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.
Highlights
Crystallographic studies of PBPs show that substrate binding often induces conformational changes, providing attractive scaffolds to make indicators[24,25,26,27]
To overcome the disadvantages of existing methods, we developed a series of single fluorescent protein (FP)-based histidine sensors with various affinities and large dynamic range by combining cpYFP with a bacterial periplasmic binding protein, HisJ
Various genetically encoded fluorescent sensors have been created to monitor cellular events and the microenvironment[17,28]. These sensors could be generally categorized into fluorescence resonance energy transfer (FRET)–based and single FP-based sensors[17,29]
Summary
Crystallographic studies of PBPs show that substrate binding often induces conformational changes, providing attractive scaffolds to make indicators[24,25,26,27]. To overcome the disadvantages (e.g., invasiveness of sample and low spatiotemporal resolution) of existing methods, we developed a series of single fluorescent protein (FP)-based histidine sensors with various affinities and large dynamic range by combining cpYFP with a bacterial periplasmic binding protein, HisJ. These sensors, dubbed FHisJ, allow for specific, sensitive, and quantitative monitoring of histidine metabolism in various subcellular compartments of mammalian cells
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