George Abraham Bobin, Sarkisyan Karen S, Mishin Alexander S, Santala Ville, Tkachenko Nikolai V, Karp Matti
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101, Tampere, Finland.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997, Moscow, Russia.
PLoS One. 2015 Aug 3;10(8):e0134436. doi: 10.1371/journal.pone.0134436. eCollection 2015.
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).
利用荧光蛋白变体的荧光共振能量转移(FRET)被广泛用于研究活细胞中的生化过程。通过荧光寿命测量进行FRET检测是测量FRET最直接、最可靠的方法。传统的基于蓝绿色荧光蛋白的FRET对正逐渐被绿色-红色荧光蛋白变体所取代。绿色-红色对能够在更长波长处激发,这在监测FRET时可减少细胞自发荧光和光毒性。尽管基于FRET的传感器取得了进展,但低FRET效率和动态范围仍使其在细胞生物学和高通量筛选中的应用变得复杂。在本文中,我们利用了NowGFP较长的寿命并筛选红色荧光蛋白变体,以开发具有高动态范围和FRET效率的FRET对。通过蛋白水解活性分析FRET变化,并通过稳态和时间分辨测量进行检测。基于这些结果,NowGFP-tdTomato和NowGFP-mRuby2作为具有大荧光寿命动态范围的FRET对显示出很高的潜力。体外测量表明,NowGFP-tdTomato对于生物研究中使用的任何基于荧光蛋白的FRET对具有最高的Förster半径。所开发的FRET对将有助于设计基于FRET的传感器以及采用荧光寿命成像显微镜(FLIM)的研究。