To help understand the nature of physical/chemical interactions in biomolecules and biomolecular assemblies, we have developed an experimental approach to study their structure (morphology) and thermodynamic properties simultaneously as a function of the length scale (spatial resolution). The method combines macroscopic osmotic swelling pressure measurements and small angle neutron scattering (SANS). Macroscopic swelling pressure measurements probe the system in the large length scale range, thus providing information on the overall thermodynamic response. The SANS measurement simultaneously provides information about the size of different structural elements and their respective contribution to the osmotic properties. Combining these measurements allows us both to separate the scattering intensity arising from thermodynamic concentration fluctuations from the intensity scattered by large static superstructures (e.g., aggregates), and to determine the length scales relevant to the macroscopic thermodynamic properties. This thermodynamic and structural information cannot be obtained by other techniques. Specifically, we have applied this approach to study interactions between multivalent cations (e.g., calcium) on various biomolecules. Such ions are ubiquitous in the biological milieu, yet existing theories do not adequately explain their effect on and interactions with charged macromolecules in general, and biomolecules in particular. Moreover, experiments to study these interactions are difficult to perform, particularly in solution, because above a low concentration threshold multivalent cations cause charged molecules to phase separate or precipitate. We have overcome this limitation by cross-linking our biopolymers. Since macroscopic phase separation does not occur in cross-linked gels, we are able to extend the range of ion concentrations in which the system remains stable and can be studied. In pilot studies, this new method has been applied to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and of DNA. Concentrated DNA solutions and DNA gels have never before been investigated using SANS in conjunction with osmotic measurements. The proposed method also provides a unique framework for analyzing the osmotic and scattering behavior of other biomolecular systems. Recently, we have begun to apply this methodology to study interactions between macromolecular constituents of extracellular matrix.
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