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

Abstract

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.