Using Solution NMR to Study the RNA-Binding Properties and Molecular Mechanisms of the RNA Helicase DDX3X

DEAD-box RNA helicases (DDXs) are a family of enzymes that participate in nearly all aspects of cellular RNA metabolism by remodeling RNA structures. Among them, DDX3X plays key roles in diverse physiological processes, including the translational regulation of gene expression, and its malfunction is frequently associated with the onset of disease. To elucidate the molecular mechanisms of DDX3X, solution nuclear magnetic resonance (NMR) spectroscopy provides unique insights into its RNA-binding properties, dynamic behavior, and intermolecular interactions, complementing conventional structural approaches aimed at determining high-resolution atomic models. In this chapter, we present a detailed protocol for preparing isotopically labeled proteins for NMR analysis, using the minimal functional core region of DDX3X as an example. The presented protocol enables the production of large amounts of purified protein, which can also be adapted for other in vitro structural and biochemical experiments used to characterize helicase function.