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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Phosphopeptide Enrichment by Covalent Chromatography After Solid Phase Derivatization of Protein Digests on Reversed Phase Supports
The isolation of the phosphopeptide constituents from phosphoprotein digests is prerequisite to facilitate the mass spectrometric characterization of phosphorylation events. Here, we describe a chemical proteomics approach which combines solid phase derivatization of phosphoprotein digests with phosphopeptide enrichment by covalent chromatography. The use of the solid phase support for derivatization ensures for speed and completeness of reactions. The isolates proved highly suitable for mapping of the sites of phosphorylation by collisionally induced dissociation (CID). The method combines robustness with simplicity of op...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Phosphopeptide Detection with Biotin-Labeled Phos-tag
Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. A series of peptides that are specific substrates of various protein kinases are immobilized on a glass slide and subjected to phosphorylation by cell lysates. The resulting phosphorylated forms of the v...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Thiol-ene-Enabled Detection of Thiophosphorylation as a Labeling Strategy for Phosphoproteins
The adenosine triphosphate (ATP) analogue adenosine 5′-O-(3-thiotriphosphate) (ATPγS) has been applied as a tool to study kinase-substrate phosphorylation. Not only does the transfer of a thiophosphate group represent a unique modification amid the phosphoproteome, but it can also be stable to phosphatase activity. However, detection of this species is complicated due to the similar chemical reactivity of thiophosphate and proteinaceous thiols. Here, we describe a novel method for detection of protein thiophosphorylation utilizing the thiol-ene reaction. By first chemoselectively capping protein thiols through ...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Measuring Cell–Cell Binding Using Flow-Cytometry
Cell–cell adhesion mediates a number of competitive and cooperative microbial interactions. Fluorescence labeling and flow cytometry techniques allow us to observe and measure these interactions rapidly and easily. Here, we describe a method to quantify cell–cell adhesion events between two differentially labeled cell populations. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
An In Vitro Assay for Substrate Translocation by FhaC in Liposomes
The two-partner secretion (TPS) pathway is used by gram-negative bacteria to secrete a large family of virulence exoproteins. Its name is derived from the fact that it involves two proteins, a secreted TpsA protein and a cognate TpsB transporter in the outer membrane. A typical TPS system is represented by the filamentous hemagglutinin FhaB (TpsA protein) and its transporter FhaC (TpsB protein) of Bordetella pertussis. Results from mutational analysis and heterologous expression experiments suggested that FhaC is essential for FhaB translocation across the outer membrane of bacteria. We have devised a cell-free biochemical...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Small Angle X-ray Scattering (SAXS) Characterization of the POTRA Domains of BamA
BamA is the central component of the BAM complex and contains a C-terminal β-barrel domain embedded in the outer membrane, and a soluble, periplasmic domain, made out of five polypeptide transport associated (POTRA) motifs. Structural characterization of the POTRA domains was carried out by a combination of crystallographic, NMR and solution Small Angle X-ray Scattering (SAXS) approaches. Despite its limited resolution, SAXS is an excellent complement to NMR and crystallography. It is well suited to validate high-resolution models in solution and is particularly useful to characterize flexible systems such as the POTR...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
An In Vitro Assay for Outer Membrane Protein Assembly by the BAM Complex
To elucidate the mechanism of a biochemical process it is often essential to reconstitute the reaction in vitro using the minimal set of factors required to drive the reaction to completion. Here, we describe a method to reconstitute the folding and membrane integration of bacterial outer membrane (OM) proteins that have a characteristic β-barrel structure. In this method the BAM complex, a heteroligomer that catalyzes the membrane integration of β-barrel proteins, is first purified and inserted into small lipid vesicles. Denatured OM proteins are then assembled and integrated into the vesicles in the presence of...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Methods to Characterize Folding and Function of BamA Cross-Link Mutants
The utility of protein engineering, both the mutation and deletion of specific amino acids, to investigate protein structure and function has been demonstrated time and time again, and intermolecular and intramolecular interactions within the BAM complex and its individual components are no exception. Extensive efforts have probed conserved and unique amino acid sequences of the Bam proteins to define their functional roles. This chapter summarizes efforts as applied to the disulfide cross-link mutants of BamA and describes experimental methods used in our studies to determine that lateral opening of the barrel domain is r...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Assessing the Outer Membrane Insertion and Folding of Multimeric Transmembrane β-Barrel Proteins
In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form β-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual β-barrels, whereas in the latter, the single autotransporter β-barrel domain is formed by three separate polypeptides. This c...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Expression and Purification of the Individual Bam Components BamB–E
BamB, BamC, BamD, and BamE are lipoproteins that, along with the integral membrane protein BamA, form the β-barrel assembly machinery (BAM) complex in the outer-membrane of Gram-negative bacteria. Elucidating the roles that these lipoproteins play in the β-barrel assembly process requires both structural and functional studies that rely on milligram quantities of pure protein. Here, we describe a simple protocol for expressing individual BamB–BamE proteins in Escherichia coli and purifying them by nickel affinity and size-exclusion chromatography. This protocol yields pure proteins in amounts that are suffi...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
The Expression, Purification, and Structure Determination of BamA from E. coli
In gram-negative bacteria, assembly of outer membrane proteins requires the multicomponent β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. To understand how BamA facilitates outer membrane protein biogenesis, it is important to obtain sufficient amounts of purified recombinant BamA protein for in vitro functional analysis and structure determination. In this chapter, we describe the protocol that we used in our laboratory for the cloning, expression, and purification of E. coli BamA and its N-terminal deletion variants for in vitro ...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Structure Determination of the BAM Complex Accessory Lipoproteins BamB–E
Outer membrane protein biogenesis is a fundamental and essential process in all Gram-negative bacteria. The key players conducting this process are organized in the β-barrel assembly machinery (BAM) complex. This complex has recently attracted a lot of attention due to its importance in cell wall generation, maintenance, and the fascinating yet partially unknown mechanism. The currently best studied example is the BAM complex from E. coli which comprises five proteins, BamA–BamE, two of which, BamA and BamD, are essential for cell survival. Four of the complex proteins, BamB–BamE, are lipoproteins and are ...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Expression, Purification, and Screening of BamE, a Component of the BAM Complex, for Structural Characterization
In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMP) are assembled by the β-barrel assembly machine complex, or BAM complex. This complex includes the essential components BamA, an OMP composed of a carboxyl terminal β-barrel domain and five polypeptide transport-associated domains (POTRA), and the lipoprotein BamD. In Escherichia coli, the complex contains an additional three lipoproteins, BamB, C and E required for efficient delivery of OMPs to the outer membrane. Here we provide methods for production, isotope labeling, purification, and functional screening of BamE for research purp...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Construction and Characterization of an E. coli bamD Depletion Strain
The central Bam components BamA and BamD are both essential genes in E. coli, a fact that often confounds genetic analysis using classical methods. The isolation of “depletion strains” in which these genes can be conditionally expressed removes this obstacle and facilitates the in vivo characterization of Bam function. This chapter describes an efficient two-step recombineering method for the construction of such a depletion strain, which contains an arabinose-inducible allele of bamD, using the λ Red system. Additionally, a simple protocol is presented for the depletion of bamD expression in live cells,...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Identification of BamC on the Surface of E. coli
In order to relate the structural architecture of the BAM complex to its function in outer membrane protein assembly, the arrangement of each component within the complex is vital. This chapter explores the structure and topology of BamC, using a range of biochemical techniques to probe the topology and surface exposure. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
