Role of associated water in stabilizing human γ‐D crystallin under shape‐dependent macromolecular crowding

Abstract The cytoplasm of a cell is inherently crowded with diverse biological macromolecules of different sizes, shapes, and compositions. These macromolecular crowders strongly influence the thermodynamics and kinetics of essential biological processes, including protein folding, association, and enzymatic activity. Therefore, in our earlier study, we examined PEG size‐dependent crowding effects on protein stability via water‐network modulation; however, excluded‐volume (entropic) contributions varied and were not systematically controlled. To address this limitation, in this work, we examined the shape‐dependent effects of crowding by selecting similarly sized crowders, PEG‐35, Ficoll‐70, and dextran‐40, to mimic the heterogeneous shapes of crystallins in the eye lens. Our results revealed distinct outcomes: PEG‐35 facilitated the thermal unfolding of HγDC, dextran‐40 counteracted unfolding, while Ficoll‐70 produced no significant effect. The thermodynamic analysis provided even more intriguing insights. Both PEG‐35 and Ficoll‐70 entropically destabilized HγDC, in direct contrast to the predictions of traditional excluded volume theory, whereas dextran‐40 exerted an entropic stabilizing effect. To rationalize these observations, we hypothesized the associated water stabilization mechanism (AWSM), which emphasizes the critical role of hydration water structure in protein stability under crowded conditions. Collectively, our findings highlight the importance of associated water in shaping protein stability and solubility, offering new perspectives on shape‐dependent crowding effects. The results not only account for entropic destabilization and entropy–enthalpy compensation, but also demonstrate that the thick, resilient hydration shell surrounding human γ‐D crystallin underpins its long‐term stability and solubility within the dense and crowded environment of the eye lens.