Electron paramagnetic resonance (EPR) protocol

Cysteine residues were introduced using site directed mutagenesis into LeuT, LeuT R375A, and LeuT R375D constructs. Experiments were conducted as in Claxton et al., 2010. The apo conformation refers to Na⁺ and leucine-free transporter, while the +Na/Leu state was obtained in 200 mM NaCl and fourfold molar excess of Leu relative to LeuT. Double Electron Electron Resonance (DEER) (Jeschke and Polyhach, 2007) was performed at 83K on a Bruker 580 pulsed EPR spectrometer operating at Q-band frequency using a standard 4-pulse sequence (Zou and Mchaourab, 2010). DEER echo decays were analyzed to obtain distance distributions (Jeschke et al., 2002).