Role of the alpha-Helical Domain Lysine Residues in Electrostatic Interaction between Intestinal Fatty Acid Binding Protein and Pospholipid Membranes.

FALOMIR LOCKHART, LISANDRO J.; LABORDE, LISANDRO; GARDA, HORACIO; STORCH, JUDITH; CÓRSICO, BETINA

Angras do Rey, Brasil

Congreso; 1st Latin American Protein Science Meeting Latin American Protein Society; 2004

Protein Society/ Latin America Protein Society

Intestinal FABP (IFABP) structure consists of ten antiparallel beta-strands that form a beta-barrel, which is capped by two short helices arranged as a helix-turn-helix segment. It is believed that this helical domain is part of a dynamic portal that regulates fatty acid (FA) entry and exit from the internal binding cavity. Previously, we demonstrated that transfer from IFABP occurs during protein-membrane-collisional interactions, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. We have also shown, using a helix-less variant of IFABP that the alpha-helical region of IFABP is involved in membrane interactions, and appears to play a primary role in the collisional mechanism of fatty acid transfer from IFABP to membranes. In the present study, the role of specific lysine residues of the portal region of IFABP was directly examined using site-directed mutagenesis. We have engineered a series of point mutant proteins of IFABP where lysine’s positive charge of the alpha-helical domain were eliminated and reverted. We also included a pair of mutants of the barrel as controls. All mutants were studied for conformational and binding site integrity and showed non relevant modifications compared to the wild type. Employing a fluorescence resonance energy transfer assay most of the alpha-helical domain mutants showed slower rates of FA transfer to zwitterionic small unilamellar vesicles (SUVs), but only a significant modification of the absolute rate was observed for mutant K27I of the alpha-II helix; which also showed a drastic modification of the FA transfer mechanism to aqueous-difusional. Sensitivity to negatively charged SUVs was also reduced; the mutants with charge reversion in the alpha-II helix were the most affected. These results demonstrate that the lysines of the alpha-helical region of IFABP participate of the protein-membrane collisional mechanism of fatty acid transfer to membranes. Furthermore, it appears that the alpha-II segment is more important in the charge-charge interaction. On the other hand, crosslinking experiments with the lipid photoactivable reagent 125I-TID-PC showed physical interaction of wild-type IFABP and all point mutants with vesicles of different composition. Only if the helical domain is completely removed, as in the Helix-Less IFABP, the protein looses the ability to interact with membranes.