Mechanistic Investigation of Green Fluorescent Protein Acquiring Energy for Emitting Light: A Theoretical Study

Green fluorescent protein (GFP) is famous for noninvasively observing the internal biological processes of cells and organisms, revolutionizing the field of cell biology. GFP was first discovered in jellyfish <i>Aequorea victoria</i> (<i>AV</i>). The GFP bioluminescence (BL) in <i>AV</i> can be divided into three stages: the first singlet excited state coelenteramide (S₁-CTD) is formed in aequorin; GFP acquires energy from S₁-CTD via an energy transfer (ET) process; and GFP emits green light. The first and final stages have been well studied, whereas the detailed mechanism of the second stage remains unclear, with only sporadic experimental evidence. The purpose of this study is to clarify how GFP acquires energy before emitting green light in <i>AV</i>. Through protein-protein docking, molecular dynamics simulations, and combined quantum mechanics and molecular mechanics calculations, we demonstrate that the ET process occurs via the Förster resonance energy transfer (FRET) mechanism. The calculated FRET rate is faster than the radiative and nonradiative decay ones of S₁-CTD, which means the ET process can occur efficiently. Additionally, the calculated fluorescence quantum yield explains the experimentally observed BL enhancement after the ET. This is the first theoretical report on the ET mechanism in BL. This study not only clearly interprets how GFP acquires energy for emitting light but also helps to understand the ET mechanism in other bioluminescent systems and sheds new light on bioluminescence resonance energy transfer.