Secondary active transporters are membrane proteins involved in moving substrates across the cellular membrane. Conformational dynamics underlie this process, allowing the transporter to sample at least two conformations, nominally grouped into inward- and outward-facing states. While studies of structure and dynamics have revealed atomistic insight into transport mechanisms and transport rates, the relative free energy differences among conformations remain underexplored. In this work, we quantified free energy differences between inward- and outward-facing conformations of the multidrug E. coli transporter EmrE using 19F NMR spectroscopy. EmrE consists of an antiparallel and asymmetric homodimer, where its quaternary structure resembles the inverted repeat structures found in larger nonoligomeric transporters. NMR experiments were performed using a minimal heterodimer of EmrE, where a single mutation was introduced into one monomer of the native EmrE homodimer. We discovered that a single conservative mutation perturbed the conformational equilibrium between inward- and outward-facing states by up to 1.5 kcal/mol. Surprisingly, we also found that as little as a single fluorine atom influenced the equilibrium by up to 0.8 kcal/mol. These measurements provide quantitative evidence that subtle changes influence the free energy landscape of a transporter, suggesting a plasticity that may be beneficial in divergent evolution to tune the transport mechanism.
Ramírez-Cordero et al. (Mon,) studied this question.