title: 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.
authors.affiliation: CNRS, Université Paris Sud, Laboratoire de Chimie Physique, Orsay, France. fabienne.merola@u-psud.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Mérola
authors.forename: Asma
authors.initials: A
authors.lastname: Fredj
authors.forename: Dahdjim-Benoît
authors.initials: DB
authors.lastname: Betolngar
authors.forename: Cornelia
authors.initials: C
authors.lastname: Ziegler
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins
chemical_list: C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins
keywords: Aquamarine; Cerulean; FLIM (fluorescence lifetime imaging microscopy); FRET (Förster resonant energy transfer); mTurquoise