Abstract
The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F(4)-TPP(+)), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure (15)N rotating-frame spin-lattice relaxation (R(1ρ)) rates of F(4)-TPP(+)-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and (1)H-detected (15)N spin-lock experiments under 55 kHz MAS, we measured (15)N R(1ρ) rates site-specifically. Many residues show spin-lock field-dependent (15)N R(1ρ) relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s(-1) at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.