INTESTINAL FATTY-ACID-BINDING PROTEIN - FOLDING OF FLUORESCEIN-MODIFIED PROTEINS

The rat intestinal fatty acid binding protein is an almost all beta-sh eet protein that encloses a large interior cavity into which the fatty acid ligand binds. The protein contains neither cysteine nor proline. In a previous report, six site-directed mutants were obtained, each h aving a single cysteine residue [Jiang, N., & Frieden, C., (1993) Bioc hemistry 32, 11015-11021] either in a turn or pointed into the cavity. In this report, each mutant has been unfolded in denaturant and modif ied with 5-iodoacetamido-fluorescein to introduce a large, bulky, and fluorescent group into the protein at a known position. In all cases, fluorescence changes indicated that the modified protein refolded, and circular dichroism measurements suggested that the refolded protein a ppeared to be mostly beta-sheet. Denaturation curves suggest that for two mutants intermediate structures exist at denaturant concentrations well below the midpoint of the unfolding curve. For each modified, fo lded protein, one- and two-dimensional H-1 NMR spectra were accumulate d and compared to the unmodified and wild-type proteins. While the spe ctra for the modified proteins showed a number of changes in chemical shifts, they were also consistent with folded proteins on the basis of the degree of chemical shift dispersion. Of the six modified mutant p roteins, two appear to have the fluorescein group located in the cavit y, but only one of these did not bind fatty acid. The remaining modifi ed proteins are capable of ligand binding. In contrast to the modified proteins which contained the fluorescein moiety in the cavity, the fl uorescein group in the other modified proteins appears to have been fo rced to the outside or to the surface. It is concluded that the beta-s heet structure and the large internal cavity of the protein allow cons iderable structural perturbations without disrupting the ability of th e protein to fold or affecting the nature of the folded structure.