NMR studies of 4-19F-phenylalanine-labeled intestinal fatty acid binding protein: evidence for conformational heterogeneity in the native state.
(19)F-Nuclear magnetic resonance (NMR) studies have been carried out after incorporation of 4-(19)F-phenylalanine into the intestinal fatty acid binding protein (IFABP), a protein composed of two beta-sheets containing a large hydrophobic cavity into which ligands bind. NMR spectra have been obtained with both the ligand-free and ligand-bound (oleate) forms. There are 29 residues involved in van der Waals or hydrophobic interactions or both to form a U-shaped ligand binding pocket (Sacchettni J. C., Scapin G., Gopaul D., and Gordon J. I. (1992) J. Biol. Chem. 267, 23534-23545). The protein contains eight phenylalanines, and all are included in those residues that line the pocket. Peak assignments were made using site-specific incorporation of 4-(19)F-phenylalanine. Fluorine is a highly sensitive probe to monitor the conformation and dynamics of the side chains in native state. We find that chemical exchange in the binding pocket exists in the native apo- and holo-state. Of the eight phenylalanine residues, Phe2, Phe47, Phe62, Phe68, and Phe93 are arranged on one side of the binding pocket, and all exist in two conformations with Phe2, Phe47, and Phe62 showing exchange cross-peaks with minor conformation in (19)F-(19)F nuclear Overhauser effect (NOESY) spectra. The line widths of Phe68 and Phe93 are broader than those of other phenylalanine residues and can be deconvoluted into two peaks. Phe47, Phe62, Phe68, Phe93, and Trp82 have been proposed to be involved in the early stage of collapse (Ropson, I. J., and Frieden, C. (1992) Proc. Natl. Acad. Sci U.S.A. 89, 7222-7226), but a temperature study suggests that Phe47 behaves differently than other residues and may be more involved in a later stage of folding, for example, side chain stabilization. In the holo-form, Phe17 shows an extra exchange cross-peak in addition to those exchange cross-peaks observed in apo-form. Holo-IFABP exhibits broader line width than the apo-form, suggesting more flexibility of the binding cavity upon ligand binding.
Li
Hua
H
Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Frieden
Carl
C
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D005461:Fluorine / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007413:Intestinal Mucosa / Q000378:metabolism; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019301:Oleic Acid / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D008024:Ligands; D005461:Fluorine; D019301:Oleic Acid; D010649:Phenylalanine