A genetically encoded toolkit for tracking live-cell histidine dynamics in space and time

Abstract

High-resolution spatiotemporal imaging of histidine in single living mammalian cells faces technical challenges. Here, we developed a series of ratiometric, highly responsive, and single fluorescent protein-based histidine sensors of wide dynamic range. We used these sensors to quantify subcellular free-histidine concentrations in glucose-deprived cells and glucose-fed cells. Results showed that cytosolic free-histidine concentration was higher and more sensitive to the environment than free histidine in the mitochondria. Moreover, histidine was readily transported across the plasma membrane and mitochondrial inner membrane, which had almost similar transport rates and transport constants, and histidine transport was not influenced by cellular metabolic state. These sensors are potential tools for tracking histidine dynamics inside subcellular organelles, and they will open an avenue to explore complex histidine signaling.

MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water

“…Conformation‐based biosensors provide more versatility than fluorescent protein variants, as various ligand binding proteins can be simply attached to fluorescent proteins . Moreover, combination of natural ligand binding proteins with fluorescent proteins allows large dynamic range . In consistent with this study, we similarly obtained a larger dynamic range compared with that of GFP mutant .…”

Glucose monitoring in living cells with single fluorescent protein-based sensors

“…1D, Table 1). Similar to other cpYFP-based sensors, 17,18 FGBP 3.1 mM has two typical excitation peaks around 420 and 500 nm and one emission peak near 515 nm (Fig. 1E).…”

“…Recently, we have reported a series of metabolite sensors, including NADH (Frex), 14 NAD+/NADH ratio (SoNar), 15,16 NADPH (iNap) 17 and histidine (FHisJ) 18 based on circularly permuted yellow uorescent protein (cpYFP). In cpYFP, the original N-and C-termini are fused by a polypeptide linker, and new termini are introduced close to the uorophore, making its uorescence highly sensitive to the protein's conformation.…”

“…In cpYFP, the original N-and C-termini are fused by a polypeptide linker, and new termini are introduced close to the uorophore, making its uorescence highly sensitive to the protein's conformation. [14][15][16][17][18] Compared with FRET-based sensors, cpYFP-based sensors oen have larger uorescence changes; in specic, Frex, 14 SoNar, 15,16 iNap 17 and FHisJ 18 sensors have more than 500% dynamic range, rendering them capable of tracking subtle metabolic changes.…”

In vivoglucose imaging in multiple model organisms with an engineered single-wavelength sensor

“…Our lab has recently developed a technique for engineering single-wavelength indicators from circularly permuted green fluorescent protein (cpGFP) and periplasmic binding proteins for a variety of small molecules: glutamate 24,25 , GABA 26 , maltose 27 , phosphonates 28 , among others. Another group has also used this technique to develop sensors for histidine 29 and recently glucose (sensor “FGBP” made using E. coli GGBP) 30 . FGBP was specific for D-glucose and D-galactose over other sugars tested, and produced a ∼200% change in fluorescence excitation ratio in E. coli cells upon addition of glucose.…”