PROBING INTERNAL WATER-MOLECULES IN PROTEINS USING 2-DIMENSIONAL F-19-H-1 NMR

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