Protein dynamics are coupled to the solvent—including lipids for membrane proteins—with functionally relevant conformational changes requiring fluctuations in the soft matter. Osmotic stress experiments are promising to investigate interactions with water, yet different osmolytes produce opposite effects on protein function. 1 We hypothesize that concentration and molar mass of osmolytes determine how they influence dynamics. A range of polymer osmolytes and their monomers were introduced to the G-protein-coupled receptor (GPCR) rhodopsin, whose activation was measured with ultraviolet-visible spectroscopy. We discovered that low concentrations of large hydrophilic polymers, such as polyethylene glycol (PEG), strongly inactivate rhodopsin, while higher concentrations have a diminished effect. By contrast, low concentrations of small osmolytes activate rhodopsin, until reaching a saturation point, beyond which inactivation occurs. We therefore separate the effects of osmolytes into different regimes, each containing osmolytes of a certain size and concentration that produce similar effects on rhodopsin. Large polymers are excluded from the interior of rhodopsin and withdraw bulk-like water, while small osmolytes penetrate into the protein core and remove structural water from microdomains. High concentrations of small osmolytes saturate the receptor interior and act similarly to large polymers. Quinary interactions occur at high concentrations of all species, reducing inactivation by stabilizing the active state. Furthermore, osmolytes with very large molar masses deviate from the trends observed for other large polymers and may interact with proteins by reducing available volume (crowding). We extend these findings to the energy-landscape model of protein dynamics, where low-energy conformational transitions are driven by movements of bulk-like water while higher energy changes involve structural water. 1 This correspondence can allow for selectively engineering the energy landscapes of proteins. 1 Bachler, Z. T. et al. (2024). Biophys. J. 123, 4167–4179.
Bachler et al. (Sun,) studied this question.