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Engineering of New Fluorescent Proteins

Yasmina Bousmah (Assistant Engineer), Marie Erard (Professor), Hélène Pasquier (Associate Professor, HDR), Théo Beguin (PhD student 2022–)
Alumni: Fabienne Merola (Senior Researcher, retired in 2020)

In this research area, we study how the structure and dynamics of fluorescent proteins influence their key properties for imaging: brightness, photochromism, photobleaching, stability under varying temperature and pH, association equilibria, and oxidation reactions. This is an essential step for proposing improved variants adapted to imaging applications. We focus primarily on cyan and yellow variants of EGFP, which are the most popular partners for FRET experiments.

In these studies, we combine site-directed mutagenesis, UV-visible spectroscopy (steady-state and time-resolved absorption and fluorescence, circular dichroism), biochemical and structural analyses (mass spectrometry, crystallography, analytical ultracentrifugation, stopped-flow), as well as molecular dynamics and modeling approaches (in collaboration with the TheoSim team at ICP).

Model and STED image of tdLanYFP fused to the cytoskeleton (top) and pH-independent FRET signal of an Aquamarine-tdLanYFP tandem (bottom).

We recently demonstrated that the yellow dimeric fluorescent protein tdLanYFP [Bousmah2021], derived from a tetrameric protein discovered in the lancelet Branchiostoma lanceolatum, exhibits exceptional properties. It stands out with a quantum yield of 0.92, an extinction coefficient of 133,000 mol⁻¹·L·cm⁻¹, a pK₁/₂ of 3.9—very low compared to other YFPs—and a photostability approximately 10 times higher than that of YFPs, both in vitro and in cells.

Thanks to these outstanding performances under irradiation, tdLanYFP is compatible not only with STED nanoscopy (stimulated emission depletion), but also with single-molecule spectromicroscopies such as FCS (fluorescence correlation spectroscopy). Thus, tdLanYFP enriches the collection of fluorescent proteins suitable for microscopy techniques requiring intense irradiation, a domain where YFPs have so far been underrepresented due to their low photostability. Additionally, tdLanYFP proves to be an effective FRET partner, whether as a donor or acceptor. Its performance was notably demonstrated in a kinase AURKA activity biosensor [Bertolin2019].

YFPs gradually lose their fluorescence as pH decreases, which limits their utility in acidic cellular compartments. We have shown that this pH sensitivity is reduced when YFPs interact with synthetic proteins such as alphaRep, both in vitro and in living cells [Bousmah2024]. Specifically, alphaRep binding reduces the pKa of Citrine by 0.75 pH units, thereby decreasing its sensitivity to pH fluctuations. This effect can be generalized to other YFPs such as Venus and EYFP in vitro.

Citrine-alphaRep complex structure model. The presence of alphaRep renders the Citrine fluorescence level pH-insensitive in cells (top) by significantly reducing its pKa (bottom).