| Autor: |
Asanova AN; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Subach OM; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Myachina SA; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Evteeva MA; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Gunitseva NM; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Borisova AA; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Patrushev MV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Vlaskina AV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Nikolaeva AY; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Yang L; School of Life Sciences, Westlake University, Hangzhou 310024, China.; Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, China.; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, China., Gabdulkhakov A; Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia., Dronova E; Moscow Insitite of Physics and Technology, Dolgoprudny 141700, Russia., Samygina VR; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Xiao X; School of Life Sciences, Westlake University, Hangzhou 310024, China.; Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, China.; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, China., Zhao H; Chinese Institute for Brain Research, Beijing 102206, China., Piatkevich KD; School of Life Sciences, Westlake University, Hangzhou 310024, China.; Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, China.; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, China., Subach FV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia. |
| Abstrakt: |
Branched-chain amino acids (BCAAs) play an important role in the functioning of mammalian cells and the central nervous system. However, available genetically encoded indicators for BCAAs are based on Förster resonance energy transfer and have a limited dynamic range. We developed a single fluorescent protein-based sensor for BCAAs, called NeIle, which is composed of circularly permutated mNeonGreen protein inserted into the leucine-isoleucine-valine binding protein (LIVBP) from Escherichia coli bacteria. In solution, the NeIle indicator displayed a positive fluorescence response to adding isoleucine, leucine, and valin amino acids with high Δ F / F dynamic ranges of 27-, 19-, and 11-fold and the corresponding affinity values of 5.0, 2.9, and 75 mM, respectively. The spectral and biochemical properties of the NeIle indicator were characterized in solution. We characterized the brightness of the NeIle indicator in living mammalian cells, including cultured neurons. Using the NeIle indicator, we successfully visualized the dynamics of isoleucine transients in different organelles of mammalian cells. We obtained and analyzed the X-ray crystal structure of the NeIle indicator in an isoleucine-bound state. Structure-guided directed mutagenesis of the NeIle indicator revealed the basis of its fluorescence response and selectivity to isoleucine. |
