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
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční mikroskopie přístrojové vybavení   metody   MeSH
• mutageneze MeSH
• protozoální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• pyrofosfatasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• racionální návrh léčiv MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rezonanční přenos fluorescenční energie přístrojové vybavení   metody   MeSH
• Saccharomyces cerevisiae MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky * MeSH
• software MeSH
• zobrazení jednotlivé molekuly přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH