NanoBRETA Novel BRET Platform for the Analysis
of Protein–Protein Interactions
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Abstract
Dynamic
interactions between proteins comprise a key mechanism
for temporal control of cellular function and thus hold promise for
development of novel drug therapies. It remains technically challenging,
however, to quantitatively characterize these interactions within
the biologically relevant context of living cells. Although, bioluminescence
resonance energy transfer (BRET) has often been used for this purpose,
its general applicability has been hindered by limited sensitivity
and dynamic range. We have addressed this by combining an extremely
bright luciferase (Nanoluc) with a means for tagging intracellular
proteins with a long-wavelength fluorophore (HaloTag). The small size
(19 kDa), high emission intensity, and relatively narrow spectrum
(460 nm peak intensity) make Nanoluc luciferase well suited as an
energy donor. By selecting an efficient red-emitting fluorophore (635
nm peak intensity) for attachment onto the HaloTag, an overall spectral
separation exceeding 175 nm was achieved. This combination of greater
light intensity with improved spectral resolution results in substantially
increased detection sensitivity and dynamic range over current BRET
technologies. Enhanced performance is demonstrated using several established
model systems, as well as the ability to image BRET in individual
cells. The capabilities are further exhibited in a novel assay developed
for analyzing the interactions of bromodomain proteins with chromatin
in living cells