Abstract
Conserved structural water molecules stabilize protein folds and modulate their function, yet remain difficult to observe in solution because exchange-based readouts favor solvent-exposed sites. Here, we introduce a protocol to detect structural water molecules under native conditions, using long-lived hyperpolarized water (HyperW) enabled by UV-induced, nonpersistent radicals. In the model protein chymotrypsin inhibitor 2, HyperW-enhanced two-dimensional NMR correlation spectra across pD 5.5-8.4 reveal strong exchange-driven enhancements at solvent-exposed residues. By contrast, a distinct group of four residues shows hyperpolarized amide signals, which disappear when through-space polarization transfer via nuclear Overhauser effect (NOE) is suppressed using a CLEANEX-PM experiment. The CLEANEX-negative/HyperW-positive signature, together with the spatial proximity of these residues to crystallographically conserved water molecules, supports NOE-mediated transfer from long-residence internal water, not distinguishable by standard NMR methods. The combined observables establish HyperW NMR as a residue-specific reporter of structural hydration and hydration-coupled dynamics under native conditions, providing a route to link conserved water observed in crystals to their roles in solution.