NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
Andrew S. Dixon,Marie K. Schwinn,Mary P. Hall,Kris Zimmerman,Paul Otto,Thomas H. Lubben,Braeden L. Butler,Brock Binkowski,Thomas Machleidt,Thomas A. Kirkland,Monika G. Wood,Christopher T. Eggers,Lance P. Encell,Keith V. Wood +13 more
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TLDR
NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF and that the reporter responds reliably and dynamically in cells.Abstract:
Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M–1 s–1, koff = 0.2 s–1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction...read more
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Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.
Philip Knuckles,Tina Lence,Irmgard U. Haussmann,Dominik Jacob,Nastasja Kreim,Sarah H. Carl,Sarah H. Carl,Irene Masiello,Tina Hares,Rodrigo Villaseñor,Rodrigo Villaseñor,Daniel Hess,Miguel A. Andrade-Navarro,Marco Biggiogera,Mark Helm,Matthias Soller,Marc Bühler,Marc Bühler,Jean-Yves Roignant +18 more
TL;DR: Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] is identified as a novel interactor of m6A methyltransferase complex components in Drosophila and mice and it is demonstrated that Flacc promotes m 6A deposition by bridging Fl( 2)d to the mRNA-binding factor Nito.
Journal ArticleDOI
Illuminating G-Protein-Coupling Selectivity of GPCRs.
Asuka Inoue,Asuka Inoue,Francesco Raimondi,Francois Marie Ngako Kadji,Gurdeep Singh,Takayuki Kishi,Akiharu Uwamizu,Yuki Ono,Yuji Shinjo,Satoru Ishida,Nadia Arang,Kouki Kawakami,J. Silvio Gutkind,Junken Aoki,Robert B. Russell +14 more
TL;DR: This work systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini, and identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which were used to develop a coupling predictor that outperforms previous methods.
Journal ArticleDOI
NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.
TL;DR: This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.
Journal ArticleDOI
Structure of the neurotensin receptor 1 in complex with β-arrestin 1.
Weijiao Huang,Matthieu Masureel,Qianhui Qu,John Janetzko,Asuka Inoue,Hideaki E. Kato,Hideaki E. Kato,Michael J. Robertson,Khanh Nguyen,Jeffrey S. Glenn,Georgios Skiniotis,Georgios Skiniotis,Brian K. Kobilka +12 more
TL;DR: It is found that phosphorylation of NTSR1 is critical for the formation of a stable complex with β-arrestin 1, and phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction are identified.
Journal ArticleDOI
Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.
Qingwen Wan,Najeah Okashah,Asuka Inoue,Rony Nehmé,Byron Carpenter,Christopher G. Tate,Nevin A. Lambert +6 more
TL;DR: Mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells and should be useful for discovering and characterizing G protein subtype–biased ligands.
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