Work

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

Year: 2025

Type: article

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 H 2 O and D 2 O. Fluorescence quenching during heating–cooling cycles indicates that unfolding begins at approximately 55 °C in H 2 O and 64 °C in D 2 O, consistent with the stabilizing effect of isotopic substitution. Circular dichroism corroborates this shift, with higher melting temperatures in D 2 O (83 vs 79 °C in H 2 O). EGFP Brownian velocity measurements, through luminescence thermometry, reveal bilinear temperature dependence with crossover temperatures of 55 °C in H 2 O and 65 °C in D 2 O, 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.

Source: The Journal of Physical Chemistry B

DOI: 10.1021/acs.jpcb.5c06143

Authors: Yongwei Guo, Fernando E. Maturi, Ramon S. Raposo Filho, Carlos D. S. Brites, Luís D. Carlos

Institutions: Laboratório Associado CICECO – Instituto de Materiais de Aveiro, University of Aveiro

Language: English

Topic: Protein Structure and Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Funders: China Scholarship Council, Fundação para a Ciência e a Tecnologia

Awards: G1067239717, G1989158764, CICECO-Instituto de Materiais de Aveiro, G5926315872