Helbling, Rachel E.Rachel E.HelblingAeschimann, WalterWalterAeschimannSimona, FabioFabioSimonaStocker, AchimAchimStocker0000-0001-6862-7887Cascella, MicheleMicheleCascella2024-10-112024-10-112012https://boris-portal.unibe.ch/handle/20.500.12422/84842"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-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 a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-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 a-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 c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."en500 - Science::570 - Life sciences; biology500 - Science::540 - Chemistryarticle10.7892/boris.1478600031123460002810.1371/journal.pone.0049195