Use of fluorescence spectroscopy for quantitative investigations of ubiquitin interactions with the ubiquitin-binding domains of NEMO

Methods in Molecular Biology
Virginie DubosclardFabrice Agou

Abstract

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 signi...Continue Reading

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