BPS2026 – X-ray footprinting/mass spectrometry provides a new, detailed view of intrinsically disordered protein structural ensembles

The functional role of an intrinsically disordered protein (IDP) is defined by its unique structural ensemble. The fast dynamics and high structural heterogeneity of IDPs makes them refractory to study by traditional structural biology methods, and additionally, by standard biophysical methods that measure the free energy of transitions between distinct conformations. In this work, X-ray footprinting coupled to mass spectrometry detection (XFMS) is demonstrated to be a robust, sensitive, and readily interpretable method to characterize the structural ensemble of IDPs in a biologically relevant, high-resolution manner. In XFMS, dilute protein samples are exposed to high-intensity-X-ray radiation which covalently modifies amino acid sidechains; these covalent modifications are identified and quantified at peptide-level or single-residue resolution by liquid chromatography coupled to tandem mass spectrometry. The rate of modification of an individual sidechain is proportional to the ensemble-averaged solvent-accessible surface area of that sidechain, providing a straightforward structural probe at high resolution. Here, we present two vignettes. First, we combined XFMS with chemical denaturation to identify regions of the short model IDP-the transcriptional activation domain of CITED2 that are buried from solvent by transient intramolecular interactions in the IDP’s native structural ensemble. Comparing CITED2 to rationally designed variants, we show that CITED2’s structural ensemble dramatically reorganizes upon perturbation, which is a signature of allostery. Next, we utilized XFMS to study the ensemble of the human transcription factor Sox2. Upon binding DNA, an intrinsically disordered region of Sox2 that contains a transcriptional activation domain becomes exposed to solvent, suggesting DNA-binding allosterically modulates Sox2’s affinity for transcriptional coactivators. In these ways, XFMS can capture biologically relevant details of an IDP’s structural ensemble at high resolution.