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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: 575, 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 ...

Sequence Alignment Using Machine Learning for Accurate Template-based Protein Structure Prediction

Authors: Shuichiro Makigaki and Takashi Ishida, date: 05/05/2020, view: 1665, Q&A: 0

[Abstract] Template-based modeling, the process of predicting the tertiary structure of a protein by using homologous protein structures, is useful when good templates can be available. Indeed, modern homology detection methods can find remote homologs with high sensitivity. However, the accuracy of template-based models generated from the ...

F-actin Bundle Sedimentation Assay

Authors: Shan-Shan Lin, Mei-Chun Chuang and Ya-Wen Liu, date: 11/05/2019, view: 2347, Q&A: 0

[Abstract] Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and ...

Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis

Authors: Sujeet Kumar and Natividad Ruiz , date: 06/20/2019, view: 2055, Q&A: 0

[Abstract] Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of ...

Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool

Authors: Felix Boos, Timo Mühlhaus and Johannes M. Herrmann, date: 09/05/2018, view: 4182, Q&A: 0

[Abstract] Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through ...

Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method

Authors: Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 3495, Q&A: 0

[Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System

Authors: Kuancheng Liu and Jianming Hu, date: 04/20/2018, view: 3960, Q&A: 0

[Abstract] The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for ...

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins

Authors: Luke Berry, Angela Patterson, Natasha Pence, John W. Peters and Brian Bothner, date: 03/20/2018, view: 5873, Q&A: 0

[Abstract] The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen ...

Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae

Authors: Kliment A. Verba and David A. Agard, date: 10/05/2017, view: 5885, Q&A: 0

[Abstract] Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of ...

Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells

Authors: Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 7672, Q&A: 0

[Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...

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