Effect of Ca²⁺ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER

Abstract

We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ.

Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER+

“…For the indirectly excited (at 372 nm) R∗O − form, a fast quenching component within the first 2 ns was detected, which was followed by a long asymptotic decay closely approaching the lifetime of the directly excited R∗O − . Further lifetime studies in D 2 O confirmed that such a lifetime increase of G-CatchER + is a result of delayed proton geminate recombination and is caused by a combination of acid-base equilibrium and arrangement differences of the proton network with the chromophore between Ca 2+ free and the Ca 2+ binding form ( Zhuo et al., 2015 ). Ca 2+ also exhibited a strong inhibition of the excited state proton transfer nonadiabatic geminate recombination in protic (versus deuteric) medium ( Figure S2 ).…”

Cytosolic NADH-NAD+Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

“…Several other nonratiometric single-FP sensors also show a substantial change in fluorescence lifetime, including the cytosolic and endoplasmic reticulum calcium sensors, RCaMP (28) and CatchER (31). Direct mutation of the betabarrel of the GFP to make it more conformationally sensitive has also successfully produced a single-FP sensor for PKA with a usable lifetime change (6).…”

Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

“…The first ER-targeted genetically-encoded Ca 2+ indicator (GECI) was an aequorin construct specifically targeted to the ER lumen that, however, has been used exclusively for measurements in cell populations [ 10 ]. As to GECIs suitable for single cell analysis, in the last decade different single fluorescent protein (FP) Ca 2+ sensors targeted to the ER have been generated: R-CEPIA1er and G-CEPIA1er [ 11 ], ER-LAR-GECO1 [ 12 ], CatchER [ 13 ], GCaMPer [ 14 ]. These non-ratiometric probes are, in general, very difficult to calibrate in live cells and their use is primarily qualitative.…”