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J. Biol. Chem., Vol. 280, Issue 3, 1782-1789, January 21, 2005

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Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

L28W MUTANT PROVIDES IMPORTANT INSIGHTS INTO LIGAND BINDING AND PHYSIOLOGICAL FUNCTION^*

Robert M. Hagan, Jane Worner-Gibbs, and David C. Wilton

From the Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom

Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.

Received for publication, June 25, 2004 , and in revised form, October 18, 2004.

^* 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.

To whom correspondence should be addressed: Div. of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK. Tel.: 44-2380-594308; Fax: 44-2380-594459; E-mail: D.C.Wilton{at}soton.ac.uk.

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