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Current Protocols in Protein Science

Vanessa Carvalho, Joachim W Pronk, Andreas H Engel
The steep increase of atomic scale structures determined by 3D cryo-electron microscopy (EM) deposited in the EMDataBank documents progress of a methodology that was frustratingly slow ten years ago. While sample vitrification on grids has been successfully used in all EM laboratories for decades, beam damage remains a road block. Developments in instrumentation and software to exploit the information carried by elastically scattered electrons made the task to achieve atomic scale resolution easier. This together with the development of fast single electron detecting cameras has resulted in unprecedented possibilities for structure determination by 3D cryo-EM...
September 10, 2018: Current Protocols in Protein Science
Aurora Paiano, Azzurra Margiotta, Maria De Luca, Cecilia Bucci
This article describes the general method to perform the classical two-hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein-protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two-hybrid assay have been proven to be binary direct interactions...
August 21, 2018: Current Protocols in Protein Science
Peggi M Angel, Kim Norris-Caneda, Richard R Drake
Tryptic peptide imaging is a primary workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and has led to new information reporting highly multiplexed protein localization. Technological advances within the last few years have produced robust tools for automated spraying of both matrix and enzymes. When combined with high-mass-resolution and high-mass-accuracy instrumentation, studies now generally result in two-dimensional mapping of well over 1,000 peptide peaks. This protocol describes sample preparation, spraying, and application of enzymes and matrices, and MALDI FT-ICR instrumental considerations for two-dimensional mapping of tryptic peptides from fresh-frozen or formalin-fixed, paraffin-embedded tissue sections...
August 16, 2018: Current Protocols in Protein Science
Huiying Zhao, Ghazaleh Taherzadeh, Yaoqi Zhou, Yuedong Yang
Protein-carbohydrate interaction is essential for biological systems, and carbohydrate-binding proteins (CBPs) are important targets when designing antiviral and anticancer drugs. Due to the high cost and difficulty associated with experimental approaches, many computational methods have been developed as complementary approaches to predict CBPs or carbohydrate-binding sites. However, most of these computational methods are not publicly available. Here, we provide a comprehensive review of related studies and demonstrate our two recently developed bioinformatics methods...
August 14, 2018: Current Protocols in Protein Science
Gang Hu, Lukasz Kurgan
Sequence similarity searching has become an important part of the daily routine of molecular biologists, bioinformaticians and biophysicists. With the rapidly growing sequence databanks, this computational approach is commonly applied to determine functions and structures of unannotated sequences, to investigate relationships between sequences, and to construct phylogenetic trees. We introduce arguably the most popular BLAST-based family of the sequence similarity search tools. We explain basic concepts related to the sequence alignment and demonstrate how to search the current databanks using Web site versions of BLASTP, PSI-BLAST and BLASTN...
August 13, 2018: Current Protocols in Protein Science
Heungnam Kim, Mitchell Ho
Heparan sulfate (HS) plays an important role in development and disease. It interacts with many growth factors, chemokines, and other ligands known to be important for cell growth, motility, and differentiation. However, isolating an antibody to HS in mice, rabbits, or humans is difficult due to the poor immunogenicity of HS. Phage display is a major antibody engineering technology that allows the selection of antibodies for poorly immunogenic or highly conserved antigens. This protocol contains detailed procedures for HS antigen preparation and isolation of a phage displayed human single-chain Fv (HS20) that binds HS on glypican-3 (GPC3), and analysis of the selected phage antibody...
August 9, 2018: Current Protocols in Protein Science
Sean R Gallagher
Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit composition, track post-translational modifications, and verify identity and homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration...
August 9, 2018: Current Protocols in Protein Science
Richard R Drake, Thomas W Powers, Kim Norris-Caneda, Anand S Mehta, Peggi M Angel
Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post-translational modification. These modifications occur most commonly as one of three major classes: N-linked glycosylation on asparagine residues, O-linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N-linked glycans, an endoglycosidase enzyme, peptide N-glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar...
August 3, 2018: Current Protocols in Protein Science
Kai Wen Teng, Pin Ren, Paul R Selvin
Methods to efficiently deliver fluorophores across the cell membrane are crucial for imaging the dynamics of intracellular proteins using fluorescence. Here we describe a simple protocol for permeabilizing living cells using streptolysin O, a bacterial toxin, which allows transient uptake of fluorescent probes for labeling specific intracellular proteins. The technique is applicable for delivering different classes of fluorescent probes with a molecular weight of <150 kDa, and it is also applicable to a variety of different cell lines...
