We measure, characterize, and codify the physical forces that govern the organization of all types of biological molecules. Basing our work on physical theory, we aim to create a practical physics of biological material through a series of measurements and analyses of the different types of forces revealed in vivo, in vitro, and in computation. In particular, we are working with DNA/lipid assemblies for gene therapy; DNA assemblies such as are seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid crystals. In all these systems, we observe the structure of packing while measuring, formulating, and computing the intermolecular forces that create such systems.
van der Waals forces
Parsegian, Petrache, Podgornik; in collaboration with French, Mkrtchian, Nagle
The source of the powerful surface tension at membrane interfaces and the dominant forces that cohere membranes and proteins, van der Waals forces are perhaps the sole attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. While writing a text intended to make abstruse physics accessible to non-physicists, we formulated a new set of solutions that have enabled us to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. We have entered into collaborations with spectroscopists who have been using our equations to design electronic devices to aid in the investigation of interaction between phospholipids and salts.
We have observed how the attraction between membranes varies when salts of different ions (chloride versus bromide) are dissolved in the intervening water. Membrane multilayers swell by 50 percent with bromide but not with chloride. We reformulated theory of van der Waals forces between membranes to show how membranes would respond to changes in solutions so as to have a strategy to control membrane assembly. Perhaps the most remarkable result of our work is that we now realize that the polarizability of ions is as much a part of ions’ personality as is their charge. Large ions have the ability to adhere to interfaces of water with non-polar materials such as hydrocarbon. Recognition of ion polarizability makes it possible to reveal sources of ion specificity in ion-protein and in ion-lipid interactions.
Parsegian VA. Van der Waals Forces: A Handbook for Biologists, Chemists, Engineers, and Physicists. Cambridge, UK: Cambridge University Press, 2005.
Podgornik R, Parsegian VA. Van der Waals interactions across stratified media. J Chem Phys 2004;120:3401-3405.
Podgornik R, Parsegian VA. Van der Waals interactions in a dielectric with continuously varying dielectric function. J Chem Phys 2004;121:4767-4773.
Cosolutes in molecular folding and association
Harries, Parsegian, Podgornik; in collaboration with Rau
A remarkable number of cellular processes are controlled by the osmotic action of small solutes, including gating of ionic channels and specific versus nonspecific 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 can control osmotic pressure. We have measured the effect of varied pressure on the association of carbohydrates with membrane protein channels. We observed a single event of beta-cyclodextrin (CD) nesting in the lumen of a maltoporin channel as a transient drop in ionic current as a consequence of the partial occlusion of the channel pore. The change in equilibrium constant of CD binding to the channel versus solution osmotic pressure translates into the number of water molecules released in the specific binding. Osmotic pressure differently affects the on and off rates of CD-ion channel binding. By changing the species of salt used to exert the osmotic stress, we can further probe the properties of the hydration water and interactions of different salts with both CD and porin. We find that, under equilibrium conditions, the degree to which a particular ion affects the binding is 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 that are unavailable for the dissolution of salt. Finally, we show that osmotic effect is not restricted to the action of salts. Short peptides that fold and unfold become more stable in the presence of excluded solutes in proportion to the difference in the number of solvating waters in the folded and unfolded states.
At the other end of the size scale, polymers can bridge and hold together large molecules. We analyzed the electrostatic bridging interactions between two macro-ions and within an assembly of many macro-ions. We showed that, in both cases, polyelectrolyte bridging confers an additional attractive component to the effective two-body interactions. We applied these theoretical results to measuring the second virial coefficient of nucleosomal core particles in ionic solutions of variable ionic strength. The progressive reduction in the magnitude of the second virial coefficient is in semi-quantitative agreement with our calculations. Our calculations of the polyelectrolyte bridging interactions in mixed DNA-cationic polyelectrolyte assemblies such as DNA-polylysine or DNA-polyarginine systems compare favorably with the reported experiments of Raedler et al. (Science 1997;275:810). We hope that some refinements in the current theory will make the comparison both quantitative and predictive, as it would be of paramount importance in assessing the stability of different genosome (DNA-lipid complex) formulations with cationic polyelectrolytes.
We analyzed the order and fluctuations in dense arrays of nucleosomal core particles that show highly exotic liquid crystalline mesophases. We showed that the origin of the mesophases is in the coupling of the orientationally varying interparticle potential with the lattice symmetry. We codified the existing phase diagram of the dense solutions of nucleosomal core particles in the framework of the symmetry components of the interaction potential. The liquid crystalline mesophases of the nucleosomal core particles should give us some clear indications as to the preferred local packing symmetry within the 30-nm chromatin fiber.
Harries D, Parsegian VA. Gibbs adsorption isotherm combined with Monte Carlo sampling to see the action of cosolutes on protein folding. Proteins 2004;57:311-321.
Podgornik R. Electrostatic contribution to the persistence length of a semiflexible dipolar chain. Phys Rev E 2004;70:031801.
