Upon publication of the LeuT crystal structure in 2005, researchers noticed a positively-charged lysine residue (K288) protruding into the hydrophobic membrane, an energetically unfavorable arrangement. Subsequently, it was found that a mutation of this residue (K288A) increased the kinetic turnover rate of LeuT. Furthermore, it has been suggested that this change in kinetics was due to the alanine mutant sampling the “rate-limiting” inward-facing conformation more frequently, possibly due to its unfavorable interactions with the membrane. This served as the basis for the generation of an “inward-facing” LeuT crystal structure in 2013. We sought to test these hypotheses in solution using EPR spectroscopy. Evidence provided in this series of experiments suggests that neither hypothesis was supported. The K288A mutant in total appears to sample an occluded-like conformation that is not found in the ensemble of wild type structures. Furthermore, as these experiments were conducted in a micelle, changes in the protein's conformation were occurring in the absence of a membrane. The result of this work cautions against the use of mutations of conserved residues to “select” for alternative conformations in structural studies.