Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. ? ? In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR. ? ?
