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.