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).
Highlights
Fluorescence Resonance Energy Transfer (FRET) between fluorescent proteins has become a popular tool to study the protein localization and biochemical conditions inside a living cell
The FRET pairs were created by cloning thrombin protease cleavage site and red fluorescent protein variants to NowGFP/ pQE-30 vector generating NowGFP-GGGSLVPRGS-RFP variant FRET pair
The R0 of these two FRET pairs is greater than the R0 of the other fluorescent protein based FRET pairs [2,24,25]
Summary
Fluorescence Resonance Energy Transfer (FRET) between fluorescent proteins has become a popular tool to study the protein localization and biochemical conditions inside a living cell. Cyan and Yellow variants of fluorescent proteins are widely used for FRET studies. With the development of Orange and Red variants of Fluorescent proteins, the CyanYellow FRET pairs are getting replaced as longer wavelength excitation and emission results in reduced cellular autofluorescence, decreased phototoxicity and lower light scattering [1,2]. Fluorescent Protein Based FRET Pairs with High Dynamic Range
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