A surface plasmon resonance investigation of the role of bilayer phospholipids in the binding and ligand interactions of the ⍺-tocopherol transfer protein.

Abstract

Vitamins are, by definition, required for maintaining the health of the mammalian body. Vitamin E, which occurs in eight different forms, has a crucial function as an antioxidant protecting polyunsaturated fatty acids. Attributable to the occurrence of alpha-tocopherol transfer protein (a-TTP), RRR-a-tocopherol is the only form that is selectively retained in mammals. a-TTP aids in secreting the RRR-a-tocopherol from liver to the rest of the body. The mechanism of the a-TTP-mediated movement of a-tocopherol is not yet fully elucidated. The opening of a-TTP's binding pocket and subsequent release of a-tocopherol are made possible by a-TTP's interaction with PM residing phosphatidylinositols. K217, a crucial amino acid, is present in the positive surface patch of a-TTP, which is essential for orchestrating this interaction. Surface Plasmon Resonance (SPR) was employed in this study to examine the absorption and desorption of wild-type a-TTP and the mutated form K217A to tethered phospholipid vesicles. SPR spectroscopy illustrated the affinity of either protein when it was presented with one or both of its preferred ligands (a-tocopherol or phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]) on tethered vesicles. Bovine serum albumin (BSA) was used to coat the side of the microcentrifuge tubes to mitigate protein loss that was discovered when protein was preincubated with its ligands. K217 was found to be instrumental in the binding of TTP to PIPs and the subsequent release of tocopherol. As K217A mutant had a lower adsorption to membranes containing PI(4,5)P2 vs wtTTP. It was determined that α-TTP binding to endosomal vesicles increased with increased concentration of the late endosome specific lipid bis(monoacylglycero)phosphate (BMP). Collections runs performed provided some evidence for the exchange of ligands when wtTTP was used but not for K217A. The results found in this work parallel literature findings and supplement the proposed mechanism of a α-TTP-mediated movement of α-tocopherol and PI(4,5)P2 in plasma membranes.