The Experts below are selected from a list of 3159 Experts worldwide ranked by ideXlab platform
Edward N. Baker - One of the best experts on this subject based on the ideXlab platform.
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Engineering a Lys-Asn Isopeptide Bond into an immunoglobulin-like protein domain enhances its stability
Scientific Reports, 2017Co-Authors: Hanna Kwon, Paul G. Young, Christopher J. Squire, Edward N. BakerAbstract:The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide Bonds, provided the redox environment is controlled. The discovery of stabilizing Isopeptide Bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such Bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an Isopeptide Bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes. Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the Bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed Isopeptide Bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains.
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A slow-forming Isopeptide Bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly.
Acta Crystallographica Section D Biological Crystallography, 2014Co-Authors: Hae Joo Kang, Chungyu Chang, Hung Ton-that, Neil G. Paterson, Chae Un Kim, Martin Middleditch, Edward N. BakerAbstract:The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn Isopeptide Bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to Bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 A resolution and of a two-domain (D2-D3) construct at 1.87 A resolution, show that each of the three Ig-like domains contains a single Lys–Asn Isopeptide-Bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide Bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its Isopeptide Bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.
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Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili
PloS one, 2011Co-Authors: Neil G. Paterson, Edward N. BakerAbstract:The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn Isopeptide Bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 A resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal Isopeptide Bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for Bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why Isopeptide Bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.
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The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing Isopeptide and disulfide Bonds
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Hae Joo Kang, Hung Ton-that, Neil G. Paterson, Andrew H. Gaspar, Edward N. BakerAbstract:Abstract Cell-surface pili are important virulence factors that enable bacterial pathogens to adhere to specific host tissues and modulate host immune response. Relatively little is known about the structure of Gram-positive bacterial pili, which are built by the sortase-catalyzed covalent crosslinking of individual pilin proteins. Here we report the 1.6-A resolution crystal structure of the shaft pilin component SpaA from Corynebacterium diphtheriae, revealing both common and unique features. The SpaA pilin comprises 3 tandem Ig-like domains, with characteristic folds related to those typically found in non-pilus adhesins. Whereas both the middle and the C-terminal domains contain an intramolecular Lys–Asn Isopeptide Bond, previously detected in the shaft pilins of Streptococcus pyogenes and Bacillus cereus, the middle Ig-like domain also harbors a calcium ion, and the C-terminal domain contains a disulfide Bond. By mass spectrometry, we show that the SpaA monomers are cross-linked in the assembled pili by a Lys–Thr Isopeptide Bond, as predicted by previous genetic studies. Together, our results reveal that despite profound dissimilarities in primary sequences, the shaft pilins of Gram-positive pathogens have strikingly similar tertiary structures, suggesting a modular backbone construction, including stabilizing intermolecular and intramolecular Isopeptide Bonds.
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intramolecular Isopeptide Bonds give thermodynamic and proteolytic stability to the major pilin protein of streptococcus pyogenes
Journal of Biological Chemistry, 2009Co-Authors: Hae Joo Kang, Edward N. BakerAbstract:The pili expressed by Streptococcus pyogenes and certain other Gram-positive bacterial pathogens are based on a polymeric backbone in which individual pilin subunits are joined end-to-end by covalent Isopeptide Bonds through the action of sortase enzymes. The crystal structure of the major pilin of S. pyogenes, Spy0128, revealed that each domain of the two domain protein contained an intramolecular Isopeptide Bond cross-link joining a Lys side chain to an Asn side chain. In the present work, mutagenesis was used to create mutant proteins that lacked either one Isopeptide Bond (E117A, N168A, and E258A mutants) or both Isopeptide Bonds (E117A/E258A). Both the thermal stability and proteolytic stability of Spy0128 were severely compromised by loss of the Isopeptide Bonds. Unfolding experiments, monitored by circular dichroism, revealed a transition temperature Tm of 85 °C for the wild type protein. In contrast, mutants with only one Isopeptide Bond showed biphasic unfolding, with the domain lacking an Isopeptide Bond having a Tm that was 30 °C lower than the unaltered domain. High resolution crystal structures of the E117A and N168A mutants showed that the loss of an Isopeptide Bond did not change the overall pilin structure but caused local disturbance of the protein core that was greater for E117A than for N168A. These effects on stability appear also to be important for pilus assembly.
Klaus Weber - One of the best experts on this subject based on the ideXlab platform.
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Synthetic peptides identify the minimal substrate requirements of tubulin polyglutamylase in side chain elongation
FEBS letters, 1999Co-Authors: Stefan Westermann, Uwe Plessmann, Klaus WeberAbstract:Abstract The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free α-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per αβ-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the Isopeptide Bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.
