Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Rational design of semisynthetic FRET biosensors (#216)

Joshua A Mitchell 1 , Jason H. Whitfield 1 , Will H. Zhang 1 , Megan L. O'Mara 1 , Colin Jackson 1
  1. The Australian National University, Acton, ACT, Australia

Ratiometric fluorescent sensors are a powerful technology for imaging and measuring small molecules in biological systems. Engineering protein biosensors is widely practiced by fusing fluorescent proteins to either terminus of a solute binding protein, and taking advantage of the conformational change associated with binding to alter the distance between fluorophores. The resulting change in Förster Resonance Energy Transfer (FRET) can yield a ratiometric sensor. While proteins that exhibit a conformational change upon substrate binding are available for a huge number of sensor targets, simply fusing fluorescent proteins to such a protein is often insufficient to develop a useful sensor; the inherent interdomain flexibility of fusion proteins is thought to introduce noise that dampens the sensor's signal in bulk solution. We have found that we can fix one fluorophore in position by replacing one of the fluorescent proteins with a chemically labelled synthetic dye. This technique allows us to rapidly engineer sensors for diverse analytes with excellent sensitivity and requiring little-to-no experimental optimisation.