Interplay of protein fluctuation and associated water dynamics in osmolyte-induced stabilization

The mechanism behind osmolyte-induced protein stabilization remains elusive despite extensive research. Among various hypotheses, the associated water-modulation hypothesis has proved to be the most effective in explaining osmolyte-induced stabilization effects. Earlier, we demonstrated that osmolytes that slow down associated water dynamics enhance protein thermal stability, whereas those that accelerate it promote destabilization. However, the molecular basis of this correlation remains unclear. Using fluorescence correlation spectroscopy, we observe that osmolyte-induced changes in the associated water dynamics directly affect the internal flexibility of the protein, which is assessed through conformational fluctuation dynamics, serving as a tool to measure the protein's internal flexibility. The osmolytes that induce retardation in the associated water dynamics make the interior of the protein less flexible, as reflected by the slowed conformational fluctuation dynamics of the protein, leading to enhanced thermal stabilization. The destabilizers induce effects that are exactly opposite to stabilizers. These findings provide new insights into the interplay of associated water dynamics, protein flexibility, and stability, contributing to a deeper understanding of osmolyte-induced protein stabilization.