Footprinting
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Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.
- MeSH
- aminokyseliny chemie metabolismus MeSH
- footprinting proteinů metody MeSH
- haptoglobiny chemie metabolismus MeSH
- hemoglobiny chemie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- hydroxylový radikál chemie MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.
Polymerázová řetězová reakce je dnes hojně využívána pro molekulárně genetickou identifikaci osob v soudním lékařství. V příspěvku jsou demonstrovány možnosti, které skýtá tato metoda V případech, kdy je třeba zjistit, zda biologické stopy jsou lidského či zvířecího původu. Jedna se o metodu rychlou a dobře proveditehiou ve standardně vybavené molekulárně genetické laboratoři. Bylo vyšetřeno 11 vzorků DNA izolované z různých zvířat a ptáků. Ve všech případech bylo možné odlišit tyto vzorky dle výsledku reakce od paralelně zpracovávané lidské DNA.
Polymerase chain reaction is often used for molecular genetic human identification in forensic medicine today. In this study, the possibilities are demonstrated, which are provided by the method in such cases, where it is necessary to determine, if the biological traces are of animal or human origin. The method is rapid and well performed in a molecular genetic laboratory with standard equipment. Eleven samples of DNA isolated from difíerent animals and birds were examined. In all cases, it was possible to distingfuish these samples according to the results of reaction from simultaneously examined human DNA.
Cíl: Prostudování genetické příbuznosti českých multirezistentních kmenů Acinetobacter baumannii a epidemických klonů ze severozápadní Evropy. Metodika: Studovaný soubor zahrnoval 70 epidemiologicky nesouvisejících multirezistentních kmenů, izolovaných v českých nemocnicích v letech 1991-2001,8 referenčních kmenů pro epidemické klony I a II ze severozápadní Evropy (Dijkshoorn L, et al. J Clin Microbiol 1996;34:1519-25) a kontrolní skupinu 15 citlivých českých kmenů. Kmeny byly studovány pomocí ribotypizace, AFLP fingerprintingu, biotypizace a vyšetřeny na citlivost k 11 antibiotikům diskovým difúzním testem (ampicilin + sulbaktam, piperacilin, ceftazidim, imipenem, ko-trimoxazol, ofloxacin, gentamycin, tobramycin, amikacin, netilmicin a tetracyklin). Výsledky: Pomoci numerické analýzy AFLP profilů a ribotypů byly české multirezistentní kmeny klasifikovány do klonu I (n = 41), klonu II (n = 21) a heterogenní skupiny ostatních kmenů (n = 8). Citlivé kmeny byly genotypově a fenotypově heterogenní a ostře odlišené od klonu I a II. Kmeny náležející k témuž klonu měly shodné nebo podobné ribotypy, odlišné od ribotypů ostatních kmenů. Kmeny klonu I náležely k biotypům U (n = 24), 6 (n = 15) a 12 (n = 1) a byly v průměru rezistentní k 7 antibiotikům; kmeny klonu II patřily k biotypu 2 a byly v průměru rezistentní k 6 antibiotikům. Závěr. České multirezistentní kmeny A. baumannii patřily převážně do dvou klonálních uskupení, prokázaných u nemocničních epidemických kmenů ze severozápadní Evropy. Skupiny A a B, dříve popsané v České republice, jsou podmnožinami těchto klonů (klonu I resp. Klonu II). Oba klony zahrnovaly epidemické i sporadické kmeny, vyskytující se nejméně od roku 1991 v České republice. Dlouhodobé panevropské rozšířeni a vnitřní typová diverzita klonů naznačuje, že jde o relativně stará uskupení, zahrnující geografické subklony. Vysoce rezistentní české kmeny klonu I náležející k biotypu 11 představují pravděpodobně jeden z těchto subklonů.
Objectives: To investigate genetic relatedness of multiresistant hospital Acinetobacter baumannii strains from the Czech Republic and epidemic A. baumannii clones from north-western Europe. Methods: Seventy epidemiologically unrelated multiresistant strains isolated in Czech hospitals between 1991 and 2001, 8 reference strains of epidemic clones 1 and II from north-western Europe (Dijkshoorn L, et al. J Clin Microbiol 1996;34:1519-25) and 15 control susceptible Czech strains were studied by AFLP fingerprinting, ribotyping with HindIII and HincII and biotyping and were tested for susceptibility to 11 antibiotics (ampicillin +sulbactam, piperacillin, ceftazidime, imipenem, co-trimoxazole, ofloxacin, gentamicin, tobramycin, amikacin, netilmicin and tetracycline) using disk diffusion test. Results: Based on numerical analysis of AFLP fingerprints and ribotypes, the Czech multiresistant strains were classified into clone (n = 41), clone II (n = 21) or a heterogeneous group of other strains (n = 8). The susceptible strains were genotypically and phenotypically heterogeneous and distinct from those of clones I and II. The strains of the same clone had identical or similar ribotypes not found in the other strains. Clone I strains belonged to biotypes 11 (n = 24), 6 (n = 15) or 12 (n = 1) and were resistant, on average to seven antibiotics; clone II strains were of biotype 2 and were resistant, on average to six antibiotics. Conclusions: The Czech multiresistant A. baumannii strains belonged mostly to two clonal lineages previously recognized among strains from north-western European hospitals. Groups A and B recently described in the Czech Republic are subgroups of these clones (clone I and clone II, respectively). Both clones comprise outbreak and sporadic strains isolated from all over the Czech Republic since 1991. Paneuropean spread over a long time and intraclonal type diversity indicate that the clones are relatively old groups with a number of geographical subclones. One of these is probably represented by highly resistant Czech clone I strains belonging to biotype 11.
[Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.
- MeSH
- deoxyribonukleasa I MeSH
- DNA footprinting MeSH
- DNA chemie metabolismus MeSH
- ethidium chemie MeSH
- financování organizované MeSH
- kompetitivní vazba MeSH
- konformace nukleové kyseliny MeSH
- pyridiny chemie MeSH
- restrikční enzymy metabolismus MeSH
- sekvence nukleotidů MeSH
- stereoizomerie MeSH
- superhelikální DNA chemie MeSH
- železnaté sloučeniny chemie MeSH
Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
- MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- bezbuněčný systém MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- DNA bakterií metabolismus MeSH
- DNA footprinting MeSH
- fungální proteiny fyziologie MeSH
- histony genetika metabolismus MeSH
- mikroskopie atomárních sil MeSH
- multiproteinové komplexy fyziologie MeSH
- nukleozomy fyziologie ultrastruktura MeSH
- plazmidy chemie MeSH
- proteiny vázající RNA fyziologie MeSH
- rekombinantní proteiny metabolismus MeSH
- restrikční endonukleasy typu II MeSH
- restrukturace chromatinu genetika fyziologie MeSH
- Saccharomyces cerevisiae metabolismus ultrastruktura MeSH
- Xenopus laevis genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo-helical 'flexicate' complexes, bimetallic triple-stranded ferro-helicates [Fe2(NN-NN)3](4+) incorporating the common NN-NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo-helical complexes for cancer chemotherapy.
- MeSH
- 2-aminopurin analýza MeSH
- amilorid analýza MeSH
- denaturace nukleových kyselin MeSH
- DNA footprinting MeSH
- DNA-lyasa (apurinová nebo apyrimidinová) antagonisté a inhibitory MeSH
- DNA chemie MeSH
- inhibitory enzymů chemie MeSH
- kalorimetrie MeSH
- poškození DNA * MeSH
- protinádorové látky chemie MeSH
- vazebná místa MeSH
- železnaté sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
