Conformational ensemble of the sodium coupled aspartate transporter

Sodium and aspartate symporter from Pyrococcus horikoshii, Glt(Ph), is a homologue of the mammalian glutamate transporters, homotrimeric integral membrane proteins controlling the neurotransmitter levels in brain synapses. These transporters function by alternating between outward and inward facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we employ double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer within distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers sampling the outward and inward facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions vary only modestly in detergent and in bilayers, but in several mutants unique conformations are stabilized by the latter.