Direct Measurement and Computation of Forces Between Membranes or Macromolecules

Child Health And Human Development

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Abstract

Physical forces between molecules link their structure with the function of biologically important complexes. They let us predict the strength and specificity of interactions among proteins, nucleic acids, lipid bilayers, and carbohydrates. We have found that water structuring plays an unexpectedly large role in the close interaction of all biological systems so far investigated. We have been able to follow up our measurement of the work required to remove water from a complex between the specific DNA binding protein EcoRI and a non-cognate DNA sequence using the osmotic stress approach and a competitive equilibrium binding assay. Our strategy is to couple force measurements between molecules in macroscopic condensed arrays with the interaction of molecules in dilute solution. Our results on complexes of EcoRI with other, nonspecific DNA sequences examined changes in dissociation constant under the osmotic stress. Now we have been able to determine the dissociation rate of the specific complex dissociation rate. The rate slows linearly with osmotic stress and is insensitive to solute identity for a wide range of sizes and chemical natures. The osmotic sensitivity of dissociation is virtually identical to the difference in osmotic sensitivity of specific and nonspecific binding modes of EcoRI. It emerges that: a) Dissociation rates can be used instead of equilibrium assays to measure changes in water sequestered by EcoRI-DNA complexes (to allow us to use much higher osmotic stresses without the complications accompanying equilibrium measurements; b) It is the dehydrated, specifically associated state that is stabilized by osmotic stress or "crowding". We have quantified the interaction of small solutes with macromolecular surfaces condensed in ordered macroscopic arrays. From the effect of these small solutes on directly measured forces between DNA double helices and between polysaccharides, we have found that small-molecule solubility varies exponentially with the distance from the macromolecular surface. This has been seen with several salts in the Hofmeister series and several neutral polyhydric or zwitterionic solutes that are known to stabilize native protein structures. The magnitude of the interaction of salts correlates with their known effect on water structure, further showing the importance and ubiquity of water structuring forces on the interaction of molecules in solution. At the same time we have been doing theoretical physical studies on polyelectrolytes, particularly DNA. We have been able to test the popular 'ion-condensation' theory of DNA and have shown that it fails qualitatively to account for DNA osmotic pressures. Our association with NASA has now developed to the point where we are installing an x-ray lens said to boost photon flux by a factor of 10 to 100 times. We will be using this new lens in our force measurements as well as testing new strategies for x-ray diffraction.