This paper introduces a groundbreaking revelation in the field of bioluminescence resonance energy transfer (BRET). It unveils, for the first time, that the enhanced BRET luminescence based on bacterial luciferase is achieved through a protein complex dimer mechanism. Within the dimeric system, the enhanced BRET luminescence outperforms the inherent bioluminescence of bacteria in both luminescence intensity and kinetics.

Abstract

Bacterial bioluminescence holds significant potential in the realm of optical imaging due to the inherent advantages of bioluminescence and ease of operation. However, its practical utility is hindered by its low light intensity. The fusion of bacterial luciferase with a highly fluorescent protein has been demonstrated to significantly enhance autonomous luminescence. Nevertheless, the underlying mechanism behind this enhancement remains unclear, and there is a dearth of research investigating the mechanistic aspects of bioluminescence resonance energy transfer (BRET) luminescence, whether it occurs naturally or can be achieved through experimental means. In this study, we investigated the phenomenon of bacterial luciferase-based BRET luminescence employing a range of computational techniques, including structural modeling, molecular docking, molecular dynamics simulations, as well as combined quantum mechanics and molecular mechanics calculations. The theoretical findings suggest that the BRET luminescence occurs through resonance energy transfer between the excited bioluminophore and the ground chromophore within the protein complex dimer. The proposed mechanism of the protein complex dimer offers a microscopic understanding of the intriguing BRET phenomenon and has the potential to inspire further practical applications in the field of optical imaging.