title: NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions.
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.
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.affiliation: ‡Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States.
authors.forename: Carolyn C
authors.initials: CC
authors.lastname: Woodroofe
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Marie K
authors.initials: MK
authors.lastname: Schwinn
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Jacqui
authors.initials: J
authors.lastname: Méndez
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Robers
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Kris
authors.initials: K
authors.lastname: Zimmerman
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Otto
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Danette L
authors.initials: DL
authors.lastname: Daniels
authors.affiliation: ‡Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kirkland
authors.affiliation: †Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States.
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps
chemical_list: D005456:Fluorescent Dyes; D049410:Luciferases, Renilla