The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter.

EmrE is an Escherichia coli H(+)-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release.

On parallel and antiparallel topology of a homodimeric multidrug transporter.

The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ion-coupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology.

Substrate-induced tryptophan fluorescence changes in EmrE, the smallest ion-coupled multidrug transporter.

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis.