Structure Characterization of a Disordered Peptide Using In-Droplet Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics

The highly dynamic nature of intrinsically disordered proteins (IDPs), those containing intrinsically disordered regions (IDRs), makes their structural characterization very difficult using traditional measurement approaches. Especially challenging is the determination of the propensity to form specific types of structure upon interacting with binding partners. Here, the reactivities of select peptides ranging from highly-structured to unstructured species have been examined with in-droplet hydrogen-deuterium exchange (HDX) techniques. Overall, the consideration of intrinsic rates (kint) of exchange for different backbone sites is shown to be a poor predictor of peptide reactivity. Here, we develop a new model for HDX prediction that combines the use of protection factors (PF) from molecular dynamics (MD) simulations with kint to obtain high-accuracy HDX predictability for peptides having various structural types. The relationship of peptide structural flexibility and HDX reactivity for different peptides is elucidated. Additionally, the model is used to characterize the degree of structural bias for the disease-relevant Nt17; although highly flexible, being primed for facile conversion to α-helical conformation upon binding with molecular partners imparts HDX protection. In the future a scale may be developed whereby HDX reactivity is associated with the degree of IDR structural flexibility and bias. Such structural resolution may ultimately be used for high-throughput screening of IDR structural transformation (including structural flexibility and bias) upon binding of drug candidates.