Dynamics of palmitic acid complexed with rat intestinal fatty acid bindingprotein

Dynamics of palmitic acid (PA), isotopically enriched with C-13 at the seco nd, seventh, or terminal methyl position, were investigated by C-13 NMR. Re laxation measurements were made on PA bound to recombinant rat intestinal f atty acid binding protein (I-FABP) at pH 5.5 and 23 degrees C, and, for com parison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho choline (MPPC) micelles, and dissolved in methanol. The C-13 relaxation dat a, T-1, and steady-state nuclear Overhauser effect (NOE) obtained at two di fferent magnetic fields were interpreted using the model-free approach [Lip ari, G., and Szabo, A. (1982) J. Am. Chem. Sec, 104, 4546-4559]. The overal l rotational correlation time of the fatty acid protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protei n itself (>6 ns). Order parameters (S-2), which are a measure of the amplit ude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were r elatively small (<0.4) in the protein complex. S-2 values for given C-H vec tors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the, view that the fatty acid is not rigidly anchored within the I- FABP binding pocket, but rather has considerable freedom to move within the pocket.