Podgornik R. Polyelectrolyte-mediated bridging interactions. J Polym Sci [B] 2005;42:3539-3556.
Podgornik R, Harries D, Strey HH, Parsegian VA. Molecular interactions in lipids, DNA and DNA-lipid complexes. In: Templeton NS, ed. Gene and Cell Therapy, 2nd edition. New York: Marcel Dekker, 2004;301-332.
Podgornik R, Saslow WM. Long-range many-body polyelectrolyte bridging interactions. J Chem Phys2005;122:204902.
Ions, lipids, and membrane-protein interactions
Petrache, Podgornik; in collaboration with Dubois, Gawrisch, Gondre-Lewis, Loh, Nagle, Porter, Zemb
Although expected from theory and simulations, depletion of ions at fuzzy biomembrane interfaces has long eluded experimental verification. We have now shown how salt exclusion can be accurately measured by surprisingly simple yet accurate bench-top techniques. Multilamellar aggregates of common phospholipids sink in low salt but float in salt solutions that are much less dense than the lipid itself. By manipulating bath and lipid densities and using heavy water and varied lipid chain length, we have obtained accurate exclusion curves over a wide range of KCl and KBr concentrations. While maintaining a constant width at low salt, the exclusion layer decreases in high salt following the Debye screening length. Consistent with interfacial accumulation of polarizable ions, bromide salts are less excluded than chloride, with an attraction of about 1 kT per bromide ion. Neglected until now in theoretical descriptions, the competition between salt exclusion and binding is critical to understanding membrane interactions and specific ionic effects.
Given that ions vary widely in their effects on biological materials, ion “specificity” beyond simple 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. Another surprising feature of ions is the tendency to stick to charged bilayers to an extent beyond what would be expected from charge-charge attraction. This 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. Our comparisons between bilayers of phosphatidylserine and phosphatidylcholine lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.
Solutes can also change membranes from within. Sterols with structures that vary by as little as the difference between neighbors on the path of metabolic synthesis can change the bending stiffness of a bilayer membrane. This difference shows up in the spacing between bilayers and, more ominously, in the impaired vesicular secretion in diseases in which normal cholesterol synthesis is blocked. Working with Peng Loh, Margorie Gondre-Lewis, and Forbes Porter, we found a strong correlation between the mechanical properties of bilayers with different sterols and the symptoms of Smith-Lemli-Opitz syndrome.
Kucerka N, Liu Y, Chu N, Petrache HI, Tristram-Nagle S, Nagle JF. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles. Biophys J 2005;88:2626-2637.
Petrache HI, Harries D, Parsegian VA. Alteration of lipid membrane rigidity by cholesterol and its metabolic precursors. Macromol Symp 2005;219:39-50.
Petrache HI, Kimchi I, Harries D, Parsegian VA. Measured depletion of ions at the biomembrane interface. J Am Chem Soc 2005;127:11546-11547.
Rajammorthi K, Petrache HI, McIntosh TJ, Brown MF. Packing and viscoelasticity of polyunsaturated w-3 and w-6 lipid bilayers as seen by 2H NMR and X-ray diffraction. J Am Chem Soc 2005;127:1576-1588.
Publications Related to Other Work
Kutnjak Z, Filipic C, Podgornik R, Nordenskiold L, Korolev N. Charge transport mechanism in native deoxyribonucleic acid. Physica Scripta 2005;T118:208-210.
Kutnjak Z, Lahajnar G, Filipic C, Podgornik R, Nordenskiold L, Korolev N, Rupprecht A. Electrical conduction in macroscopically oriented deoxyribonucleic and hyaluronic acid samples. Phys Rev E 2005;71:041901.
Lorman V, Podgornik R, Zeks B. Correlated and decorrelated positional and orientational order in the nucleosomal core particle mesophases. Europhys Letts 2005;69:1017-1023.
1Six months per year
collaborators
David Andelman, PhD, Tel Aviv University, Tel Aviv, Israel
Joel Cohen, PhD, University of the Pacific, San Francisco, CA
Monique Dubois, PhD, CEA-Saclay, Gif-sur-Yvette, France
Evan A. Evans, PhD, Boston University, Boston, MA, and University of British Columbia, Vancouver, Canada
Roger French, PhD, University of Pennsylvania, Philadelphia, PA, and Dupont Research Laboratories, Wilmington, DE
Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Rockville, MD
William Gelbart, PhD, University of California Los Angeles, Los Angeles, CA
Marjorie Gondre-Lewis, PhD, Section on Cellular Neurobiology, NICHD, Bethesda, MD
Sol M. Gruner, PhD, Cornell University, Ithaca, NY
Per Lyngs Hansen, PhD, Syddansk Universitet, 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
Stephanie Tristram-Nagle, PhD, Carnegie-Mellon University, Pittsburgh, PA
Thomas Zemb, PhD, CEA-Saclay, Gif-sur-Yvette, France
For further information and publications, contact aparsegi@helix.nih.gov.