Exploring Green Fluorescent Protein Brownian Motion: Temperature and Concentration Dependencies Through Luminescence Thermometry

Abstract

Luminescent nanothermometry emerges as a powerful tool for studying protein dynamics. This technique was employed to perform the first measurement of the temperature dependence of protein Brownian velocity, showcasing the illustrative example of enhanced green fluorescent protein (EGFP) across physiologically relevant temperatures (30−50 °C) and concentrations (40, 60, and 80 × 10−3 kg m−3). EGFP exhibited a concentration‐dependent decrease in Brownian velocity, from (1.47 ± 0.09) × 10−3 m s−1 to (0.35 ± 0.01) × 10−3 m s−1, at 30 °C, mimicking crowded cellular environments. Notably, the protein Brownian velocity increased linearly with temperature. These results demonstrate the suitability of concentrated suspensions for modeling intracellular crowding and validate luminescent nanothermometry for protein Brownian motion studies. Furthermore, the observed linear relationship between the logarithm of the protein Brownian velocity and concentration indicates that EGFP motion is not primarily driven by diffusion, but more of a ballistic transport.