Engineering tocopherol selectivity in α-TTP: a combined in vitro/in silico study.

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Tocopherol transfer protein (TTP) plays a crucial role in vitamin E metabolism.
• Understanding TTP's selectivity for different tocopherol isomers is essential for vitamin E research and applications.

Purpose of the Study:

• To elucidate the molecular basis for the selectivity of TTP towards alpha-tocopherol over gamma-tocopherol.
• To engineer TTP variants with altered selectivity profiles.

Main Methods:

• Combined in vitro and in silico approaches, including molecular dynamics simulations and free energy perturbation calculations.
• Differential scanning fluorimetry and in vitro competitive binding assays were used for experimental validation.

Main Results:

• Computational methods predicted a significantly lower binding free energy for alpha-tocopherol compared to gamma-tocopherol with wild-type TTP.
• The A156L mutation was predicted to reverse selectivity, favoring gamma-tocopherol binding.
• Experimental assays confirmed the predicted affinity changes and the critical role of residue A156.

Conclusions:

• Residue A156 is a key determinant of TTP's tocopherol selectivity.
• Engineered TTP variants with modulated binding properties have potential industrial applications in vitamin E purification.
• The study provides insights into the evolutionary mechanisms of tocopherol selection in animals.