August 2018: Current Protocols in Protein Science
Zoltan Nagy, Shane Comer, Albert Smolenski
Phos-tag gels are recent tools to dissect protein phosphorylation that operate by inducing a shift in the electrophoretic mobility of phosphorylated proteins compared to their nonphosphorylated counterparts. This article describes the preparation and electrophoresis of Zn2+ -Phos-tag gels along with electrotransfer of the separated phospho- and nonphosphoproteins onto a PVDF membrane using either wet-tank or semidry transfer. We also discuss the theory behind the technology with critical parameters to keep in mind for its successful application...
August 2018: Current Protocols in Protein Science
Guillaume Lenoir, Thibaud Dieudonné, Anaïs Lamy, Maylis Lejeune, José-Luis Vazquez-Ibar, Cédric Montigny
Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent...
August 2018: Current Protocols in Protein Science
Minoru Tamura
Actin is one of the most abundant proteins in the cytoplasm of eukaryotic cells and plays important roles in a variety of cellular functions. However, it has been difficult to produce actin in substantial amounts using bacterial expression systems. In this article, a new method is described for the production of recombinant actin in bacterial cells. Human β-actin (His-tagged) can be expressed using a cold shock vector, pCold, in a bacterial expression system and then separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin...
August 2018: Current Protocols in Protein Science
Kirsten N Swonger, Anne S Robinson
Determining ligand binding kinetics provides an indirect route to probe the functional capabilities of the binding pocket of a membrane protein receptor. Presented in this unit are four ligand-binding protocols that provide data useful for characterizing membrane proteins, including equilibrium binding, thermostability, competitive ligand binding, and kinetic ligand binding. These techniques use fluorescence anisotropy, which is safer, less costly, and simpler to execute than radioactive ligand binding. Each protocol may be used on its own or in combination with others to quantify a number of ligand binding constants...
August 2018: Current Protocols in Protein Science
Javier Manzella-Lapeira, Joseph A Brzostowski
This updated unit compares three methods to acquire Förster Resonance Energy Transfer (FRET) data in living cells using a confocal microscope: Acceptor photobleaching, Acceptor-sensitized emission FRET, and Donor fluorescence lifetime imaging. Detailed protocols for live cell husbandry, image acquisition, and data analysis are provided. In addition to providing instructions for manufacturer's analysis tool sets, we provide an easy-to-use, MATLAB-based code to calculate FRET efficiency from data obtained using the Acceptor photobleaching or Acceptor-sensitized emission method, which can be freely downloaded...
August 2018: Current Protocols in Protein Science
Ziyun Ding, Daisuke Kihara
Understanding protein-protein interactions (PPIs) in a cell is essential for learning protein functions, pathways, and mechanism of diseases. PPIs are also important targets for developing drugs. Experimental methods, both small-scale and large-scale, have identified PPIs in several model organisms. However, results cover only a part of PPIs of organisms; moreover, there are many organisms whose PPIs have not yet been investigated. To complement experimental methods, many computational methods have been developed that predict PPIs from various characteristics of proteins...
August 2018: Current Protocols in Protein Science
Helena Nevalainen, Peter Bergquist, Valentino Setoa Junior Te'o
This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation...
April 2018: Current Protocols in Protein Science
Clelton A Santos, Anete P Souza
Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production...
April 2018: Current Protocols in Protein Science
Erik Walinda, Daichi Morimoto, Kenji Sugase
Proteins and nucleic acids are central to all biological processes. NMR spectroscopy has proven to be excellent for studying the dynamics of these macromolecules over various timescales. Relaxation rates and heteronuclear nuclear Overhauser-effect values can resolve motion on pico- to nanosecond timescales, residual dipolar couplings provide information on submicro- to millisecond timescales, and even slower dynamics over seconds to hours can be resolved by hydrogen-exchange experiments. Relaxation dispersion experiments are especially valuable because they resolve motion on micro- to millisecond timescales, encompassing biomolecular motions associated with ligand binding, enzymatic catalysis, and domain-domain opening...
April 2018: Current Protocols in Protein Science
Stéphane Jaisson, Aurore Desmons, Manon Doué, Laëtitia Gorisse, Christine Pietrement, Philippe Gillery
Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts...
April 2018: Current Protocols in Protein Science
Helena Nevalainen, Robyn Peterson, Natalie Curach
Filamentous fungi are lower eukaryotes increasingly used for expression of foreign proteins ranging from industrial enzymes originating from other fungi and bacteria to proteins of mammalian origin, such as antibodies and growth factors. Their strengths include an excellent capacity for protein secretion and their eukaryotic protein processing machinery. Proteins secreted from filamentous fungi are modified in the secretory pathway, with folding, proteolytic processing, and addition of glycans being the main modifications...
April 2018: Current Protocols in Protein Science
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