Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging

Abstract

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent performances on the popular form ECFP, leading to Aquamarine (QY = 0.89, τ(f) = 4.12 ns). Besides an impressive pH stability (pK(1/2) = 3.3), Aquamarine shows a very low general sensitivity to its environment, and undetectable photoswitching reactions. Aquamarine gives efficient and bright expression in different mammalian cell systems, with a long and single exponential intracellular fluorescence lifetime mostly insensitive to the fusion or the subcellular location of the protein. Aquamarine was also able to advantageously replace the CFP donor in the FRET biosensor AKAR for ratiometric measurements of protein kinase A activity. The performances of Aquamarine show that only two rounds of straightforward single point mutagenesis can be a quick and efficient way to optimize the donor properties in FRET-based biosensors.

Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9

“…Control or treated cells were plated on glass cover slips and incubated with 4 μM of the dye for 10 min at 37 • C. Its fluorescence variations upon addition of MβCD (10 mM, 10 min) were easily detectable in Fluorescence Lifetime Imaging or FLIM (Owen et al, 2006). Fluorescence lifetimes were measured with a time-resolved laser scanning TCSPC microscopy setup developed on a TE2000 microscope equipped with a 60x, NA 1.2 water immersion objective (Erard et al, 2012). The pulsed laser diode provided the excitation at 440 nm (PicoQuant) and the fluorescence was collected above 530 nm (Long Pass, Omega).…”

Shigella flexneridisruption of host cell-cell tension promotes intercellular spread

“…HeLa cells expressing t-farnesyl-RFP were seeded at 6×10 5 cells per well on coverslips. The next day, cells were infected at an MOI of 100, as above, with designated strains expressing pROEX-Aqua [Addgene; plasmid #42889, (Erard et al, 2013)]. At 30 minutes of infection, media was replaced with DMEM supplemented with 0.45% glucose, 1.2% arabinose, 25 µg/mL gentamicin, 1 mM IPTG, and 10% FBS.…”

Environmental Effects on Reactive Oxygen Species Detection—Learning from the Phagosome

“…Aquamarine and EGFP are proteins derived from the GFP of the jellyfish Aequorea victoria while mCherry and mStrawberry were engineered from DsRed, a protein of the coral Discosoma sp. (Table 1) (15,21). Even if they share the same 3D structure, FPs derived from different species have low sequence identity and may exhibit different reactivity toward ROS.…”

“…At pHs lower than about two pH units below pK a , the fluorescence itself and its variations can be significantly decreased (4,9,21,22). Green and yellow FPs (GFP, YFP, Venus, Citrine) that bear a tyrosine in their chromophore have pK a values between pH 5.7 and 6.9 (64) that may increase in presence of high chloride concentrations (6).…”

“…We focused on the CFP, Aquamarine, and on the two RFPs mCherry and mStrawberry, which all have a pK a below 4.5 (Table 1) (21,64). EGFP has been successfully used as a HOCl biosensor expressed in the cytosol of E.…”