|dc.description.abstract||Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general
tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention
of a-tocopherol in the human circulation system is regulated by the a -Tocopherol
Transfer Protein (a-TIP).
Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our
laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to
monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results
implied that NBD-a-Toe simply diffused from 6-His-a-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-a-hTTP from the metal affinity
column as the source of the problem.
In order to completely avoid OmpF contamination, a GST -a-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 a-hTTP utilizes a
collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol
transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s)
indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and
the packing density of phospholipid head groups within acceptor membranes.
Based on the calculated free energy change (dG) when a protein is transferred
from water to the lipid bilayer, a model was generated to predict the orientation of a-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 F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP
mutants were directly correlated to their membrane binding capabilities, indicating that
membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc.
The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.||en_US