Use of Fluorescence Spectroscopy for Quantitative Investigations of Ubiquitin Interactions with the Ubiquitin-Binding Domains of NEMO

Virginie Dubosclard (1) , Elisabeth Fontan (1) , Fabrice Agou (1)

1 SMAC - Signalisation Moléculaire et Activation Cellulaire

Ubiquitin serves as a signal for a variety of cellular processes and its specifi c interaction with ubiquitin-binding domain (UBD) regulates key cellular events including protein degradation, cell-cycle control, DNA repair, and kinase activation. Several binding mechanisms for isolated UBDs have been reported in recent years. However, little is known about the mechanism through which proteins containing multiple-UBDs achieve specifi city for a particular oligomer of polyUb. The NF-κB essential modulator (NEMO, also known IKKγ), which plays a key role in the NF-κB signaling pathway, belongs to the latter family of proteins since it contains two distal NOA (also known UBAN/CC2-LZ/NUB) and ZF UBDs, separated by an unstruc-tured proline-rich linker of about 40 residues in length. Here, we show a new procedure for fast purifi ca-tion of this bipartite domain. We also describe the use of intrinsic fl uorescence spectroscopy for quantitative investigations of ubiquitin interactions between two distal ubiquitin-binding domains of NEMO (NOA and ZF). This spectroscopic method has many advantages over other techniques like GST pulldown and Biacore's SPR for monitoring avid interactions between two UBDs, especially when UBDs are located at signifi cant distance from each other within the protein.

Mots clés: Intrinsic fluorescence spectroscopy; NEMO; Ubiquitin-binding domains; NF-κB; Polyubiquitin chains; Protein purification

Domaines: Sciences du Vivant [q-bio]; Biophysique; Physique Nucléaire Expérimentale [nucl-ex]

Virginie Dubosclard, Elisabeth Fontan, Fabrice Agou. Use of Fluorescence Spectroscopy for Quantitative Investigations of Ubiquitin Interactions with the Ubiquitin-Binding Domains of NEMO. NF-Kappa B Methods and Protocols, 1280, pp.321-337, 2015, NF-Kappa B Methods in Molecular Biology, ⟨10.1007/978-1-4939-2422-6_19⟩. ⟨pasteur-01128925⟩