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Journal articleFrench PMW, Görlitz F, Kelly D, et al., 2017,
Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy
, Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087XWe present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
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Journal articlePerdios L, Lowe AR, Saladino G, et al., 2017,
Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET
, Scientific Reports, Vol: 7, ISSN: 2045-2322Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.
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Journal articleBarlow NE, Bolognesi G, Flemming AJ, et al., 2016,
Multiplexed droplet Interface bilayer formation
, Lab on a Chip, Vol: 16, Pages: 4653-4657, ISSN: 1473-0197We present a simple method for the multiplexed formation ofdroplet interface bilayers (DIBs) using a mechanically operatedlinear acrylic chamber array. To demonstrate the functionality ofthe chip design, a lipid membrane permeability assay is performed.We show that multiple, symmetric DIBs can be created andseparated using this robust low-cost approach.
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Journal articleBoyd C, Parsons ES, Smith RAG, et al., 2016,
Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation
, Scientific Reports, Vol: 6, ISSN: 2045-2322The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant β-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.
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Journal articleSalzmann CG, Slater B, Radaelli PG, et al., 2016,
Detailed crystallographic analysis of the ice VI to ice XV hydrogen ordering phase transition
, JOURNAL OF CHEMICAL PHYSICS, Vol: 145, ISSN: 0021-9606- Author Web Link
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- Citations: 27
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Journal articleGoncalves V, Brannigan JA, Laporte A, et al., 2016,
Structure-guided optimization of quinoline inhibitors of Plasmodium N-myristoyltransferase
, MedChemComm, Vol: 8, Pages: 191-197, ISSN: 2040-2511The parasite Plasmodium vivax is the most widely distributed cause of recurring malaria. N-myristoyltransferase (NMT), an enzyme that catalyses the covalent attachment of myristate to the N-terminal glycine of substrate proteins, has been described as a potential target for the treatment of this disease. Herein, we report the synthesis and the structure-guided optimization of a series of quinolines with balanced activity against both Plasmodium vivax and Plasmodium falciparum N-myristoyltransferase (NMT).
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Journal articleJohnson S, Rahmani R, Drew DR, et al., 2016,
Truncated latrunculins as actin inhibitors targeting plasmodium falciparum motility and host-cell invasion
, Journal of Medicinal Chemistry, Vol: 59, Pages: 10994-11005, ISSN: 0022-2623Polymerization of the cytosolic protein actin is critical to cell movement and host-cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins, with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesised and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with latrunculin B against Plasmodium falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.
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Journal articleWard JA, McLellan L, Stockley M, et al., 2016,
Quantitative Chemical Proteomic Profiling of Ubiquitin Specific Proteases in Intact Cancer Cells
, ACS Chemical Biology, Vol: 11, Pages: 3268-3272, ISSN: 1554-8937Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activity-based profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.
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Journal articleRodgers U, Lanyon-Hogg T, Masumoto N, et al., 2016,
Characterization of hedgehog acyltransferase inhibitors identifies a small molecule probe for hedgehog signaling by cancer cells
, ACS Chemical Biology, Vol: 11, Pages: 3256-3262, ISSN: 1554-8937The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.
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Journal articleChan CL, Bolognesi G, Bhandarkar A, et al., 2016,
DROPLAY: laser writing of functional patterns within biological microdroplet displays
, Lab on a Chip, Vol: 16, Pages: 4621-4627, ISSN: 1473-0197In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblieswith user-defined re-writable two-dimensional patterns of functional droplets. Light responsive water-in-oil dropletscapable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays in amicrofluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to ‘write’ userdefined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent)droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biologicalmonitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a largepopulation of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissuemimics and biological display materials.
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