Cyan and Yellow Super Fluorescent Proteins with Improved Brightness, Protein Folding, and FRET Förster Radius^,

Abstract

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.

Arabidopsis BIRD Zinc Finger Proteins Jointly Stabilize Tissue Boundaries by Confining the Cell Fate Regulator SHORT-ROOT and Contributing to Fate Specification

“…For mammalian constructs, JKD, BIB, SCR, and SHR coding sequences (CDSs) were amplified using primers described in Supplemental Table 1 and subcloned by ligation into mammalian vector containing mTurquoise, SYFP2, or mCherry under the constitutive cytomegalovirus promoter (Kremers et al, 2006;Goedhart et al, 2007Goedhart et al, , 2010. The resulting clones are listed in Supplemental Table 2.…”

PECTIN METHYLESTERASE48 Is Involved in Arabidopsis Pollen Grain Germination

“…A ligationindependent cloning method was used to insert the promoter of the PME48 gene (pPME48) upstream of the super YFP (Kremers et al, 2006) in a ligationindependent cloning binary vector, named pPLV06 (GenBank accession no. JF909459) and belonging to the set of plant ligation-independent cloning vectors made by De Rybel et al (2011).…”

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

“…This laser emits a continuous spectrum from 470 to 670 nm, within which any individual excitation wavelength in 1-nm increments can be selected. For excitation of sCFP3A and sYFP2 (Kremers et al, 2006), a diode laser (440 nm) or the 514-nm line of an argon laser was used, respectively. Confocal imaging was performed using internal filter-free spectral photomultiplier tube detectors.…”