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.
Hecker et al. (Wed,) studied this question.