Ratiometric imaging of flux dynamics of cobalt with an optical sensor

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

“…This is because the FRET is dependent on the distance, the orientation of the fluorophores (dipole–dipole orientation), and the overlap of the spectral lines from the donor’s emission and acceptor’s absorption. − The N- and C-termini of SeBP, which are located at the distal extremities of the two lobes, should come together upon selenium binding, enhancing the fluorescence emission intensity ratio. Therefore, as ligands are added, the construct should move, which could alter the relative orientation of the connected fluorophores’ transition dipoles and change the FRET ratio. , A change in the fluorescent protein’s emission spectrum after selenium addition was detected by spectral analysis, indicating that the selenium-binding domain’s conformation changed to bring the two fluorophores together and cause FRET. In the absence of selenium, no modification in the analysis of the emission spectra was observed.…”

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…202 CobOS sensors utilized the wild-type CbiK P protein (CobOS-1.3μ), a mutant of the Glu Co 2+ -binding residue to Leu (CobOS-1.0μ), and a mutant of a His residue outside of the binding site to Leu (CobOS-86n). 170 Each mutation was chosen because it was reported to increase cobaltochelatase activity. 203 The three sensors had similar affinities for Co 2+ (K d = 0.86−1.3 μM) and similar in vitro dynamic ranges (∼1.1− 1.2), but chelator-induced reversibility was not demonstrated.…”

“…We described how promising metal ion-binding domains have been identified and implemented in initial FRET-based Co 2+ and Ni 2+ sensors, but further optimization is needed, as those discussed here have low dynamic ranges and reversibility was not evaluated. 170,171 In general, the discovery and testing of more metal ion-binding domains and structure-guided design of suitable metal ionbinding sites are needed to generate a wider variety of proteinbased sensors for essential metal ions beyond Ca 2+ and Zn 2+ . Here, researchers could take advantage of progress in the metalloprotein design field.…”

“…Only select bacteria and archaea can perform the 30-step synthesis of cobalamin, and humans must obtain it from their diets (as vitamin B12) . There is one reported series of fluorescent protein-based Co 2+ sensors . Here, FRET-based Co 2+ sensors (CobOS) were developed by combining ECFP and Venus with a cobalt-binding cobaltochelatase, CbiK P (and mutants), from D. vulgaris Hildenborough .…”

“…2 There is one reported series of fluorescent protein-based Co 2+ sensors. 170 Here, FRET-based Co 2+ sensors (CobOS) were developed by combining ECFP and Venus with a cobalt-binding cobaltochelatase, CbiK P (and mutants), from D. vulgaris Hildenborough. 201 CbiK P is a periplasmic protein that inserts cobalt into sirohydrochlorin, an intermediate in the synthesis of cobalamin.…”