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 host’s 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 AdiC’s four conformations by monitoring the emission-polarization changes of a fluorophore attached to an α-helix that adopts conformation-specific orientations and, furthermore, quantified their probabilities in a series of arginine concentrations. The K_D values determined for arginine in four individual conformations are statistically comparable to the previously reported overall K_D 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 transporter’s function, as well as those of other membrane proteins.