The phase behavior of several medium-chain (10- and 12-carbon) and long-chain (18-carbon) fatty acids in water was examined as a function of the ionization state of the carboxyl group. Equilibrium titration curves were generated above and below fatty acid and acid-soap chain melting temperatures and critical micelle concentrations, and the phases formed were characterized by X-ray diffraction, 13C NMR spectroscopy, and phase-contrast and polarized light microscopy. The resulting titration curves were divided into five regions: (i) at pH values less than 7, a two-phase region containing oil or fatty acid crystals and an aqueous phase; (ii) at pH approximately 7, a three-phase region containing oil, lamellar, and aqueous (or fatty acid crystals, 1:1 acid-soap crystals, and aqueous) phases; (iii) between pH 7 and 9, a two-phase region containing a lamellar fatty acid/soap (or crystalline 1:1 acid-soap) phase in an aqueous phase; (iv) at pH approximately 9, a three-phase region containing lamellar fatty acid-soap (or crystalline 1:1 acid-soap), micellar, and aqueous phases; and (v) at pH values greater than 9, a two-phase region containing micellar and aqueous phases. Interpretation of the results using the Gibbs phase rule indicated that, for oleic acid/potassium oleate, the composition of the lamellar fatty acid/soap phase varied from approximately 1:1 to 1:3 un-ionized to ionized fatty acid species. In addition, constant pH regions observed in titration curves were a result of thermodynamic invariance (zero degrees of freedom) rather than buffering capacity. The results provide insights into the physical states of fatty acids in biological systems.(ABSTRACT TRUNCATED AT 250 WORDS)

The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements. The amide 1H/15N resonances for apo and holo I-FABP were assigned at 25 degrees C, and gradient- and sensitivity-enhanced 2D experiments were employed to measure l5N T1, T2, and [1H]15N NOE values and relative 1H saturation transfer rates. The 15N relaxation parameters were analyzed using five different representations of the spectral density function based on the Lipari and Szabo formalism. A majority of the residues in both apo and holo I-FABP were characterized by relatively slow hydrogen exchange rates, high generalized order parameters, and no conformational exchange terms. However, residues V26-N35, S53-R56, and A73-T76 of apo I-FABP were characterized by rapid hydrogen exchange, low order parameters, and significant conformational exchange. These residues are clustered in a single region of the protein where variability and apparent disorder were previously observed in the chemical shift analyses and in the NOE-derived NMR structures of apo I-FABP. The increased mobility and discrete disorder in the backbone of the apo protein may permit the entry of ligand into the binding cavity. We postulate that the bound fatty acid participates in a series of long-range cooperative interactions that cap and stabilize the C-terminal half of helix II and lead to an ordering of the portal region. This ligand-modulated order-disorder transition has implications for the role of I-FABP in cellular fatty acid transport and targeting.

The three-dimensional structure of the unliganded form of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) has been determined using triple-resonance three-dimensional nuclear magnetic resonance (3D NMR) methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 33 degrees C and used to determine the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an eight-stage iterative procedure was used to assign the 3D 13C- and 15N-resolved NOESY spectra, yielding a total of 3335 interproton distance restraints or 26 restraints/residue. The tertiary structures were calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization to achieve improved sampling and convergence. The final ensemble of NMR structures exhibited a backbone conformation generally consistent with the beta-clam motif described for members of the lipid-binding protein family. However, unlike holo-I-FABP, the structure ensemble for apo-I-FABP exhibited variability in a discrete region of the backbone. This variability was evaluated by comparing the apo- and holoproteins with respect to their backbone 1H and 13C chemical shifts, amide 1H exchange rates, and 15N relaxation rates. Together, these results established that the structural variability represented backbone disorder in apo-I-FABP. The disorder was most pronounced in residues K29-L36 and N54-N57, encompassing the distal half of alpha-helix II, the linker between helix II and beta-strand B, and the reverse turn between beta-strands C and D. It was characterized by a destablization of long-range interactions between helix II and the C-D turn and a fraying of the C-terminal half of the helix. Unlike the solution-state NMR structure, the 1.2-A X-ray crystal structure of apo-I-FABP did not exhibit this backbone disorder. In solution, the disordered region may function as a dynamic portal that regulates the entry and exit of fatty acid. We hypothesize that fatty acid binding shifts the order-disorder equilibrium toward the ordered state and closes the portal by stabilizing a series of cooperative interactions resembling a helix capping box. This proposed mechanism has implications for the acquisition, release, and targeting of fatty acids by I-FABP within the cell.

Fatty acid binding proteins (FABPs) exhibit a ␤-barrel topology, comprising 10 antiparallel ␤-sheets capped by two short ␣-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of f luorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusionmediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the ␣-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.