Integrative 4D-conformational mechanisms of single AdiC transporter molecules

Abstract

To understand the mechanism of counter-transport of substrates by the amino-acid transporter AdiC, we used a state-of-the-art polarization-microscope to investigate conformation-specific changes of the emission polarization of a fluorophore attached to individual AdiC molecules. This capability enabled us to determine the lifetimes of two energetic states of each of AdiCs four conformations in the absence and presence of its two natural substrates, totaling 24 states. From these lifetimes and relative state-to-state transition frequencies, we further determined 60 rate constants of all state transitions and the 4 KD values for the two substrates to interact with both sides of AdiC, quantitatively defining a 24-state model that satisfactorily predicts previously observed transporting behaviors of AdiC. Combining this temporal information and the existing structural information, we have successfully built a fully experiment-based integrative 4D-model to capture and exhibit the complex spatiotemporal mechanisms of a facilitated counter-transport of an amino acid and its metabolite. Thus, a combination of the present method and existing structural techniques serves as an effective means to help transition structural biology, which has thus far been highly successful in the investigation of individual static structures, to an integrative form of dynamic structural biology.