PROJECT SUMMARY Intrinsically disordered proteins (IDPs), proteins that do not assume a preferred folded conformation in solution, make up around one-third of the entire human proteome, and their misfolding is a causative agent of several serious diseases. Although an array of biophysical tools has been applied to the structural analysis of IDPs, there is still a significant gap between the need to accurately measure IDP structures and the available tools. Current tools provide either not enough information content to gain significant insight on the many IDP structures present in the overall ensemble or can only provide an ensemble average due to interconversion of conformations over the time frame of the experiment. Therefore, the goal of this application is to apply native ion mobility/mass spectrometry (IM/MS) tools developed in the Webb laboratory for analyzing well-folded proteins in combination with molecular dynamics to characterize the structural ensembles of IDPs and intrinsically disordered regions (IDRs) with conformational and proteoform specificity and to determine the effect on those structural ensembles of post-translational modifications (PTMs). The proposed work in this application is expected to answer the following biological questions: 1.) What are the IDP conformations in the overall ensemble? Crosslinked and labeled IDPs/IDRs will be identified under native-like conditions to determine structural ensembles as a function of solution conformation (by IM and MS). Crosslinking/labeling identifications will be used to determine structural ensembles of IDPs/IDRs with molecular dynamics. 2.) How do post translational modifications and ligand binding affect IDP structure? These methods will be used to measure how PTMs and small molecule binding affects conformational ensembles of IDPs, linking structural changes to their functions and to aggregation. 3.) Which conformational states and proteoforms of histones bind epigenetic partners? Histone tails are IDRs decorated with many combinations of PTMs that regulate access to DNA. We will use our tools on individual histones, on the intact nucleosome, and on nucleosomes upon binding modifier and reader proteins to measure changes in structural ensembles. This research represents a substantive departure from the status quo by using a combined solution (crosslinking and labeling) and gas-phase MS structural toolkit to provide structural details of IDPs/IDRs measured independently by proteoform and conformation. The research is significant because it is expected to bridge the gap between the tools available for the conformational study of IDPs and the fundamental and biomedical need to characterize their structures. Ultimately, IDP conformational characterization is likely to lead to a much broader understanding of cellular biology and the importance of IDPs/IDRs as interaction hubs and regulators and a better understanding of their roles in disease and treatment.