abstract
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 degrees C in H2O and 64 degrees 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 degrees C in H2O). EGFP Brownian velocity measurements, through luminescence thermometry, reveal bilinear temperature dependence with crossover temperatures of 55 degrees C in H2O and 65 degrees 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.
keywords
GREEN FLUORESCENT PROTEIN; HYDROGEN-BONDS; STABILITY; DYNAMICS; CELL; SCATTERING; BEHAVIOR; STATES; GFP
subject category
Chemistry
authors
Guo, YW; Maturi, FE; Raposo, RS; Brites, CDS; Carlos, LD
our authors
Carlos Brites
Associate Professor
Fernando Eduardo Maturi
Research Fellowship
Guo Yongwei
PhD Student
Luís Carlos
Full professor
Ramon S. Raposo Filho
PhD StudentProjects
Collaboratory for Emerging Technologies, CoLab (EMERGING TECHNOLOGIES)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
acknowledgements
This work was developed within the scope of the project CICECO - Aveiro Institute of Materials, UID/50011/2025 & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020) and The Shape of Water: Part II - Unveiling the Hidden Architecture 2023.18028.ICDT, financed by national funds through the FCT/MCTES (PIDDAC). Y.G. and R.S.R.F. acknowledge the financial support from the China Scholarship Council (No. 202306650010) and FCT (2024/01558/BDFCT), respectively.