NMR Analysis of the Interactions and Conformational Plasticity of Dynein Intermediate Chain

Cytoplasmic dynein complexes play crucial roles in intracellular transport of cellular organelles. While the motor domain of dynein is well characterized by techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM), structural representations of dynein usually include only the more packed and easily resolved regions and omit the long flexible and poorly structured regions. One such flexible region is the N-terminal half of the intermediate chain (IC), which contains almost 300 amino acids that are predicted to be disordered. This level of disorder makes IC impossible to study by X-ray crystallography and Cryo-EM, but amenable to study by solution nuclear magnetic resonance (NMR), a powerful technique that can elucidate residue-specific information in a dynamic ensemble of structures, and transient binding interactions of associated proteins. Here, we describe the methods we use to characterize flexible and disordered proteins including protein expression, purification, sample preparation, and NMR data acquisition and analysis.