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

ABSTRACT: The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/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-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemi

SUBMITTER: Chen W 

PROVIDER: S-EPMC10688598 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature