BRET, like FRET, is also used to investigate conformational changes
and/or protein–protein interactions. However, BRET uses bioluminescent
luciferase enzymes as donor molecules rather than fluorophores and does
not require excitation from an external light source – a process that
can cause photobleaching, autofluorescence, and light scattering. This
means BRET has a very low background and increased signal-to-noise ratio
compared to FRET (Dale et al., 2019). Renilla luciferase (RLuc),
isolated from the Renilla reniformis sea pansy, was one of the
first luciferase enzymes to be used in BRET (Dale et al., 2019). In the
case of RLuc, the luciferase enzyme catalyses the ATP-independent
oxidation of a luciferase substrate, coelenterazine (Dale et al., 2019).
The luciferase donor molecule non-radiatively transfers energy to an
acceptor fluorophore if the pair is in close proximity, in a manner
similar to FRET (Figure 1B). One of the latest developments in BRET has
been the introduction of a superior luciferase enzyme called
Nanoluciferase (NLuc) (Dale et al., 2019). NLuc is a 19 kDa subunit
derived from a larger, multi-subunit luciferase isolated from the
deep-sea shrimp Oplophorus gracilirostris (Hall et al., 2012).
NLuc has several advantages over RLuc: it is more stable; it is
approximately 150 times brighter; and its smaller size means steric
hindrance is less likely (Dale et al., 2019).
NLuc has been further exploited to produce an NLuc-derived split
luciferase reporter system termed NanoBiT. NanoBiT is composed of two
NLuc derived fragments: an 11 amino acid-long small bit (SmBiT) or high
bit (HiBiT) fragment; and a second large bit fragment (LgBiT) (17.6
kDa). These fragments are genetically fused to separate parts of the
same protein or two distinct proteins. When in proximity, the two
fragments can reconstitute and produce a luminescent signal. HiBiT is
commonly used for ligand binding assays (Soave et al., 2016, Stoddart et
al., 2015), whereas SmBiT is better suited to investigate
protein–protein interactions due to its lower affinity for LgBiT
(Figure 1C) (Lohse et al., 2012).