Harborne, SPD, Strauss, J orcid.org/0000-0001-7482-6567, Turku, A et al. (4 more authors) (2018) Chapter Three - Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET. In: Methods in Enzymology 0076-6879. Elsevier , Oxford, UK , pp. 93-130.
Abstract
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.
Metadata
Item Type: | Book Section |
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Authors/Creators: |
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Keywords: | mPPase; smFRET; smALEX; Cysteine mutagenesis; Molecular dynamics simulation; Ion pumping; Conformational change |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Physics and Astronomy (Leeds) > Theoretical Physics (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/M021610/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 04 Jun 2018 10:19 |
Last Modified: | 05 Feb 2019 09:06 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/bs.mie.2018.04.017 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:131618 |