Effect of enhanced Renilla luciferase and fluorescent protein variants on the Foerster distance of Bioluminescence resonance energy transfer (BRET)
- CSIRO Food Futures Flagship and Ecosystem Sciences, Canberra (Australia)
- Laboratory for Molecular Endocrinology-GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, The University of Western Australia, Perth (Australia)
Highlights: Black-Right-Pointing-Pointer First experimental determination of Foerster distance (R{sub 0}) for enhanced BRET systems. Black-Right-Pointing-Pointer Effect of brighter BRET components RLuc2, RLuc8 and Venus was assessed. Black-Right-Pointing-Pointer Using brighter BRET components substantially increased (25%) R{sub 0} of the BRET{sup 1} system. Black-Right-Pointing-Pointer Using brighter BRET components marginally increased (2-9%) R{sub 0} of the BRET{sup 2} system. Black-Right-Pointing-Pointer Brighter BRET components improve the different weaknesses of BRET{sup 1} and BRET{sup 2} 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. Foerster distance (R{sub 0}), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R{sub 0} 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 R{sub 0} for BRET{sup 1} 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 R{sub 0} of the original BRET{sup 1} system. R{sub 0} for BRET{sup 2} systems combining green fluorescent proteins (GFP{sup 2}) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET{sup 2} system despite being {approx}30-fold brighter.
- OSTI ID:
- 22210219
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 425, Issue 3; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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