Fluorine-19 NMR studies on the acid state of the intestinal fatty acid binding protein.

The intestinal fatty acid binding protein (IFABP) is composed of two beta-sheets with a large hydrophobic cavity into which ligands bind. After eight 4-(19)F-phenylalanines were incorporated into the protein, the acid state of both apo- and holo-IFABP (at pH 2.8 and 2.3) was characterized by means of (1)H NMR diffusion measurements, circular dichroism, and (19)F NMR. Diffusion measurements show a moderately increased hydrodynamic radius while near- and far-UV CD measurements suggest that the acid state has substantial secondary structure as well as persistent tertiary interactions. At pH 2.8, these tertiary interactions have been further characterized by (19)F NMR and show an NOE cross-peak between residues that are located on different beta-strands. Side chain conformational heterogeneity on the millisecond time scale was captured by phase-sensitive (19)F-(19)F NOESY. At pH 2.3, native NMR peaks are mostly gone, but the protein can still bind fatty acid to form the holoprotein. An exchange cross-peak of one phenylalanine in the holoprotein is attributed to increased motional freedom of the fatty acid backbone caused by the slight opening of the binding pocket at pH 2.8. In the acid environment Phe128 and Phe17 show dramatic line broadening and chemical shift changes, reflecting greater degrees of motion around these residues. We propose that there is a separation of specific regions of the protein that gives rise to the larger radius of hydration. Temperature and urea unfolding studies indicate that persistent hydrophobic clusters are nativelike and may account for the ability of ligand to bind and induce nativelike structure, even at pH 2.3.