title: Engineering tocopherol selectivity in α-TTP: a combined in vitro/in silico study.
abstract: We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of [Formula: see text]-tocopherol transfer protein ([Formula: see text]-TTP) towards [Formula: see text]-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for [Formula: see text]-tocopherol to [Formula: see text]-TTP 8.26[Formula: see text]2.13 kcal mol[Formula: see text] lower than that of [Formula: see text]-tocopherol. Our calculations show that [Formula: see text]-tocopherol binds to [Formula: see text]-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of [Formula: see text]-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to [Formula: see text]-tocopherol, with striking structural similarities to the wild-type-[Formula: see text]-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of [Formula: see text]-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a [Formula: see text]-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for [Formula: see text]-tocopherol selection in omnivorous animals.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Helbling
authors.forename: Walter
authors.initials: W
authors.lastname: Aeschimann
authors.forename: Fabio
authors.initials: F
authors.lastname: Simona
authors.forename: Achim
authors.initials: A
authors.lastname: Stocker
authors.forename: Michele
authors.initials: M
authors.lastname: Cascella
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D019295:Computational Biology; D005470:Fluorometry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D058767:Protein Unfolding; D013816:Thermodynamics; D024505:Tocopherols / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D050505:Mutant Proteins; C017763:alpha-tocopherol transfer protein; D024505:Tocopherols