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Beta-tubulin of bull sperm is polyglycylated
FEBS Letters, 1995Co-Authors: Manfred Ruediger, Uwe Plessmann, Andreas Ruediger, Klaus WeberAbstract:Abstract Car☐y-terminal fragments of α and β tubulin from bull sperm were isolated and characterized by automated sequencing and mass spectrometry. About 60% of sperm α tubulin is polyglycylated. The lateral chain, which can reach 13 residues in length, is covalently attached via an Isopeptide Bond. The fully detyrosinated sperm α tubulin lacks polyglycylation. Thus mammalian sperm microtubules differ from the ciliary axonemal microtubules of the protozoan Paramecium for which others have documented a complete polyglycylation of both α and β tubulin.
Mark Howarth - One of the best experts on this subject based on the ideXlab platform.
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Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox
Nature Publishing Group, 2019Co-Authors: Irsyad Khairil N. A. Anuar, Anusuya Banerjee, Anthony H. Keeble, Alberto Carella, Georgi I. Nikov, Mark HowarthAbstract:Peptide tags are important for protein purification but have minimal benefits afterwards. Here the authors present Spy&Go, which uses rational engineering of the SpyDock protein to capture SpyTag peptides without requiring an irreverisble Isopeptide Bond
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dual plug and display synthetic assembly using orthogonal reactive proteins for twin antigen immunization
Bioconjugate Chemistry, 2017Co-Authors: Karl D Brune, Can M Buldun, Iona Taylor, Florian Brod, Sumi Biswas, Mark HowarthAbstract:Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an Isopeptide Bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an Isopeptide Bond with SnoopTag peptide through transamidation. SpyCatcher–IMX–SnoopCatcher provides a modular platform, whereby SpyTag–antigen and SnoopTag–antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization...
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peptide tag forming a rapid covalent Bond to a protein through engineering a bacterial adhesin
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Bijan Zakeri, Jacob O Fierer, Emrah Celik, Emily Chittock, Ulrich Schwarzlinek, Mark HowarthAbstract:Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous Isopeptide Bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide Bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent Bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous Isopeptide Bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
Sebyung Kang - One of the best experts on this subject based on the ideXlab platform.
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plug and playable fluorescent cell imaging modular toolkits using the bacterial superglue spytag spycatcher
Chemical Communications, 2016Co-Authors: Hyojin Moon, Sebyung KangAbstract:Simple plug-and-playable fluorescent cell imaging modular toolkits are established using the bacterial superglue SpyTag/SpyCatcher protein ligation system. A variety of affibody–fluorescent protein conjugates (AFPCs) are post-translationally generated via the Isopeptide Bond formation, and each AFPC effectively recognizes and binds to its targeting cells, visualizing them with selective colors on demand.
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Plug-and-playable fluorescent cell imaging modular toolkits using the bacterial superglue, SpyTag/SpyCatcher
Chemical Communications, 2016Co-Authors: Hyojin Moon, Sebyung KangAbstract:Simple plug-and-playable fluorescent cell imaging modular toolkits are established using the bacterial superglue SpyTag/SpyCatcher protein ligation system. A variety of affibody–fluorescent protein conjugates (AFPCs) are post-translationally generated via the Isopeptide Bond formation, and each AFPC effectively recognizes and binds to its targeting cells, visualizing them with selective colors on demand.
Ewald Hannappel - One of the best experts on this subject based on the ideXlab platform.
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Thymosin β4 and Tissue Transglutaminase. Molecular Characterization of Cyclic Thymosin β4
Protein Journal, 2013Co-Authors: Jana Knop, Thomas Huff, Heinrich Sticht, Ewald HannappelAbstract:Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an e-amino group of thymosin β4 serves also as an aminyl substrate and an intramolecular Bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming Isopeptide Bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β4 only one Isopeptide Bond between Lys16 and Gln36 was formed (cyclic thymosin β4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β4 × G-actin complex.
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Thymosin β_4 and Tissue Transglutaminase. Molecular Characterization of Cyclic Thymosin β_4
The Protein Journal, 2013Co-Authors: Christine App, Thomas Huff, Jana Knop, Heinrich Sticht, Ewald HannappelAbstract:Thymosin β_4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β_4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β_4 serves also as an aminyl substrate and an intramolecular Bond is formed concomitantly NH_3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β_4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming Isopeptide Bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β_4 only one Isopeptide Bond between Lys16 and Gln36 was formed (cyclic thymosin β_4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β_4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β_4 × G-actin complex.
