Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity.
The tocopherol transfer protein (TTP) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[1,2,5]oxadiazol-4-ylamino)nonyl]chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue shifted, and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analogue binds to TTP reversibly and with high affinity (K(d) = 8.5 +/- 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the ataxia with vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (K(d) = 71 +/- 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high-resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction.
Morley
Samantha
S
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
Cross
Valerie
V
Cecchini
Matt
M
Nava
Phil
P
Atkinson
Jeffrey
J
Manor
Danny
D
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004530:Egg Yolk / Q000201:enzymology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D024505:Tocopherols; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
C509411:2,5,7,8-tetramethylchroman-2-(9-(7-nitrobenz(1,2,5)oxadiazol-4-ylamino)nonyl)chroman-6-ol; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D011993:Recombinant Fusion Proteins; C017763:alpha-tocopherol transfer protein; D014810:Vitamin E; D009327:4-Chloro-7-nitrobenzofurazan; D024505:Tocopherols