Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

• MeSH
• Bacterial Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Enzyme Assays instrumentation   methods   MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence instrumentation   methods   MeSH
• Mutagenesis MeSH
• Protozoan Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Pyrophosphatases chemistry   genetics   isolation & purification   metabolism   MeSH
• Drug Design MeSH
• Recombinant Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Fluorescence Resonance Energy Transfer instrumentation   methods   MeSH
• Saccharomyces cerevisiae MeSH
• Sequence Alignment MeSH
• Molecular Dynamics Simulation * MeSH
• Software MeSH
• Single Molecule Imaging instrumentation   methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH