NeIle, a Genetically Encoded Indicator for Branched-Chain Amino Acids Based on mNeonGreen Fluorescent Protein and...

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Branched-chain amino acids (BCAAs) are crucial for mammalian cell and central nervous system function.
• Existing genetically encoded BCAA indicators have limitations, particularly a restricted dynamic range.

Purpose of the Study:

• To develop a novel, single fluorescent protein-based sensor for BCAAs with an improved dynamic range.
• To characterize the sensor's properties and visualize BCAA dynamics in living cells.

Main Methods:

• Development of the NeIle sensor by fusing circularly permuted mNeonGreen with the leucine-isoleucine-valine binding protein (LIVBP).
• Biochemical and spectral characterization of NeIle in solution and in living mammalian cells.
• X-ray crystallography to determine the structure of NeIle in an isoleucine-bound state.

Main Results:

• NeIle demonstrated a positive fluorescence response to isoleucine, leucine, and valine with high dynamic ranges (27-, 19-, and 11-fold).
• The sensor exhibited high affinity for leucine (2.9 mM) and isoleucine (5.0 mM).
• NeIle successfully visualized isoleucine dynamics in various organelles within living mammalian cells, including neurons.

Conclusions:

• NeIle is a sensitive and versatile fluorescent sensor for BCAAs, overcoming limitations of previous indicators.
• The sensor's structure-guided design provides insights into its response mechanism and selectivity.
• NeIle enables real-time monitoring of BCAA dynamics in cellular and subcellular compartments.