Biology subjects

Lewis, J. H.

Publications and source records attributed to Lewis, J. H..

2 recordsLinked to original sources

bioRxiv2022

Integrative 4D-conformational mechanisms of single AdiC transporter molecules

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.

biophysics↗

bioRxiv2022

Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules

The AdiC transporter facilitates the movement of arginine and its metabolite across the membrane of pathogenic enterobacteria, enabling them to evade a hosts highly acidic gastric defense barrier to reach the intestines. Like other transporters, AdiC undergoes a series of necessary conformational changes. Detection of these changes, which occur on angstrom-and- millisecond scales, remains extremely challenging. Here, using a high-resolution polarization-microscopic method, we have successfully resolved AdiCs four conformations by monitoring the emission-polarization changes of a fluorophore attached to an -helix that adopts conformation-specific orientations and, furthermore, quantified their probabilities in a series of arginine concentrations. The KD values determined for arginine in four individual conformations are statistically comparable to the previously reported overall KD determined using isothermal titration calorimetry. This demonstrated strong resolving power of the present polarization-microscopy method will enable an acquisition of the quantitative information required for understanding the expected complex conformational mechanism underlying the transporters function, as well as those of other membrane proteins.

biophysics↗