| Autor: |
Subach OM; 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., Agapova YK; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Korzhenevskiy DA; Laboratory of Electrophysiology, Federal Center of Brain Research and Neurotechnologies, Ostrovityanova Str. 1, Bld. 10, Moscow 125367, Russia., Nikolaeva AY; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., Varizhuk AM; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.; Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia., Subach MF; Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia., Patrushev MV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia., 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., Boyko KM; Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld. 2, Moscow 119071, Russia., Subach FV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', Moscow 123182, Russia. |
| Abstrakt: |
NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca 2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively. |
