PHYSICAL FORCES ORGANIZING BIOMOLECULES
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V. Adrian Parsegian, PhD, Head, Section on Molecular Biophysics Daniel Harries, PhD, Visiting Fellow |
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| Despite the remarkable advances in molecular biology, the tools for describing how biomolecules do their work remain primitive. We therefore measure, characterize, and codify the forces that govern the organization of a wide variety of biological molecules. Our undertaking is strengthened by its strong connection with physical theory. Our long-term goal is to build a practical physics of biological material. To that end, we undertake a series of measurements and analyses of the different types of forces as 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 simultaneously observe the structure of packing and measure intermolecular forces or interaction energies. Ions, lipids, and membrane-protein interactions Petrache, Podgornik; in collaboration with Dubois, Gawrisch, Nagle, Zemb 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 polarizability, 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 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 transmembrane ionic channels. Our comparisons between bilayers of phosphatidylserine (PS) and phosphatidylcholine (PC) lipids with the same chains and at the same temperature enable us to focus on the effects of these headgroups on bilayer properties. Using X-ray diffraction and NMR spectroscopy, we found that, in the presence of sodium ions, negatively charged PS bilayers have lateral areas much smaller than the areas of corresponding neutral PC analogs. The shrinkage occurs despite the extra electrostatic repulsion expected for charged PS headgroups. The condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. We find that the charged bilayers repel as expected when interlamellar forces are measured by osmotic stress and X-ray diffraction. The questions arise as to why the same repulsion does not occur between charged groups on the same bilayer and why the charged lipids are stiffer against bending. The culprit is likely to be 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. Even more intriguing is the observation that, when these same lipids are used as a scaffold for protein channels, exposure to protons shifts channel gating. Petrache HI, Zuckerman DM, Sachs JN, Killian JA, Koeppe RE, Woolf TB. Hydrophobic matching mechanism investigated by molecular dynamics simulations. Langmuir 2002;18:1340-1351. Podgornik R., Hansen PL. Membrane pinning on a disordered substrate. Europhys Lett. 2003;62:124-130. Podgornik R. Two-body polyelectrolyte-mediated bridging interactions. Chem Phys 2003;118:11286-11296. Sachs JN, Petrache HI, Woolf TB. Interpretation of small angle x-ray measurements guided by molecular Sachs JN, Petrache HI, Zuckerman DM, Woolf TB. Molecular dynamics simulations of ionic concentration Van der Waals forces Parsegian, Petrache, Podgornik; in collaboration with French, Nagle It is not generally appreciated how much work is done among membranes and macromolecules by bonds that are weaker than covalent bonds, electrical-charge interactions, charge fluctuation, or van der Waals forces. The main force that coheres membranes and proteins and a source of the powerful surface tension at membrane interfaces, van der Waals forces are the dominant, perhaps sole, attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. This past year, we have learned to formulate, measure, and modify these neglected interactions. The key has been to begin with the elements of physical theory and to relate the polarizability of materials to the fluctuations of charges within them. From this, we have been able to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. We have been able to see how adding even the simplest salts to water around lipid bilayers will change how these membranes assemble into multilayers. For example, we have seen how the attraction between membranes varies when salts of different ions (e.g., chloride versus bromide) are dissolved in the intervening water. Membrane multilayers will swell by 50 percent with bromide but not with chloride. We have reformulated van der Waals forces between membranes to show how they would respond to changes in solutions as a strategy to control membrane assembly. One unexpected byproduct has been a 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 that can be used to compute van der Waals forces. Another unexpected result relates to what was known about the friction between an ion and the water through which it moves. Just as a charge-water interaction creates a drag, there is a charge-fluctuation source of drag on a moving particle. Kutnjak Z, Filipic C, Podgornik R, Nordenskiold L, Korolev N. Electrical conduction in native Podgornik R, Hansen PL, Parsegian VA. On a reformulation of the theory of Lifshitz-van der Waals interactions in multilayered systems. J Chem Phys 2003;119:1070-1077. Cosolutes in molecular folding and association Harries, Parsegian; in collaboration with Rau Driven by the conditions set by smaller solutes, proteins "fold" and "unfold." Experimentally, these conditions are stated as intensive variables, i.e., pH and other chemical potentials, as though small solutes were infinite resources that come at an independently varied price. Computationally, the finite spaces of simulation allow only fixed small numbers of the solutes. By combining the analytic Gibbs adsorption isotherm with the computational Monte Carlo sampling of polymer configurations, we have been able to overcome an inherent limitation of computer simulation. The key is to compute analytically the free energy changes wrought by solutes on each particular configuration. Then, numerical computation is needed only to sample the set of configurations as efficiently as though no bathing solute were present. The result is a more accurate computation procedure 500 times faster than earlier simulations that had to count all possible positions of the cosolutes in the bathing solution. To monitor how small, adherent molecules affect molecular association, we measured the changes of binding free energy versus change in water activity for the specific binding of cyclodextrin with an adamantane derivative. Using different salts and neutral agents, we found that the dependence of the binding constant on osmotic pressure 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 with added solute indicates that these osmolytes are interacting primarily enthalpically with these surfaces. Hansen PL, Cohen JA, Podgornik R, Parsegian VA. Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws. Biophys J 2003;84:350-355. Harries D, Ben-Shaul A, Szliefer I. Enveloping of charged proteins by lipid bilayers. 2003; in press. Harries D, May S, Ben-Shaul A. Curvature and charge modulations in lamellar DNA-lipid complexes. J Phys Chem 2003;107:3624-3630. Parsegian VA. Protein-water interactions. In: Zeuthen T, Stein WD, eds. International Review of Cytology: a Survey of Cell Biology; Molecular Mechanisms of Water Transport across Biological Membranes. New York: Academic Press, 2002;215:1-30. Podgornik R, Harries D, Strey HH, Parsegian VA. Molecular interactions in lipids, DNA, and lipid- DNA complexes. In: Smyth, TN, ed. Gene Therapy - Therapeutic Mechanisms and Strategies. 2nd ed. 2003; in press. COLLABORATORS Joel Cohen, PhD, University of the Pacific, San Francisco CA Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Rockville MD Per Lyngs Hansen, PhD, South Denmark University, Odense, Denmark John Nagle, PhD, Carnegie Mellon University, Pittsburgh PA Peter Rand, PhD, Brock University, St Catharines, Canada Donald Rau, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda MD Wayne Saslow, PhD, Texas A & M University, Arlington TX Thomas Zemb, PhD, Service de Chimie Moléculaire, CEA Saclay, France *Six months per year For further information, contact aparsegi@helix.nih.gov
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