Exploring Water Beyond the Solvent: Insights into Density Fluctuations and EGFP Unfolding via Luminescence Thermometry

Water plays an active role in protein stability, but directly probing its density fluctuations at the protein interface remains challenging. Here, we use enhanced green fluorescent protein (EGFP) to investigate how low-density (LD) and high-density (HD) water motifs modulate unfolding in H₂O and D₂O. Fluorescence quenching during heating-cooling cycles indicates that unfolding begins at approximately 55 °C in H₂O and 64 °C in D₂O, consistent with the stabilizing effect of isotopic substitution. Circular dichroism corroborates this shift, with higher melting temperatures in D₂O (83 vs 79 °C in H₂O). EGFP Brownian velocity measurements, through luminescence thermometry, reveal bilinear temperature dependence with crossover temperatures of 55 °C in H₂O and 65 °C in D₂O, indicating that LD motifs persist longer in heavy water. Together, these results establish a fully optical strategy that directly links hydration-water structure to protein stability, providing a new route to study hydration-mediated dynamics in biomolecules.