Thomas H Sharp

Leiden University Medical Centre, Leyden, South Holland, Netherlands

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Publications (9)106.51 Total impact

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    ABSTRACT: Synthetic anion transporters (anionophores) have potential as biomedical research tools and therapeutics. However, the efficient and specific delivery of these highly lipophilic molecules to a target cell membrane is non-trivial. Here, we investigate the delivery of a powerful anionophore to artificial and cell membranes using a coiled-coil-based delivery system inspired by SNARE membrane fusion proteins. Incorporation of complementary lipopeptides into the lipid membranes of liposomes and cell-sized giant unilamellar vesicles (GUVs) facilitated the delivery of a powerful anionophore into GUVs, where its anion transport activity was monitored in real time by fluorescence microscopy. Similar results were achieved using live cells engineered to express a halide-sensitive fluorophore. We conclude that coiled-coil driven membrane fusion is a highly efficient system to deliver anionophores to target cell membranes.
    No preview · Article · Jan 2016 · Chemical Science
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    ABSTRACT: An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrels-i.e., pre-assembled bundles of α-helices with central channels-can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric and heptameric α-helical barrels sequester hydrophobic dye within their lumens.
    No preview · Article · Jul 2015 · Journal of the American Chemical Society
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    ABSTRACT: Olfactomedin-1 (Olfm1; also known as noelin, pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell-surface bound receptors to induce cell signaling processes. Using a combined approach of X-ray crystallography, solution scattering, analytical ultracentrifugation and electron microscopy we determine that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the ″V″ is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V-legs consists of a parallel dimeric disulfide-linked coiled coil with at the tips a C-terminal β-propeller dimer. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1(coil-Olf)), which reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and informs on the conformation of several other olfactomedin domain family members. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Apr 2015 · Journal of Biological Chemistry
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    ABSTRACT: The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue, and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers, and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)2, was confirmed by biochemical analysis, transmission electron microscopy and single-channel electrical recording. We show that one of two β-barrels of (α7)2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. The (α7)2 pores spanning two bilayers were also observed by transmission electron microscopy.
    Full-text · Article · Apr 2013 · Nature Communications
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    ABSTRACT: An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals, and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back-to-back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.
    No preview · Article · Apr 2013 · Science
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    ABSTRACT: The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide- and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.
    Full-text · Article · Aug 2012 · Journal of the American Chemical Society
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    ABSTRACT: Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.
    Full-text · Article · Jul 2012 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer-membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer-membrane/periplasmic proteins, including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.
    Full-text · Article · Jul 2012 · Cell host & microbe
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    ABSTRACT: Membrane traffic between organelles is essential for a multitude of processes that maintain cell homeostasis. Many steps in these tightly regulated trafficking pathways take place in microdomains on the membranes of organelles, which require analysis at nanometer resolution. Electron microscopy (EM) can visualize these processes in detail and is mainly responsible for our current view of morphology on the subcellular level. This review discusses how EM can be applied to solve many questions of intracellular membrane traffic, with a focus on the endosomal system. We describe the expansion of the technique from purely morphological analysis to cryo-immuno-EM, correlative light electron microscopy (CLEM), and 3D electron tomography. In this review we go into some technical details of these various techniques. Furthermore, we provide a full protocol for immunolabeling on Lowicryl sections of high-pressure frozen cells as well as a detailed description of a simple CLEM method that can be applied to answer many membrane trafficking questions. We believe that these EM-based techniques are important tools to expand our understanding of the molecular details of endosomal sorting and intracellular membrane traffic in general.
    No preview · Article · Dec 2010 · Methods in cell biology

Publication Stats

201 Citations
106.51 Total Impact Points

Institutions

  • 2016
    • Leiden University Medical Centre
      • Department of Molecular Cell Biology
      Leyden, South Holland, Netherlands
  • 2015
    • Leiden University
      • Molecular Cell Biology Group
      Leyden, South Holland, Netherlands
  • 2013
    • University of Oxford
      Oxford, England, United Kingdom
  • 2010-2013
    • University of Bristol
      • School of Chemistry
      Bristol, England, United Kingdom