| Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general term used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the alpha-Tocopherol Transfer Protein (alpha-TTP).;In order to completely avoid OmpF contamination, a GST-alpha-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that alpha-hTTP utilizes a collisional mechanism to deliver its ligand. Furthermore, a higher rate of alpha-tocopherol transfer to small unilamellar vesicles (SUVs) versus large unilamellar vesicles (LUVs) indicated that transfer is sensitive to membrane curvature. These findings suggest that alpha-hTTP mediated alpha-Toc transfer is dominated by the hydrophobic nature of alpha-hTTP and the packing density of phospholipid head groups within acceptor membranes.;Based on the calculated free energy change (AG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of alpha-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F165, F169 and I202. In addition, the rates of ligand transfer of the alpha-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in alpha-TTP mediated transfer of alpha-Toc. The propensity of alpha-TTP for highly curved membrane provides a connection to its colocalization with alpha-Toc in late endosomes.;Using a fluorescently labelled alpha-tocopherol (NBD-alpha-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by alpha-hTTP in lipid vesicles. Preliminary results implied that NBD-alpha-Toc simply diffused from 6-His-alpha-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli, outer membrane porin F (OmpF) that co-purified with 6-His-alpha-hTTP from the metal affinity column as the source of the problem. |
