Abstract:
One of the various functions of proteins in biological systems is the transport of small
molecules, for this purpose proteins have naturally evolved special mechanisms to allow both
ligand binding and its subsequent release to a target site; a process fundamental to many
biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to
membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage.
In this research, the ligand binding characteristics of several members of the CRALTRIO
family of lipid binding proteins was examined; the recombinant human a-Tocopherol
Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein
(TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer
protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli
for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic
a-tocopherol.
Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed
determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol
and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding
interactions observed.
The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and
SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same
affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216)
and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by
a-TIP.
Efforts to adequately represent biologically active SPFff AP involved investigation of
tocopherol binding for several different recombinant proteins derived from different constructs
and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these
conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP
serves as the physiological mediator of a-tocopherol, yet structural homology between
proteins allows common recognition of similar ligand features.
In addition, several photo-affmity analogs of a-tocopherol were evaluated for their
potential utility in further elucidation of a-TTP function or identification of novel tocopherol
binding proteins.
