Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET)

Highlights: ► First experimental determination of Förster distance (R0) for enhanced BRET systems. ► Effect of brighter BRET components RLuc2, RLuc8 and Venus was assessed. ► Using brighter BRET components substantially increased (25%) R0 of the BRET1 system. ► Using brighter BRET components marginally increased (2–9%) R0 of the BRET2 system. ► Brighter BRET components improve the different weaknesses of BRET1 and BRET2 systems. -- Abstract: Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R0), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R0 provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor–acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R0 for BRET1 systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R0 of the original BRET1 system. R0 for BRET2 systems combining green fluorescent proteins (GFP2) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2–9% greater than the original BRET2 system despite being ∼30-fold brighter.