NMR Approaches to Identify Transient Structure and Interactions of Intrinsically Disordered Dynein Intermediate Chain

Nuclear magnetic resonance (NMR) spectroscopy is widely recognized for its ability to provide atomic-level resolution of structures and interactions in intrinsically disordered proteins (IDPs). However, its application is often limited when studying large proteins that contain both structured and disordered regions. This challenge arises due to the broad peaks exhibited by structured regions in such proteins, which result from local compaction and restricted motions, complicating spectral analysis. Additionally, broadening in IDP complexes caused by exchange between free and bound states and/or the large size of the bound state, further obscures NMR signals and hinders the mapping of interaction sites. Moreover, IDPs are highly sensitive to proteolytic cleavage, necessitating careful handling and optimization during expression, purification, and data collection. In this study, we demonstrate how we successfully overcame these hurdles using examples from our work on the N-terminal region of the dynein intermediate chain (IC), which contains both ɑ-helical and intrinsically disordered regions. By employing paramagnetic relaxation enhancement (PRE) NMR to probe conformational dynamics, water-amide chemical exchange to measure solvent accessibility, and saturation transfer difference (STD) NMR to map specific interactions with p150^Glued and Nudel, we identified novel transient structures and interaction networks within IC. Our findings highlight the utility of these advanced NMR techniques in elucidating the dynamic behavior of IDPs and their complexes, providing valuable insights into their structural and functional roles.