Intestinal Fatty Acid-Binding Protein: The Structure and Stability of a Helix-less Variant ² Keehyuk Kim, David P. Cistola,* and Carl Frieden* Department of Biochemistry and Molecular Biophysics, Washington UniVersity School of Medicine, St. Louis, Missouri 63110-1093 ReceiVed December 11, 1995; ReVised Manuscript ReceiVed March 6, 1996 X ABSTRACT: The structure of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) exhibits a -clam topology comprised of two five-stranded antiparallel -sheets surrounding a large solvent- filled cavity into which the ligand binds. It also contains two R-helices that span residues E15-A32 and join -strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed Δ17-SG, has a high -sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of Δ17-SG revealed patterns similar to those observed for wild-type I-FABP, except for the selective absence of resonances and through-space interactions assigned to the helical domain. The Δ17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of Δ17-SG were essentially identical to those of wild-type. We conclude that Δ17-SG is a helix-less, essentially all--sheet variant of I-FABP and that the helical domain is not a required element of the -clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., & Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding. Intestinal fatty acid-binding protein (I-FABP) 1 belongs to a family of predominately -sheet proteins that bind a diverse group of polar lipids, such as fatty acids, retinoids, and sterols (Sacchettini & Gordon, 1993; Banaszak et al., 1994; Veerkamp & Maatman, 1995). To date, the three-dimen- sional structures of 11 members of this family have been determined by X-ray crystallography and NMR (Banaszak et al., 1994; Haunerland et al., 1994; Kleywegt et al., 1994; Lassen et al., 1995), 2 and all exhibit essentially the same backbone fold. This fold consists primarily of two five- stranded antiparallel -sheets surrounding a large cavity into which the ligand binds (Figure 1). Common to these structures is a small helix-turn-helix motif that is interspersed in the sequence between the first and second -strands. The role of this motif is unknown except that it serves to cover one end of the ligand-binding ² This work was supported by National Institutes of Health Grants DK13332 (to C.F.) and DK48046 (to D.P.C.). D.P.C. was supported by a Johnson & Johnson/Merck Research Scholar Award from the American Digestive Health Foundation. * To whom correspondence should be addressed: Campus Box 8231, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: 314-362-3344 or -4382. Fax: 314-362- 7183. X Abstract published in AdVance ACS Abstracts, May 15, 1996. 1 Abbreviations: I-FABP, recombinant rat intestinal fatty acid- binding protein; Δ17-SG, a variant of I-FABP engineered by deleting 17 residues (E15-G31) and inserting Ser-Gly after residue Y14; CD, circular dichroism; TOCSY, two-dimensional 1 H total correlation spectroscopy; NOESY, two-dimensional 1 H nuclear Overhauser and exchange spectroscopy; HSQC, two-dimensional heteronuclear single quantum correlation spectroscopy; Gdn, guanidine hydrochloride. 2 The NMR solution structure of wild-type I-FABP complexed with palmitate has recently been determined on the basis of 3889 distance constraints derived from three-dimensional 13 C- and 15 N-resolved NOESY data (M. E. Hodsdon, J. W. Ponder, and D. P. Cistola, manuscript in preparation). FIGURE 1: Backbone fold of wild-type intestinal fatty acid-binding protein complexed with palmitate. This ribbon diagram is based on the 2.0 Å X-ray crystal structure (Brookhaven file 2ifb; Sacchettini et al., 1989) and the NMR chemical shift-derived location of secondary structure elements (Hodsdon et al., 1995). To generate the Δ17-SG deletion mutant, amino acids in the R-helical region from residue 15 through 31 (indicated by the arrows) were deleted and replaced by the dipeptide Ser-Gly. The resulting mutant protein appears to be an essentially helix-less, all -sheet variant of I-FABP. The positions of the two tryptophan residues (W6 and W82) are noted. This diagram was prepared using MOLSCRIPT (Kraulis, 1991). 7553 Biochemistry 1996, 35, 7553-7558 S0006-2960(95)02911-4 CCC: $12.00 © 1996 American Chemical Society