protein cross-linking Dotaz Zobrazit nápovědu
Studium proteinů a zvláště jejich 3D struktury či protein -proteinových interakcí hraje v dnešním biochemickém výzkumu nezanedbatelnou roli. Řada alternativních metod tohoto výzkumu (např. PIXL, FRET) využívá biotechnologické postupy pro zavedení nepřirozených aminokyselin či jejich strukturních analogů do proteinové sekvence během jejich rekombinantní přípravy. Předkládaná práce uvádí několik biotechnologických přístupů inkorporace foto -methioninu (pMet, L-2-amino-5,5-azi -hexanová kyselina) do sekvence dvou modelových savčích proteinů.
The study of proteins and especially their 3D structure or protein -protein interactions plays significant role in contemporary biochemical research. Many alternative methods of the research (e.g. PIXL, FRET) employing different biotechnology techniques to introduce the non -natural amino acids or their structural analogues within protein sequence during its recombinant expression. This study presents several biotechnology approaches to introduce photo -methionine (pMet, L-2-amino-5,5-azi -hexanoic acid) into the sequence of two model mammalian proteins.
- Klíčová slova
- světlem iniciované síťování, foto-methionin,
- MeSH
- aminoacyl-tRNA-synthetasy metabolismus MeSH
- aminokyseliny chemie metabolismus MeSH
- biotechnologie MeSH
- diazomethan MeSH
- konformace proteinů * MeSH
- mapování interakce mezi proteiny * MeSH
- methionin MeSH
- posttranslační úpravy proteinů * MeSH
- proteiny chemie MeSH
- proteosyntéza MeSH
- techniky in vitro MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- práce podpořená grantem MeSH
Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- kalmodulin analýza chemie MeSH
- konformace proteinů MeSH
- mapování interakce mezi proteiny metody MeSH
- reagencia zkříženě vázaná chemie MeSH
- sekundární struktura proteinů MeSH
- skot MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
1st ed. 2340 s. : il.
The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.
Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
- MeSH
- hmotnostní spektrometrie přístrojové vybavení metody normy MeSH
- konformace proteinů MeSH
- lidé MeSH
- mapování interakce mezi proteiny metody MeSH
- mezinárodní spolupráce MeSH
- proteiny ultrastruktura MeSH
- proteomika přístrojové vybavení metody normy MeSH
- reagencia zkříženě vázaná chemie MeSH
- reprodukovatelnost výsledků MeSH
- směrnice jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- kongresy MeSH
- práce podpořená grantem MeSH
Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
- MeSH
- arginin chemie MeSH
- DNA chemická syntéza chemie MeSH
- histony chemie MeSH
- ketony chemická syntéza chemie MeSH
- nádorový supresorový protein p53 chemie MeSH
- peptidy chemie MeSH
- proteiny chemie MeSH
- reagencia zkříženě vázaná chemická syntéza chemie MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- thiminnukleotidy chemická syntéza chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chemical cross-linking mass spectrometry has become a popular tool in structural biology. Although several algorithms exist that efficiently analyze data-dependent mass spectrometric data, the algorithm to identify and quantify intermolecular cross-links located at the interaction interface of homodimer molecules was missing. The algorithm in LinX utilizes high mass accuracy for ion identification. In contrast with standard data-dependent analysis, LinX enables the elucidation of cross-linked peptides originating from the interaction interface of homodimers labeled by 14N/15N, including their ratio or cross-links from protein-nucleic acid complexes. The software is written in Java language, and its source code and a detailed user's guide are freely available at https://github.com/KukackaZ/LinX or https://ms-utils.org/LinX. Data are accessible via the ProteomeXchange server with the data set identifier PXD023522.
Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.
- MeSH
- fotochemické procesy MeSH
- lidé MeSH
- mapování interakce mezi proteiny metody MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.
- MeSH
- hmotnostní spektrometrie metody MeSH
- karbodiimidy chemie MeSH
- konformace proteinů MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- proteiny 14-3-3 chemie genetika izolace a purifikace metabolismus MeSH
- reagencia zkříženě vázaná chemie MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- vodík-deuteriová výměna metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nucleotides, 2'-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).
