Molecular Biology


    Protocols in Current Issue
    Determination of Microtubule Lattice Parameters from Cryo-electron Microscope Images Using TubuleJ
    Authors:  Siou Ku, Cédric Messaoudi, Charlotte Guyomar, Charles Kervrann and Denis Chrétien, date: 11/05/2020, view: 685, Q&A: 0

    The α-β tubulin heterodimer undergoes subtle conformational changes during microtubule assembly. These can be modulated by external factors, whose effects on microtubule structure can be characterized on 2D views obtained by cryo-electron microscopy. Analysis of microtubule images is facilitated if they are straight enough to interpret

    Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
    Authors:  Andrea Saponaro, Vincenzo Maione, Alexandre M. J. J. Bonvin and Francesca Cantini, date: 10/20/2020, view: 713, Q&A: 0
    [Abstract] This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ability to incorporate experimental data, such as NMR Chemical Shift Perturbations and biochemical ...
    BRIDGE: An Open Platform for Reproducible Protein-Ligand Simulations and Free Energy of Binding Calculations
    Authors:  Tharindu Senapathi, Christopher B. Barnett and Kevin J. Naidoo, date: 09/05/2020, view: 2737, Q&A: 2
    [Abstract] Protein-ligand binding prediction is central to the drug-discovery process. This often follows an analysis of genomics data for protein targets and then protein structure discovery. However, the complexity of performing reproducible protein conformational analysis and ligand binding calculations, using vetted methods and protocols can be a ...
    Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues
    Authors:  Siou Ku, Claire Heichette, Laurence Duchesne and Denis Chrétien, date: 08/20/2020, view: 925, Q&A: 0
    [Abstract] Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, ...
    Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins
    [Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...
    In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance
    [Abstract] The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...
    Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
    Authors:  Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 1426, Q&A: 0
    [Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...
    Generation, Analyzing and in-vivo Drug Treatment of Drosophila Models with IBMPFD
    Authors:  Ting Zhang, Bruce A. Hay and Ming Guo, date: 05/20/2020, view: 1436, Q&A: 0
    [Abstract] Missense mutations of p97/cdc48/Valosin-containing protein (VCP) cause inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative diseases. The pathological mechanism of IBMPFD is not clear and there is no treatment. We generated Drosophila models of IBMPFD in adult flight muscle in ...
    Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus
    Authors:  Simon Goto, Kotaro Ishida, Ryosuke Suzuki and Eiji Morita, date: 05/05/2020, view: 1071, Q&A: 0
    [Abstract] Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such ...
    Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
    Authors:  Océane Marescal, Frieder Schöck and Nicanor González-Morales, date: 04/05/2020, view: 1957, Q&A: 0
    [Abstract] Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two ...

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