Transient Helicity in the Intrinsically Disordered Protein ACTR Measured by Hydrogen Exchange

Intrinsically disordered proteins (IDPs) play essential roles in cellular signaling and regulation, often relying on transient structural elements to mediate interactions. NMR chemical shifts are widely used to detect secondary structures in IDPs, but complementary methods are needed to validate and refine these measurements. Hydrogen exchange is a powerful probe of local structure and dynamics in folded proteins, yet its accuracy for detecting small differences in transient helicity in IDPs remains understudied. Here, we systematically evaluate hydrogen exchange measured by NMR and MS (HDX-MS) in four variants of the activator for thyroid hormone and retinoid receptors (ACTR) activation domain that differ in helical propensity. Using NMR-based exchange rates, we introduce pseudo-protection factors referenced to the wild-type protein, enabling a robust comparison among variants without relying on "intrinsic" peptide-based chemical exchange rates. These pseudo-protection factors correlate strongly with helicity derived from chemical shifts, demonstrating that hydrogen exchange can resolve subtle structural differences in highly dynamic regions and vice versa. Our findings establish hydrogen exchange as a sensitive and reproducible method for characterizing transient structure in IDPs, complementing NMR chemical shift analysis.