Use of fluorescence spectroscopy for quantitative investigations of ubiquitin interactions with the ubiquitin-binding domains of NEMO.
Ubiquitin serves as a signal for a variety of cellular processes and its specific 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 specificity 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 unstructured proline-rich linker of about 40 residues in length. Here, we show a new procedure for fast purification of this bipartite domain. We also describe the use of intrinsic fluorescence 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 significant distance from each other within the protein.
Unité de Signalisation Moléculaire et Activation Cellulaire, Département de Biologie Cellulaire et Infection, Institut Pasteur, 25 rue du Dr. Roux, 75015, Paris, France.
Virginie
V
Dubosclard
Elisabeth
E
Fontan
Fabrice
F
Agou
D002942:Circular Dichroism; D051550:I-kappa B Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D025801:Ubiquitin / Q000302:isolation & purification / Q000378:metabolism*
C491731:IKBKG protein, human; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D051550:I-kappa B Kinase