CD and porin. We found that, under equilibrium conditions, the degree to which a particular ion affected the binding was related to the ion's ranking in the Hofmeister series. In fact, by using osmometry, we have been able to determine that CD itself is hydrated by waters unavailable for the dissolution of salt. Finally, we showed that osmotic effect is not restricted to the action of salts.

Publications Related to Other Work

¹ Itamar Kimchi, former Summer Student

COLLABORATORS

David Andelman, PhD, _Tel Aviv University, Tel Aviv, Israel
Luc Belloni, PhD, CNRS, CEA, Saclay, Gif-sur-Yvette, France_
Sergey Bezrukov, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Joel Cohen, PhD, University of the Pacific, San Francisco, CA
Monique Dubois, PhD, CEA Saclay, Gif-sur-Yvette, France
Roger French, PhD, University of Pennsylvania, Philadelphia, PA
William Gelbart, PhD, University of California Los Angeles, Los Angeles, CA
Marjorie Gondre-Lewis, PhD, Office of the Scientific Director, NICHD, Bethesda, MD
Philip Gurnev, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Per Lyngs Hansen, PhD, South Denmark University, Odense, Denmark
Charles Knobler, PhD, University of California Los Angeles, Los Angeles, CA
Y. Peng Loh, PhD, Office of the Scientific Director, NICHD, Bethesda, MD
Vanik Mkrtchian, PhD, Institute of Physics, National Academy of Sciences, Ashtarak, Armenia
John F. Nagle, PhD, Carnegie-Mellon University, Pittsburgh, PA
Forbes Porter, MD, Heritable Disorders Branch, NICHD, Bethesda, MD
Donald Rau, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Jonathan Sachs, PhD, NIST, Gaithersburg, MD
Wayne Saslow, PhD, Texas A&M University, College Station, TX
Stephanie Tristram-Nagle, PhD, Carnegie-Mellon University, Pittsburgh, PA
Gerard Wong, PhD, University of Illinois at Urbana-Champaign, Urbana, IL
Thomas Zemb, PhD, CEA Saclay, Gif-sur-Yvette, France

For further information, contact aparsegi@helix.nih.gov.