Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

PLoS One. 2015 Aug 3;10(8):e0134436. doi: 10.1371/journal.pone.0134436. eCollection 2015.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins
  • Escherichia coli
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins*
  • Luminescent Proteins*
  • Microscopy, Fluorescence / methods*
  • Red Fluorescent Protein

Substances

  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Luminescent Proteins
  • Green Fluorescent Proteins

Grants and funding

LASKEMO graduate school is acknowledged for financial support by BGA. MK acknowledges the sabbatical year funding from Finnish Cultural Foundation (Suomen Kulttuurirahasto). KSS and ASM are supported by Russian Foundation for Basic Research (grant 13-04-01878-А). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.