Improving FRET dynamic range with bright green and red fluorescent proteins

By A. Lam, F. St-Pierre, Yiyang Gong, Jesse D. Marshall, Paula J. Cranfill, M. A. Baird, Michael R Mckeown, J. Wiedenmann, Michael W. Davidson, Mark J. Schnitzer and 2 more. Nature Methods, 2012

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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 CFPs and YFPs, 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. 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 with these two proteins 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.

Subfields: Biophysics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/cd361ae4a66f1b124be8767fad62b690e34eeaa5

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Citations in the corpus

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis (2018, background): “Davidson (74): Addgene plasmid 54561], pCytERM-mScarlet-N1 [gift from D.”
Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity* (2016, methodology): “We generated mutant proteins in the context of fluorescent fusions of Shrm SD2 (His10-mRuby2-1427–1610) and Rock SBD (His10-Clover707–946) (46).”
PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals (2017, methodology): “Full-length human PEBP1 and related mutants were cloned into a pET21-derived (EMDMillipore, Billerica, MA) bacterial expression plasmid modified to express PEBP1 with N-terminal His10- and mRuby2 tags (Lam et al., 2012).”
A Guide to Fluorescent Protein FRET Pairs (2016, methodology): “For example, RhoA activation in neuronal growth cones during ephrinA-stimulated retraction exhibits only a 5% FRET change with Clover-mRuby2 [2].”
A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum (2013, background): “1); EB3 (7 aa), coding sequence cloned between BglII and BamHI (EB3; cDNA source: L. Cassimeris, Lehigh University; NM_012326.”
Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics (2013, background): “RCaMP1d was found to contain the mutation Ala270Val; the corresponding mRuby mutation, Ala120Val, was independently observed recently during the generation of mRuby2 (Lam et al., 2012), which exhibits greater brightness and photostability.”
The growing and glowing toolbox of fluorescent and photoactive proteins (2016, methodology): “mClover3 was engineered from avGFP by combining key insights accumulated from years of evolution, including identification of positions that influenced the H-bonding network surrounding the chromophore, stacking residues that shift the energy of emission, and residues that influence photostability a”