Phenylalanine side chain behavior of the intestinal fatty acid-binding protein: The effect of urea on backbone and side chain stability

The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by ¹⁹F-NMR after incorpora tion of 4-fluorophenylalanine and by pulsed field gradient diffusion ¹H-NMR. At low urea concentrations (0-3 M) but prior to the global unfolding that begins at 4 M urea, the protein exhibits dynamic motion in the backbone and an expanded hydrodynamic radius with no major change in the side chain orientation. As monitored by two-dimensional ¹⁹F-¹⁹F nuclear Overhauser effect, the distance between two phenylalanine residues (Phe ⁶⁸ and Phe⁹³) located in the two different β-sheets that enclose the internal cavity did not change up to 4 M urea. Additionally, the chemical shifts of these two residues changed almost identically as a function of denaturant. At all urea concentrations, as well as in the native protein, multiple conformations exist. These conformers interconvert at different rates under different conditions, ranging from slow exchange by showing separate peaks in the native state to intermediate exchange at intermediate urea concentrations. Residual structure persisted around Phe ⁶² even at very high concentrations of denaturant, suggesting that region as a nucleation site during folding. The results were compared with previous studies examining the backbone behavior (Hodsdon, M. E., and Frieden, C. (2001) Biochemistry 40, 732-742) and suggest that the side chains show more stability than the backbone prior to global unfolding of the protein.