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Publisher Summary This chapter presents specific examples to describe procedures and use osmotic stress with maximum ease and efficiency. These examples are systems where intentionally varied water activity has revealed a connection between different functional states of proteins or other large molecules with different amounts of water associated with them. The method––osmotic stress on macromolecules–– is applied to four kinds of processes, which are (1) ionic channel opening/closing, (2) enzyme/ substrate association and turnover, (3) molecular binding, and (4) longrange interaction. Through these examples, a thermodynamic relations is developed that is useful in the interpretation of osmotic stress data. Osmotic stress experiments, in explicitly recognizing the importance of water activity, are designed to buffer it so that it is as well defined as other more familiar solution variables. Osmotic stress measurements have revealed a surprisingly large influence of water activity on macromolecular reactions and conformational transitions. It even appears that small solutes, typically thought to affect macromolecules only through direct binding, show an indirect effect through their influence on water activity.

The physical properties of chromatin have been explored at the level of individual nucleosomes, extended polynucleosome filaments, and folded polynucleosome fibers. To compare the physical properties of bulk chromatin with the properties of chromatin in transcriptionally active genes, we have examined the stability of the 30 nm chromatin fiber in the neighborhood of the adult s-globin gene in chicken red cells. We find that the DNA in the neighborhood of this gene is indistinguishable from bulk chromatin in its degree of compaction and in its reaction with the HMG proteins HMG 14/17.

Publisher Summary This chapter focuses on the practical use of osmotic stress (OS) to measure macromolecular forces and chemical potentials. It also reviews several examples of its application. Osmotic stress is applied to a wide set of charged or electrically neutral membranes, to the arrays of muscle protein, to tobacco mosaic virus particles, to ordered arrays of DNA double helices, to sickle cell hemoglobin undergoing polymerization, to the aqueous cavities of water-in-oil liquid crystals, and to ionic channels through bilayer membranes. OS permits ascertaining the properties of boundary water under thermodynamically well-defined conditions. Three equivalent ways in which OS is consistently applied are also discussed in the chapter. The most tedious aspect of OS is getting accurate osmotic pressures of the stressing polymer solutions. The OS measurement of forces between DNA double helices demonstrates the utility of the method for examining an entire class of linear macromolecules, such as collagen triple helices and xanthan polysaccharides.