Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging

Abstract

Cyan fluorescent proteins (CFPs) derived from Aequorea victoria green fluorescent protein are the most widely used Förster resonant energy transfer (FRET) donors in genetically encoded biosensors for live-cell imaging and bioassays. However, the weak and complex fluorescence emission of cyan variants, such as enhanced cyan fluorescent protein (ECFP) or Cerulean, has long remained a major bottleneck in these FRET techniques. Recently, several CFPs with greatly improved performances, including mTurquoise, mTurquoise2, mCerulean3, and Aquamarine, have been engineered through a mixture of site-directed and large-scale random mutagenesis. This review summarizes the engineering and relative merits of these new cyan donors, which can readily replace popular CFPs in FRET imaging protocols, while reaching fluorescence quantum yields close to 90%, and unprecedented long, near-single fluorescence lifetimes of about 4 ns. These variants display an increased general photostability and much reduced environmental sensitivity, notably towards acid pH. These new, bright, and robust CFPs now open up exciting outlooks for fluorescence lifetime imaging microscopy and advanced quantitative FRET analyses in living cells. In addition, the stepwise engineering of Aquamarine shows that only two critical mutations in ECFP, and one in Cerulean, are required to achieve these performances, which brings new insights into the structural bases of their photophysical properties.

Intravital FRET: Probing Cellular and Tissue Function in Vivo

“…The acquisition of both donor and acceptor fluorescence decays leads back to unpredictable effects of a variable ddSNR also affecting the evaluation of the decay curves. New FRET constructs based on fluorescent proteins (as proposed by several groups in the last few years) with a well-characterized, mono-exponential fluorescence lifetime of the donors are one possible solution for this challenge [28,29]. Further efforts are currently being done (and are necessary) to translate the expertise on these constructs—gained under in vitro conditions—to transgenic mouse technology.…”

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

“…In the cyan variants (CFPs) derived from GFPs as Aquamarine, the aromatic residue is a tryptophan ( Figure 5A) (15,44). The photophysical and chemical properties of the chromophore can be influenced by modifications on the β-barrel in the chromophore pocket itself but also on more remote positions that may for example modify the overall protein flexibility or sensitivity to its environment.…”

“…An extended pH range should be tested for their use in the phagosome and a detailed characterization with ratiometric but also lifetime based methods might be useful to dissect the behavior of OxyFRET and PerFRET at low pHs. Those biosensors might be improved by replacing the donor, Cerulean, with less pH sensitive FPs such as Aquamarine or mTurquoise (44). In the meantime, a useful advice is to combine the use of H 2 O 2 biosensor with a pH sensor.…”

Molecular-Size Fluorescence Emitters

“…They include biological labeling to track and quantify individual or multiple protein species, probing to monitor protein-protein interactions, sensing to describe intracellular events and detecting different targets. Different applications have been realized using these proteins as FRET donors and acceptors in fluorescence lifetime imaging microscopy (FLIM) (Mérola et al 2014). However, their relatively large size (~26-28 kDa) may create problems by interference with the expression and folding of fused protein partners and affecting interactions with other molecules.…”