title: Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli.
abstract: Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ∼0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.
authors: University of Michigan, Department of Chemistry, Ann Arbor, Michigan 48109, USA. wangda@umich.edu
authors: Da
authors: D
authors: Wang
authors: Tamiika K
authors: TK
authors: Hurst
authors: Richard B
authors: RB
authors: Thompson
authors: Carol A
authors: CA
authors: Fierke
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods; D015032:Zinc / Q000032:analysis* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D002256:Carbonic Anhydrases; D008164:Luminescent Proteins; D015032:Zinc; D000097573:Red Fluorescent Protein