title: Genetically encoded FRET-based nanosensor for in vivo measurement of leucine.
abstract: Besides fundamental role in protein synthesis, leucine has metabolic roles as energy substrates, precursors for synthesis of other amino acids and as a modulator of muscle protein synthesis via the insulin-signaling pathway. Leucine concentration in cell and tissue is temporally dynamic as the metabolism of leucine is regulated through multiple enzymes and transporters. Assessment of cell-type specific activities of transporters and enzymes by physical fractionation is extremely challenging. Therefore, a method of reporting leucine dynamics at the cellular level is highly desirable. Given this, we developed a series of genetically encoded nanosensors for real-time in vivo measurement of leucine at cellular level. A leucine binding periplasmic binding protein (LivK) of Escherichia coli K12 was flanked with CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) at N-terminus and C-terminus, respectively. The constructed nanosensors allowed in vitro determination of fluorescence resonance energy transfer (FRET) changes in a concentration-dependent manner. These sensors were found to be specific to leucine, and stable to pH-changes within a physiological range. Genetically encoded sensors can be targeted to a specific cell type, and allow dynamic measurement of leucine concentration in bacterial and yeast cells.
authors.affiliation: Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Hamdard University, New Delhi 110062, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Mohsin
authors.forename: M Z
authors.initials: MZ
authors.lastname: Abdin
authors.forename: Lata
authors.initials: L
authors.lastname: Nischal
authors.forename: Hemant
authors.initials: H
authors.lastname: Kardam
authors.forename: Altaf
authors.initials: A
authors.lastname: Ahmad
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007930:Leucine / Q000032:analysis* / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C024913:LivK protein, E coli; D008164:Luminescent Proteins; D033902:Periplasmic Binding Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D007930:Leucine
keywords: FRET; Fluorescent protein; Genetically encoded nanosensor; Leucine; Periplasmic binding protein