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An evaluation of genetically encoded FRET-based biosensors for quantitative metabolite analyses in vivo

Paper ID Volume ID Publish Year Pages File Format Full-Text
23022 43409 2014 10 PDF Available
Title
An evaluation of genetically encoded FRET-based biosensors for quantitative metabolite analyses in vivo
Abstract

•The apparent Kd of FRET biosensors is sensitive to various environmental parameters.•The fluorescent proteins and the central metabolite binding domains are influenced.•Application for intracellular metabolites analyses requires careful calibration.•The in vitro calibration systems should mimic the in vivo system as far as possible.

A broad range of genetically-encoded fluorescence biosensors has been developed, allowing the detection of signaling intermediates and metabolites in real time. Many of these biosensors are based on Foerster Resonance Energy Transfer (FRET). The two biosensors of the well-known “Venus-flytrap” type exemplarily studied in this work are composed of a central sugar binding protein flanked by two green fluorescent protein derivatives, namely ECFP as well as Citrine and EYFP, respectively. In order to evaluate FRET-based biosensors as an in vivo tool for quantitative metabolite analyses, we have thoroughly studied the effects of pH, buffer salts, ionic strength, temperature and several intracellular metabolites on the signal intensity of both biosensors and both fluorescence proteins. Almost all micro-environmental variations led to considerably different FRET signals, because either the fluorescent proteins or the metabolite binding domains were affected by the tested parameters. This resulted not only in altered FRET ratios between the apo state and the saturated state but also in significant shifts of the apparent binding constant. This underlines the necessity of careful controls in order to allow reliable quantitative measurements in vivo.

Keywords
AxP, AMP ADP ATP; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; FP, fluorescent proteinGFP; EYFP; ECFP; Citrine; Intracellular metabolite analysis
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An evaluation of genetically encoded FRET-based biosensors for quantitative metabolite analyses in vivo
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 250–259
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us