Probing internal water molecules in proteins using two-dimensional ¹⁹F-¹H NMR

A simple approach for detecting internal water molecules in proteins in solution is described. This approach combines ¹⁹F-detected heteronuclear Overhauser and exchange spectroscopy (HOESY) with site-specific ¹⁹F substitution. The model system employed was intestinal fatty acid-binding protein complexed with [2-mono-¹⁹F]-palmitate. An intense cross peak was observed between the fluorine and a buried water molecule, as defined in the 1.98 Å crystal structure of the complex. From HOESY spectra, the fluorine-water distance was estimated to be 2.1 Å, in agreement with the crystal structure. This approach may be applicable to macromolecules that are too large for ¹H-detected NMR methods.