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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.The β-Barrel Assembly Machinery Complex
The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of β-barrel structure, and critical for bacterial survival. These β-barrel proteins rely on the β-barrel assembly machinery (BAM) complex for their integration into the outer membrane as folded species. The central and essential subunit of the BAM complex, BamA, is a β-barrel protein conserved in all gram-negative bacteria and also found in eukaryotic organelles derived from bacterial endosymbionts. In Escherichia coli, BamA docks with four peripheral lipoproteins, BamB, BamC, BamD and BamE, partner subun...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA
TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a te...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Strategies for the Analysis of Bam Recognition Motifs in Outer Membrane Proteins
Well-structured proteins interact with other proteins through surface–surface interactions. In such cases, the residues that form the interacting surface are not necessarily neighboring residues on the level of protein sequence. In contrast, unfolded or partially unfolded proteins can interact with other proteins through defined linear motifs. In the case of the β-barrel assembly machinery (BAM) in the outer membrane of Gram-negative bacteria, unfolded β-barrel proteins are recognized through a C-terminal linear motif, and are inserted into the membrane. While the exact mechanism of recognition is still und...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Summary and Future Directions
β-barrel outer membrane proteins (OMPs) are found in the outer membranes (OMs) of all gram-negative bacteria, yet exactly how they are folded and inserted remains unknown. The last decade has provided a wealth of discovery including the identification of the BAM complex, a multicomponent complex responsible for the biogenesis of all OMPs into the OM. It is anticipated that the next decade will further advance our knowledge of how the BAM complex is able to perform its unique and interesting function. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Experimental Methods for Studying the BAM Complex in Neisseria meningitidis
Neisseria meningitidis is a human pathogen. It is intensively studied for host–pathogen interactions and vaccine development. However, its favorable growth properties, genetic accessibility, and small genome size also make it an excellent model organism for studying fundamental biological processes, such as outer membrane biogenesis. Indeed, the first component of the assembly machinery for outer-membrane proteins, the BAM complex, was identified in N. meningitidis. Here, we describe protocols to inactivate chromosomal genes and to express genes from a well-controlled promoter on a plasmid in N. meningitidis. Togethe...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Yeast Mitochondria as a Model System to Study the Biogenesis of Bacterial β-Barrel Proteins
Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial β-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Heat Modifiability of Outer Membrane Proteins from Gram-Negative Bacteria
β-barrel membrane proteins are somewhat unique in that their folding states can be monitored using semi-native SDS-PAGE methods to determine if they are folded properly or not. This property, which is commonly referred to as heat modifiability, has been used for many years on both purified protein and on whole cells to monitor folded states of proteins of interest. Additionally, heat modifiability assays have proven indispensable in studying the BAM complex and its role in folding and inserting β-barrel membrane proteins into the outer membrane. Here, we describe the protocol our lab uses for performing the heat ...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
The Role of a Destabilized Membrane for OMP Insertion
Here we describe the procedures used in our laboratory for the in vitro investigation of the apparent folding kinetics as well as the folding efficiencies of outer membrane proteins (OMPs). Because microbial OMPs display a change in their gel migration upon folding, the usage of traditional gel electrophoresis is a standard method of folding analysis. Additional aspects of the method we detail herein include the preparation and storage of OMP stocks, the setup procedures for a folding reaction, and the analysis of fraction folded from scanned gel images. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System
The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of β-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular “passenger” domain is linked to a β-barrel that inserts in the OM. Despite their structural and functiona...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP326) and Localization of Rare Outer Membrane Proteins
The noncultivable spirochete Treponema pallidum subspecies pallidum (T. pallidum) is the etiological agent of venereal syphilis. In contrast to the outer membranes (OMs) of gram-negative bacteria, the OM of T. pallidum lacks lipopolysaccharide, contains a paucity of integral membrane proteins, and is extremely labile. The lability of the T. pallidum OM greatly hinders efforts to localize the bacterium’s rare outer membrane proteins (OMPs). To circumvent this problem, we developed the gel microdroplet method in which treponemes are encapsulated in porous agarose beads and then probed with specific antibodies in the ab...
Source: Springer protocols feed by Protein Science - October 6, 2015 Category: Biochemistry Source Type: news
Identification of SUMO E3 Ligase-Specific Substrates Using the HuProt Human Proteome Microarray
The functional protein microarray is a powerful and versatile systems biology and proteomics tool that allows the rapid activity profiling of thousands of proteins in parallel. We have recently developed a human proteome array, the HuProt array, which includes ~80 % of all the full-length proteins of the human proteome. In one recent application of the HuProt array, we identified numerous SUMO E3 ligase-dependent SUMOylation substrates. For many SUMO E3 ligases, only a small number of substrates have been identified and the target specificities of these ligases therefore remain poorly defined. In this protocol, we outline ...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
A Bead-Based Multiplex Sandwich Immunoassay to Assess the Abundance and Posttranslational Modification State of β-Catenin
A system-wide analysis of cell signaling involves detecting and quantifying a range of proteins and their posttranslational modification states in the same cellular sample. We propose a protocol for a miniaturized, bead-based array and describe its efficiency in characterizing the different forms and functions of β-catenin. The protocol provides detailed instructions for cell culture and bead array assays that enable insights into complex networks at the systems level. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Three-Dimensional Electrophoresis for Quantitative Profiling of Complex Proteomes
Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post-translational modifications (PTMs) detection. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Sample Preservation Through Heat Stabilization of Proteins: Principles and Examples
Due to post-sampling changes, caused by residual enzyme activity in the sample, levels of analytes can change from their in vivo levels so that they no longer accurately reflect conditions in the living system. The Stabilizor™ system accomplishes elimination of enzyme activity through heat-induced denaturation of enzymes by permanently altering the 3D protein structure of the enzymes. Heat stabilization can be introduced in the workflow either directly after sampling, with the instrument just next to where the sample is taken, or prior to sample homogenization and extraction, when samples are heat denatured directly ...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Free Flow Electrophoresis for Separation of Native Membrane Protein Complexes
This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate membrane protein complexes for proteome analysis. FFE is a highly versatile technology applied in the field of protein analysis. It is superior to native PAGE due to its fast continuous processing of sample at high resolution. Additionally, the dynamic separation range from ions, peptides, to proteins, protein complexes, up to organelles, and whole cells makes it the method of choice in the analysis of proteins. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifie...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
