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J. Biol. Chem., Vol. 280, Issue 46, 38556-38561, November 18, 2005

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Phenylalanine Side Chain Behavior of the Intestinal Fatty Acid-binding Protein

THE EFFECT OF UREA ON BACKBONE AND SIDE CHAIN STABILITY^*

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by ¹⁹F-NMR after incorporation 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 (Phe⁶⁸ and Phe⁹³) located in the two different -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 Phe⁶² 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.

Received for publication, May 18, 2005 , and in revised form, August 10, 2005.

^* This work was supported by National Institutes of Health Grant DK13332. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental TABLE ONE (relative distance change between 4-¹⁹F-Phe⁶⁸ and 4-¹⁹F-Phe⁹³ as a function of urea concentration), supplemental Fig. 1 (intensity and chemical shift change of Phe⁶² as a function of urea concentration), and supplemental Fig. 2 (¹⁹F-NMR spectra of apo-IFABP with ¹⁹F-label on both Phe⁶⁸ and Phe⁹³ as a function of urea concentration.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110. Tel.: 314-362-3344; Fax: 314-362-7183; E-mail: frieden{at}biochem.wustl.edu.

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