image: 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
Mitochondrial calcium signaling is crucial for energy metabolism and calcium homeostasis, but monitoring it accurately is challenging due to pH fluctuations. Existing GECIs are often sensitive to pH changes, leading to artifacts and inaccurate measurements. Additionally, the limited availability of cyan fluorescent GECIs hinders multiplexed imaging.
Building on the bright cyan fluorescent protein mTurquoise2, TurCaMP offers an inverse response to Ca2+ transients and is insensitive to pH changes within the physiological range. This makes it a valuable tool for studying mitochondrial calcium signaling with high signal-to-noise ratio and minimal background noise.
TurCaMP's pH insensitivity and cyan fluorescence enable multiplexed imaging of calcium signals in different cellular compartments, opening new avenues for studying complex calcium-dependent physiological events.
This research showcases the potential of computational modeling and experimental validation in developing novel GECIs. TurCaMP represents a significant advancement in calcium imaging tools and holds great promise for advancing our understanding of mitochondrial calcium signaling and its role in various cellular processes and diseases.
The work entitled “A bright cyan fluorescence calcium indicator for mitochondrial calcium with minimal interference from physiological pH fluctuations”was published on Biophysics Reports (published on Oct., 2024).
Journal
Biophysics Reports
Method of Research
Experimental study
Subject of Research
Not applicable
Article Title
A bright cyan fluorescence calcium indicator for mitochondrial calcium with minimal interference from physiological pH fluctuations
Article Publication Date
3-Oct-2024