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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. 

Cytoplasmic dynein is a motor protein that plays a role in a number of cellular processes including retrograde transport. In many cases, dynein needs to interact with another protein, dynactin, to be fully active. An important step in the assembly of the dynein/dynactin complex is the interaction between the N‐terminal portion of the intermediate chain (IC) subunit of dynein and the coiled‐coil 1B (CC1B) region of the p150^Glued subunit of dynactin. Despite evidence for this interaction from binding studies, the exact location of where these proteins bind has remained elusive due to the dynamic nature of the interaction and the presence of intrinsically disordered regions in IC. By using intermolecular paramagnetic relaxation enhancements, we have been able to constrain the location of IC binding on p150^Glued to a position that is different from what has recently been hypothesized in a model of the dynein/dynactin complex based on cryo‐electron microscopy (cryo‐EM) data and AlphaFold predictions. In addition, although phosphorylation is important for regulating dynein/dynactin interactions, we show that a phosphomimetic mutation of IC is not sufficient to alter binding with p150^Glued. 

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers. 

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.