Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

The transcription factor NF-kappaB is a key mediator of inflammation, the immune response, and cell death and survival. In response to activating signals, NF-kappaB translocates from the cytosol to the nucleus where it binds DNA sites in the promoter and enhancer regions of target genes. Through interactions with co-activator proteins, it activates the transcription of hundreds of response genes. NF-kappaB contains a natively-folded Rel-homology domain (RHD) which binds DNA and an intrinsically disordered transactivation domain (TD) which interacts with co-activator proteins. Previous biophysical studies of NF-kappaB have used truncated versions of the protein investigate RHD-DNA interactions and small segments of the TD to investigate coactivator interactions. Cell-based studies of full-length NF-kappaB have raised important questions regarding how NF-kappaB achieves robust and specific transcription activation that cannot be answered through in vitro studies using truncated forms of the protein. For example, it remains an open question whether the target DNA sequence bound by NF-kappaB can influence the recruitment of coactivator proteins. We have recently been able to purify full-length NF-kappaB including the TD, enabling biophysical characterization of the protein in its physiological form. We investigated interactions between full-length NF-kappaB and target DNA sites to better understand how the TD influences interactions with DNA. We also characterized interactions between NF-kappaB and the co-activators CBP-KIX and RPS3. Use of the full-length protein enables characterization of the formation of ternary NF-kappaB-DNA-coactivator complexes, providing a more complete understanding of the biophysical mechanism of transcription activation by NF-kappaB.