The emergence of pathogens resistant to powerful antibiotics is among the most serious problems for the treatment of infectious diseases in the developing world, such as tuberculosis and malaria. The rapid rise of carbapenem-resistant Enterobacteriacea (CRE) such as Klebsiella in the hospital setting is a growing threat to public health in the US. A common mechanism by which multidrug resistance occurs in bacteria involves the action of multidrug resistance (MDR) protein transporters, which bind various cytotoxic compounds and actively extrude them out of the cell. MDR transporters from the small multidrug resistance (SMR) family are widely distributed in bacteria, including the pathogens M. Tuberculosis, B. Pertussis, N. Meningitis, B. Anthracis and S. Aureus. Therefore, successful inhibitors of SMR will be important in the treatment of a broad range of bacterial infections, especially in combination with antibiotic drugs. The objective of the present research is to design small peptides that will interfere with the function of the EmrE transporter of the SMR family. Specifically, peptide analogs will be designed to inhibit the formation of the active EmrE dimer in the bacterial membrane. In the first stage of the project, a low resolution X-ray crystal structure and cryoelectron microscopy (EM) maps of EmrE will be used together with molecular dynamics (MD) and free energy computer simulation methods to construct and validate an atomic-resolution dimer structure of EmrE inside the lipid membrane. To establish the physiological relevance of the structure, ligand dissociation constants will be computed by free energy simulations and compared with experiments. In the second stage, the atomic structure will be used to create models of EmrE-peptide inhibition complexes. The models will be optimized by directed in silico mutagenesis using MD and Monte-Carlo simulations generate peptide inhibitors with the highest affinity for EmrE. The optimized peptides will be stabilized to resist proteolysis by all-hydrocarbon cross-links. Cross-linked peptides are especially attractive candidates for the present problem because they are nontoxic, resistant to proteolysis, and use hydrophobic cross-links compatible with a high membrane permeability required for EmrE binding. The final peptide inhibitors designed in silico will be tested experimentally.