The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.

• MeSH
• 2D gelová elektroforéza MeSH
• acylace MeSH
• acyltransferasy chemie   genetika   metabolismus   MeSH
• adenylátcyklasový toxin * MeSH
• Bordetella pertussis enzymologie   MeSH
• erytrocyty účinky léků   metabolismus   patologie   MeSH
• faktory virulence rodu Bordetella chemie   genetika   metabolismus   toxicita   MeSH
• hemolýza účinky léků   MeSH
• histidin genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kyselina palmitová metabolismus   MeSH
• lysin genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• ovce MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• serin genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• acyltransferasy MeSH
• adenylátcyklasový toxin * MeSH
• faktory virulence rodu Bordetella MeSH
• histidin MeSH
• kyselina palmitová MeSH
• lysin MeSH
• peptidové fragmenty MeSH
• serin MeSH

Adamantane, the smallest diamondoid molecule with a symmetrical cage, contains two distinct carbon sites, CH and CH2. The ionization/excitation of the molecule leads to the cage opening and strong structural reorganization. While theoretical predictions suggest that the carbon site CH primarily causes the cage opening, the role of the other CH2 site remains unclear. In this study, we used advanced experimental Auger electron-ion coincidence techniques and theoretical calculations to investigate the fragmentation dynamics of adamantane after resonant inner-shell photoexcitation. Our results demonstrate that some fragmentation channels exhibit site-sensitivity of the initial core-hole location, indicating that different carbon site excitations could lead to unique cage opening mechanisms.

• Klíčová slova
• AE–PICO/PIPICO coincidence, adamantane, inner-shell fragmentation, site-selectivity,
• Publikační typ
• časopisecké články MeSH

The ion selective electrode (ISE)-based potentiometric approach is shown to be an effective means of characterizing the anion recognition sites in the molecular receptor calix[2]pyridino[2]pyrrole (CPP). In particular, potentiometric pH-measurements involving the use of experimental PVC-membranes based on CPP revealed the existence of both mono- and diprotonated forms of the receptor under readily accessible conditions. Based on these analyses, apparent surface protonation constants for this heterocalixarene were found to lie between 8.5-8.9 (pK(B1)) and 3.3-3.8 (pK(B2)). CPP was found to interact with targeted anionic analytes based on both coulombic and hydrogen bond interactions, as inferred from varying the kinds of ionic sites present within the membrane phase. Potentiometric selectivity studies revealed that CPP preferred "Y-shaped" anions (e.g. acetate, lactate, benzoate) over spherical anions (e.g. fluoride and chloride), fluoride over chloride within the set of spherical anions, and the ortho-isomer over the corresponding meta- and para-isomers in the case of hydroxybenzoate (salicylate and congeners). In the context of this study, the advantages of potentiometric determinations of acetylsalicylic acid using optimized PVC-membranes based on CPP relative to more conventional PVC-membrane ISEs based on traditional anion exchanger were also demonstrated.

• MeSH
• anionty chemie   MeSH
• Aspirin analýza   MeSH
• chemické modely MeSH
• chemické techniky analytické metody   MeSH
• iontově selektivní elektrody * MeSH
• ionty MeSH
• kalixareny chemie   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina salicylová chemie   MeSH
• polymery chemie   MeSH
• potenciometrie metody   MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• anionty MeSH
• Aspirin MeSH
• ionty MeSH
• kalixareny MeSH
• kyselina salicylová MeSH
• polymery MeSH

Herein we introduce 3-vinyl-1,2,4-triazines derivatives as dual-reactive linkers that exhibit selectivity towards cysteine and specific strained alkynes, enabling conjugate addition and inverse electron-demand Diels-Alder (IEDDA) reactions. This approach facilitates site-selective bioconjugation of biologically relevant peptides, followed by rapid and highly selective reactions with bicyclononyne (BCN) reagents.

• MeSH
• alkyny MeSH
• cykloadiční reakce MeSH
• elektrony MeSH
• peptidy * MeSH
• triaziny * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkyny MeSH
• peptidy * MeSH
• triaziny * MeSH

The mechanisms whereby the sodium and potassium binding sites of heart sacrolemmal Na+/K+-ATPase (EC 3.6.1.3) distinguished between monovalent cations were investigated using methods of enzyme kinetics. The properties of the sodium binding sites were studied in the presence of 2,4,6-trinitrobenzenesulfonic acid in concentrations completely inhibiting the action of potassium on the enzyme. To test the selectivity of potassium binding sites, K+-p-nitrophenylphosphatase activity was employed as a model. The results suggest that the selectivity of Na+- and K+-binding sites of Na+/K+-ATPase may be due to two independent mechanisms: (i) The principle of key and lock (formation of coordination bounds); (ii) Optimal difference between solvatation energy (in the specific binding site) and hydration enthalpy of the respective cation.

• MeSH
• chemické modely MeSH
• draslík metabolismus   MeSH
• krysa rodu Rattus MeSH
• matematika MeSH
• myokard enzymologie   MeSH
• sarkolema enzymologie   MeSH
• sodík metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík MeSH
• sodík MeSH
• sodíko-draslíková ATPasa MeSH

Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome-wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.

