Confronting the Invisible: Assignment of Protein ¹H^N Chemical Shifts in Cases of Extreme Broadening

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where ¹H^N spectra cannot be obtained. The approach uses a weak ¹H B₁ field, searching for elusive ¹H^N resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional ¹H^α-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate ¹H^N chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s^-1.