Prokaryotes

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    Protocols in Current Issue
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 72, Q&A: 0

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves

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    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 132, Q&A: 0

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are

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    In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
    Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 59, Q&A: 0

    Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the

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    Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP
    Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 64, Q&A: 0

    Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its

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    Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP
    Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 64, Q&A: 0
    [Abstract]

    Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the

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    In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
    Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 59, Q&A: 0
    [Abstract]

    Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 132, Q&A: 0
    [Abstract]

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 72, Q&A: 0
    [Abstract]

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

    ...
    Tracking the Subcellular Localization of Surface Proteins in Staphylococcus aureus by Immunofluorescence Microscopy
    Authors:  Salvatore J. Scaffidi, Mac A. Shebes and Wenqi Yu, date: 05/20/2021, view: 2949, Q&A: 0
    [Abstract]

    Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment,

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    ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates
    Authors:  Thurston Herricks, Magdalena Donczew, David R. Sherman and John D. Aitchison, date: 05/20/2021, view: 1616, Q&A: 0
    [Abstract]

    Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of

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    A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins
    Authors:  Javier Aguilera, Salvador Vazquez-Reyes and Jianjun Sun, date: 05/20/2021, view: 2631, Q&A: 0
    [Abstract]

    Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have

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    Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins
    Authors:  Rezwan Siddiquee and Ann H Kwan, date: 05/20/2021, view: 2066, Q&A: 0
    [Abstract]

    Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins)

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    Chromatographic Assays for the Enzymatic Degradation of Chitin
    Authors:  Sophanit Mekasha, Tina R. Tuveng, Gustav Vaaje-Kolstad and Vincent G. H. Eijsink, date: 05/05/2021, view: 1696, Q&A: 0
    [Abstract]

    Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product

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    Assessing Swarming of Aerobic Bacteria from Human Fecal Matter
    Authors:  Arjun S. Byju, Deeti Patel, Weijie Chen and Sridhar Mani, date: 05/05/2021, view: 1373, Q&A: 0
    [Abstract]

    Swarming – swift movement across a surface via flagella propulsion – is a unique property of many bacteria. The role of swarming, particularly among bacterial populations of the human gut microbiome, is not yet fully understood; although, it is becoming an area of increased scientific and clinical inquiry. To further characterize

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