Osmolyte depletion viewed in terms of the dividing membrane and its work of expansion against osmotic pressure.

How osmolytes enhance the folding, binding, and self-assembly of biological macromolecules at a microscopic scale has long been a matter of debate. Ambiguities persist on the key interpretive concepts, such as the "effective membrane" (which marks the boundary of the volume from which osmolytes are excluded) and the "free energy of exclusion" of osmolytes from biomolecular surfaces. In this paper, we formulate these elusive concepts based upon chemical thermodynamics and rigorous statistical thermodynamics (the Kirkwood-Buff theory). Positioning of the membrane at the osmotic dividing surface is crucial in order not to affect the thermodynamics of solvation. The notion of the free energy (work) of excluding osmolytes is refined to the expansion work against the osmotic pressure, which indeed describes the change of solvation free energy at dilute osmolyte concentrations.