Water molecules in the binding cavity of intestinal fatty acid binding protein: Dynamic characterization by water O-17 and H-2 magnetic relaxation dispersion

The hydration of intestinal fatty acid binding protein (IFABP) in apoform a nd complexed with palmitate, oleate, and 1-anilino-8-naphthalene sulfonate (ANS) has been studied by water O-17 and H-2 magnetic relaxation dispersion (MRD) measurements. These ligands bind in a large internal cavity, displac ing most of the crystallographically identified cavity water molecules. Unl ike most other proteins, IFABP gives rise to MRD profiles with two dispersi on steps. The low-frequency dispersion yields a correlation time of 7 ns at 300 K, matching the known tumbling time of IFABP. The dispersion amplitude requires only three (apo) or four (holo) long-lived and ordered water mole cules (residence time 0.01-4 mu s at 300 K). Comparison of MRD profiles fro m the different complexes indicates that the displaced cavity water molecul es are short-lived. The few long-lived (>10 ns) water molecules required by the MRD data are tentatively assigned to crystallographic hydration sites on the basis of accessibility, positional order, and H-bonding. The amplitu de of the high frequency dispersion corresponds to 10-20 moderately ordered water molecules, with a correlation time of ca. 1 ns that may reflect a tr ansient opening of the cavity required for exchange with external water. (C ) 1999 Academic Press.