Fluorine-19 NMR Studies on the Acid State of the Intestinal Fatty Acid Binding Protein

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• 作者：Hua Li and Carl Frieden
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：May 23, 2006
• 年：2006
• 卷：45
• 期：20
• 页码：6272 - 6278
• 全文大小：187K
• 年卷期：v.45,no.20(May 23, 2006)
• ISSN：1520-4995

文摘

The intestinal fatty acid binding protein (IFABP) is composed of two LIGN="middle">-sheets with a largehydrophobic cavity into which ligands bind. After eight 4-¹⁹F-phenylalanines were incorporated into theprotein, the acid state of both apo- and holo-IFABP (at pH 2.8 and 2.3) was characterized by means of¹H NMR diffusion measurements, circular dichroism, and ¹⁹F NMR. Diffusion measurements show amoderately increased hydrodynamic radius while near- and far-UV CD measurements suggest that theacid state has substantial secondary structure as well as persistent tertiary interactions. At pH 2.8, thesetertiary interactions have been further characterized by ¹⁹F NMR and show an NOE cross-peak betweenresidues that are located on different -strands. Side chain conformational heterogeneity on the millisecondtime scale was captured by phase-sensitive ¹⁹F-¹⁹F NOESY. At pH 2.3, native NMR peaks are mostlygone, but the protein can still bind fatty acid to form the holoprotein. An exchange cross-peak of onephenylalanine in the holoprotein is attributed to increased motional freedom of the fatty acid backbonecaused by the slight opening of the binding pocket at pH 2.8. In the acid environment Phe128 and Phe17show dramatic line broadening and chemical shift changes, reflecting greater degrees of motion aroundthese residues. We propose that there is a separation of specific regions of the protein that gives rise tothe larger radius of hydration. Temperature and urea unfolding studies indicate that persistent hydrophobicclusters are nativelike and may account for the ability of ligand to bind and induce nativelike structure,even at pH 2.3.