• MeSH
• Chlamydomonas reinhardtii genetika   MeSH
• fotosystém II (proteinový komplex) genetika   MeSH
• geneticky modifikované rostliny genetika   MeSH
• genom plastidový genetika   MeSH
• homologní rekombinace MeSH
• mutageneze cílená metody   MeSH
• rostlinné proteiny genetika   MeSH
• selekce (genetika) MeSH
• serin genetika   MeSH
• substituce aminokyselin MeSH
• Synechocystis genetika   MeSH
• termotolerance genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• photosystem II, psbA subunit MeSH Prohlížeč
• rostlinné proteiny MeSH
• serin MeSH

Fucosylated compounds are abundantly present in nature and are associated with many biological processes, therefore carrying great potential for use in medicine and biotechnology. Efficient ways to modify fucosylated compounds are still being developed. Promising results are provided by glycosyl hydrolases with transglycosylating activities, such as α-l-fucosidase isoenzyme 2 from Paenibacillus thiaminolyticus (family GH151 of Carbohydrate-Active enZYmes). Currently, there is no 3D structure representing this glycoside hydrolase family and only a few members have been investigated. Here, we present the first structure-function study of a GH151 member, providing the key insights into its specific oligomerization and active site properties. According to the crystal structure, small-angle X-ray scattering data and catalytic investigation, this enzyme functions as a tetramer of a new type and represents the second known case of active site complementation among all α-l-fucosidases. Mutation of the active site-complementing residue histidine 503 to alanine confirmed its influence on α-l-fucosidase activity and, specifically, on substrate binding. Several unique features of GH151 family α-l-fucosidases were revealed, including the oligomerization pattern, active site accessibility and complementation, and substrate selectivity. Some common properties of GH151 glycosyl hydrolases then would be the overall three-domain structure and conservation of the central domain loop 2 function, including its complementation role and the formation of the carbohydrate-binding platform in the active site vicinity.

• Klíčová slova
• GH151, active site complementation, crystal structure, α-l-fucosidase,
• MeSH
• alfa-L-fukosidasa * chemie   genetika   metabolismus   MeSH
• katalytická doména MeSH
• katalýza MeSH
• sacharidy * MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alfa-L-fukosidasa * MeSH
• sacharidy * MeSH

Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcription. CDK7 plays a pivotal role in cell division and proliferation, and the CDK7 gene often exhibits mutations or copy number loss in cancer. Pharmacological targeting of CDK7 has been proposed as a cancer treatment strategy and several inhibitors are currently in clinical trials. As opposed to CDK2, the use of structure-assisted drug design for CDK7 has been limited. We present here CDK2m7, a CDK2-based CDK7 mimic created by mutagenesis of the CDK2 active site pocket. CDK2m7 can be produced in E. coli in a fully active complex with cyclin A2 in high yield and purity. CDK2m7 exhibits a shift in inhibitor selectivity from CDK2 to CDK7 and readily crystallizes. Therefore, it can be used in structure-assisted design of CDK7 inhibitors, as demonstrated by the crystal structure of the complex with inhibitor SY5609. CDK2m7 thus represents a simple and affordable platform for CDK7 rational drug development.

• Klíčová slova
• Cyclin-dependent kinase, Inhibitor, SY5609, Selectivity, Structure-assisted inhibitor design, X-ray crystallography,
• MeSH
• cyklin-dependentní kinasa 2 * chemie   genetika   metabolismus   antagonisté a inhibitory   MeSH
• cyklin-dependentní kinasy * chemie   antagonisté a inhibitory   metabolismus   genetika   MeSH
• inhibitory proteinkinas * farmakologie   chemie   MeSH
• katalytická doména MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární mimikry MeSH
• molekulární modely MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CDK2 protein, human MeSH Prohlížeč
• CDK7 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 * MeSH
• cyklin-dependentní kinasy * MeSH
• inhibitory proteinkinas * MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH

Liquid-jet photoelectron spectroscopy was applied to determine the first acid dissociation constant (pKa) of aqueous-phase glucose while simultaneously identifying the spectroscopic signature of the respective deprotonation site. Valence spectra from solutions at pH values below and above the first pKa reveal a change in glucose's lowest ionization energy upon the deprotonation of neutral glucose and the subsequent emergence of its anionic counterpart. Site-specific insights into the solution-pH-dependent molecular structure changes are also shown to be accessible via C 1s photoelectron spectroscopy. The spectra reveal a considerably lower C 1s binding energy of the carbon site associated with the deprotonated hydroxyl group. The occurrence of photoelectron spectral fingerprints of cyclic and linear glucose prior to and upon deprotonation are also discussed. The experimental data are interpreted with the aid of electronic structure calculations. Our findings highlight the potential of liquid-jet photoelectron spectroscopy to act as a site-selective probe of the molecular structures that underpin the acid-base chemistry of polyprotic systems with relevance to environmental chemistry and biochemistry.

• Publikační typ
• časopisecké články 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
• Názvy látek
• 2-aminopurin MeSH
• amilorid MeSH
• APEX1 protein, human MeSH Prohlížeč
• DNA-lyasa (apurinová nebo apyrimidinová) MeSH
• DNA MeSH
• inhibitory enzymů MeSH
• protinádorové látky MeSH
• železnaté sloučeniny MeSH