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J. Biol. Chem., Vol. 281, Issue 20, 13979-13989, May 19, 2006

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Protein-Membrane Interaction and Fatty Acid Transfer from Intestinal Fatty Acid-binding Protein to Membranes

SUPPORT FOR A MULTISTEP PROCESS^*

Lisandro J. Falomir-Lockhart¹, Lisandro Laborde, Peter C. Kahn, Judith Storch^¶2, and Betina Córsico³

From the Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CONICET-UNLP, Facultad de Ciencias Médicas, Calles 60 y 120, 1900-La Plata, Argentina and the Departments of ^¶Nutritional Sciences and Biochemistry and Microbiology, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901-8525

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the -helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the -helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the -helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the ₂ helix, Lys²⁷, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the ₂ helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the ₂ segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Received for publication, November 4, 2005 , and in revised form, March 20, 2006.

^* This work was supported by National Institutes of Health Grant DK 38389 (to J. S.) and Grant TW01100-01 (to J. S. and B. C.) and Fundación Antorchas Grant 14116-224 (to B. C.). 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.

¹ Recipient of a Doctoral Fellowship from the National Research Council (Consejo Nacional de Investigaciones Científicas y Técnicas) of Argentina.

² To whom correspondence may be addressed. Tel.: 732-932-1689; Fax: 732-932-6837; E-mail: storch{at}aesop.rutgers.edu. ³ To whom correspondence may be addressed. Tel.: 54-221-482-4894; Fax: 54-221-425-8988; E-mail: bcorsico{at}atlas.med.unlp.edu.ar.

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