Journal of BiomolecularNMR, 6 (1995) 198-210 ESCOM J-Bio NMR 284 1H, 13C and assignments and chemical shift-derived secondary structure of intestinal fatty acid-binding protein Michael E. Hodsdon, James J. Toner and David E Cistola* Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8231, St. Louis, MO 63110-1010, U.S.A. Received 9 January 1995 Accepted 21 March 1995 Keywords. Triple-resonance3D NMR; Lipid-bindingproteins; lsotope enrichment; Resonanceassignments; Chemical shift; Protein secondary structure Summary Sequence-specificIH, 13C and 15N resonance assignments have been established for rat intestinal fatty acid-binding protein complexed with palmitate (15.4 kDa) at pH 7.2 and 37 ~ The resonance assign- ment strategy involved the concerted use of seven 3D triple-resonance experiments (CC-TOCSY, HCCH- TOCSY, HNCO, HNCA, lSN-TOCSY-HMQC, HCACO and HCA(CO)N). A central feature of this strategy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The CC-TOCSY experiment provided the unambiguous links between the side-chain spin systems observed in HCCH-TOCSY and the backbone correlations observed in the other experiments. Assignments were established for 124 of the 131 residues, although 6 of the 124 had missing amide IH resonances, presumably due to rapid exchange with solvent under these experimental conditions. The assignment database was used to determine the solution secondary structure of the complex, based on chemical shift indices for the 1H~, 13C~, ~3C~ and z3CO atoms. Overall, the secondary structure agreed well with that determined by X-ray crystallography [Sacchettini et al. (1989) J.. Mol. Biol., 208, 327-339], although minor differences were observed at the edges of secondary structure elements. Introduction Intestinal fatty acid-binding protein (I-FABP) belongs to a family of soluble, intracellular proteins that are thought to facilitate the transport and trafficking of polar lipids (Glatz and Van der Vusse, 1990; Kaikaus et al., 1990; Veerkamp et al., 1991; Bass, 1993; Banaszak et al., 1994). This family includes at least 20 distinct fatty acid-, retinoid- and sterol-binding proteins which have been identified in a variety of vertebrate and invertebrate or- ganisms. In mammals, different tissues within the same organism often express distinct lipid-binding proteins, implying that these proteins have functions that are tailored to the particular cell types in which they are found. To date, three-dimensional structures have been deter- mined for nine of the family members (Banaszak et al., 1994; Haunerland et al., 1994). Despite a variable degree of sequence identity, ranging from 19 to 64%, these struc- tures exhibit essentially the same backbone fold. The root-mean-square (rms) differences for their c~-carbon co- ordinates are 0.6 to 2.4 A. In contrast, there are striking differences between the ligand-binding specificities for a number of these proteins. For example, I-FABP is specific for long-chain fatty acids, while cellular retinol-binding protein-II (CRBP-II) binds retinol and retinaldehyde, but not fatty acids. In addition, some proteins are able to discriminate between lipids within the same class, e.g., retinol versus retinal (Li et al., 1991). In spite of the sub- stantial body of structural information for these proteins, the determinants of ligand-binding specificity and affinity in this protein family remain unclear. The lipid-binding proteins from the small intestine are unusual in that several homologues are abundantly ex- pressed in the same cell type, the enterocyte. In addition to I-FABP and CRBP-II, these homologues include liver *To whom correspondenceshould be addressed. 0925-2738/$ 6.00 + 1.00 9 1995 ESCOM SciencePublishers B.V.