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      A Guide to Fluorescent Protein FRET Pairs


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          Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

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          Most cited references102

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          A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

          Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.
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            Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

            Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
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              Improving FRET dynamic range with bright green and red fluorescent proteins

              A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.

                Author and article information

                Role: Academic Editor
                Role: Academic Editor
                Role: Academic Editor
                Sensors (Basel)
                Sensors (Basel)
                Sensors (Basel, Switzerland)
                14 September 2016
                September 2016
                : 16
                : 9
                : 1488
                [1 ]Medical Scientist Training Program, University of California, Los Angeles, CA 90095, USA; BBajar@ 123456mednet.ucla.edu
                [2 ]Harvard College, Cambridge, MA 02138, USA; emilyswang@ 123456college.harvard.edu
                [3 ]Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China; shu.zhang@ 123456siat.ac.cn
                [4 ]Departments of Bioengineering and Neurobiology, Stanford University, CA 94305, USA; mzlin@ 123456stanford.edu
                Author notes
                [* ]Correspondence: jun.chu@ 123456siat.ac.cn ; Tel.: +86-755-8639-2220

                These authors contributed equally to this work.

                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                : 21 July 2016
                : 25 August 2016

                Biomedical engineering
                fluorescence resonance energy transfer (fret),biosensors,fluorescent proteins


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