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      A Guide to Fluorescent Protein FRET Pairs

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          Abstract

          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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          A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

          Nathan Shaner, Gerard Lambert, Andrew Chammas … (2013)
          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.

            David Zacharias, Jonathan D Violin, Alexandra C Newton … (2002)
            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

              Amy Lam, François St-Pierre, Yiyang Gong … (2012)
              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.
              Article 0 comments Cited 311 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Niko Hildebrandt: Role: Academic Editor
                Igor Medintz: Role: Academic Editor
                Russ Algar: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Sensors (Basel)
                Journal ID (iso-abbrev): Sensors (Basel)
                Journal ID (publisher-id): sensors
                Title: Sensors (Basel, Switzerland)
                Publisher: MDPI
                ISSN (Electronic): 1424-8220
                Publication date (Electronic): 14 September 2016
                Publication date Collection: September 2016
                Volume: 16
                Issue: 9
                Electronic Location Identifier: 1488
                Affiliations
                [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.

                Article
                Publisher ID: sensors-16-01488
                DOI: 10.3390/s16091488
                PMC ID: 5038762
                PubMed ID: 27649177
                SO-VID: 4c2872e9-2f0f-4d11-ae31-3b3fdd14bfe7
                Copyright © © 2016 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                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/).

                History
                Date received : 21 July 2016
                Date accepted : 25 August 2016
                Categories
                Subject: Review

                ScienceOpen disciplines: Biomedical engineering
                Keywords: fluorescence resonance energy transfer (fret),biosensors,fluorescent proteins
                Data availability:
                ScienceOpen disciplines: Biomedical engineering
                Keywords: fluorescence resonance energy transfer (fret), biosensors, fluorescent proteins

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