title: Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.
abstract: Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).
authors.affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101, Tampere, Finland.
authors.forename: Bobin
authors.initials: B
authors.lastname: George Abraham
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Sarkisyan
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mishin
authors.affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101, Tampere, Finland.
authors.forename: Ville
authors.initials: V
authors.lastname: Santala
authors.affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101, Tampere, Finland.
authors.forename: Nikolai V
authors.initials: NV
authors.lastname: Tkachenko
authors.affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101, Tampere, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Karp
mesh_terms: D001426:Bacterial Proteins; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein