Intestinal fatty acid binding protein: folding of fluorescein-modified proteins.
The rat intestinal fatty acid binding protein is an almost all beta-sheet 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 having a single cysteine residue [Jiang, N., & Frieden, C., (1993) Biochemistry 32, 11015-11021] either in a turn or pointed into the cavity. In this report, each mutant has been unfolded in denaturant and modified 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 appeared 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, folded protein, one- and two-dimensional 1H NMR spectra were accumulated and compared to the unmodified and wild-type proteins. While the spectra 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 proteins, two appear to have the fluorescein group located in the cavity, but only one of these did not bind fatty acid. The remaining modified proteins are capable of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Frieden
C
C
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Jiang
N
N
Cistola
D P
DP
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005452:Fluoresceins; D007413:Intestinal Mucosa / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005452:Fluoresceins; D008024:Ligands; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C015296:5-iodoacetamidofluorescein; D003545:Cysteine