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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. 

Motor proteins are the freight trains of the cell, transporting large molecular cargo from one location to another using an array of ‘roads’ known as microtubules. These hollow tubes are oriented, with one extremity (the plus-end) growing faster than the other (the minus-end). While over 40 different motor proteins travel towards the plus-end of microtubules, just one is responsible for moving cargo in the opposite direction. This protein, called dynein, performs a wide range of functions which must be carefully regulated, often through changes in the shape and interactions of various dynein segments. The intermediate chain is one of the essential subunits that form dynein, and it acts as a binding site for a range of molecular actors. In particular, it connects the three other dynein subunits (known as the light chains) to the dynein heavy chain containing the motor domain. It also binds to two non-dynein proteins: NudE, which helps to organise microtubules, and the p150 Glued region of dynactin, a protein required for dynein activity. Despite their distinct roles, p150 Glued and NudE attach to the same region of the intermediate chain, a highly flexible ‘unstructured’ segment which is difficult to study. How the binding of p150 Glued and NudE is regulated has therefore remained unsolved. In response, Jara et al. decided to investigate how the three dynein light chains may help to control interactions between the intermediate chain and non-dynein proteins. They used more stable versions of dynein, NudE and dynactin (from a fungus that grows at high temperatures) to produce the various subcomplexes formed by the intermediate chain, the three dynein light chains, and parts of p150 Glued and NudE. A suite of biophysical techniques was applied to study these structures, as they are challenging to capture using traditional approaches. This revealed that the unstructured region of the intermediate chain can fold back on itself, bringing together its two extremities; such folding blocks the p150 Glued and NudE binding site. This obstruction is cleared when the light chains bind to the intermediate chain, demonstrating how these three subunits can regulate dynein activity. In humans, mutations in dynein are associated with a range of serious neurological and muscular diseases. The work by Jara et al. brings new insight into the way this protein works; more importantly, it describes how to combine several biophysical techniques to study non-structured proteins, offering a blueprint that is likely to be relevant for a wide range of scientists.