Site-specific incorporation of biophysical probes into NF-κB with non-canonical amino acids

The transcription factor NF-κB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-κB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-κB/IκBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-κB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IκBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-κB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-κB to include p-benzoylphenylalanine (pBpa) for UV cross-linking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-κB RelA subunit which includes the intrinsically disordered transactivation domain.