Image: mTurquoise2-based GECI with GCaMP-like design (TurCaMP0.1) and its variants showed minimal Ca2+-dependent changes. (IMAGE)
Caption
A Diagram showing the design of the TurCaMP0.1 sensor, generated by replacing cpEGFP in GCaMP6m with circularly permutated mTurquoise2 (cpmTq2). B In HEK293 cells transiently expressing TurCaMP0.1, typical traces of Ca2+ release and subsequent SOCE responses induced by 1 μmol/L thapsigargin (TG) are shown (n = 3). C–F Molecular mechanism of Ca2+-triggered fluorescence modulation in TurCaMP0.1 as revealed by molecular dynamics (MD) simulations. Superposition of Ca2+-free TurCaMP0.1 (blue) and Ca2+-bound (red) aligned using the β-barrel segment. Two of the β-sheets are set to be transparent to avoid obscuring the chromophore (C). The N-O distance between Nε1 on the indole ring of the chromophore and the O atom of E134 or water (D). The probability density distribution of the N-O distances between the chromophore’s Nε1 and O of water & E134. Without Ca2+ binding (middle panel), a dominant distribution is attributed to E134 (highlighted in the bottom-right corner). The probability density distribution of the N-O distance between the chromophore Nε1 and the carboxylate moiety of E134 is shown in the left panel (E). Close-up view of the conformational differences around the chromophore in Panel C, indicating the assignment of water molecules to the Ca2+-bound structure. Structural elements and residue in the Ca2+-free TurCaMP0.1 structure are labelled with a prime symbol and color in blue. With Ca2+ bound, the chromophore interacts with water and color in red. In Ca2+-free conditions, the chromophore is more likely to interact with E134 (F). G Computed fluorescence spectra of the chromophore in different chemical environments and protonation states using ab initio calculations. H Fluorescence excitation and emission spectra of TurCaMP0.1 at pH 7.2, in the presence (red line, 39 μmol/L CaCl2) and absence (blue line, 10 mmol/L EGTA) of Ca2+. I The LUMO of the excited state of the chromophore. Both the neutral (left) and deprotonated (right) states of the chromophore are shown. J Typical traces showing TurCaMP0.1-E134 variants lost fluorescence and responsiveness to Ca2+ in HEK293 cells. Data were from three independent biological replicates, and traces are shown as mean ± SEM
Credit
Wenjia Gu, Yuqin Yang, Yuqing Wang, Jia Li, Wanjie Li, Xiaoyan Zhang, Hao Dong, Youjun Wang
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