Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.

• Klíčová slova
• lectins, molecular dynamics, non-canonical amino acids, protein engineering, synthetic glycobiology,
• MeSH
• galektin 1 * chemie   MeSH
• galektiny metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• oligosacharidy chemie   MeSH
• tryptofan * MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• galektin 1 * MeSH
• galektiny MeSH
• ligandy MeSH
• oligosacharidy MeSH
• tryptofan * MeSH

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.

• Klíčová slova
• de novo protein design, enzyme catalysis, enzyme tunnels engineering, haloalkane dehalogenases, protein engineering,
• MeSH
• alkany chemie   metabolismus   MeSH
• biokatalýza MeSH
• halogenované uhlovodíky chemie   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• katalytická doména genetika   MeSH
• kinetika MeSH
• ligandy MeSH
• molekulární struktura MeSH
• mutageneze cílená metody   MeSH
• proteinové domény MeSH
• proteinové inženýrství metody   MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkany MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• halogenované uhlovodíky MeSH
• hydrolasy MeSH
• ligandy MeSH

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

• Klíčová slova
• G-quadruplex, Phen-DC3, dynamics, ligand binding, smFRET,
• MeSH
• chinoliny chemie   metabolismus   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• ligandy * MeSH
• rezonanční přenos fluorescenční energie MeSH
• simulace molekulární dynamiky MeSH
• telomery chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chinoliny MeSH
• ligandy * MeSH

Cytotoxic complexes containing molybdenum are widely studied as a potential substitution for commercially used drugs that often suffer from pronounced side effects and cellular resistance. Compounds of the type [(η5 -Cp')Mo(CO)2 (N,N L)][BF4 ], where Cp is cyclopentadienyl and N,N L is a bidentate ligand, are well known for their strong anticancer activity. It is a generally accepted paradigm that the nature of the coordinated N,N L ligand has a major impact on the cytotoxicity. In this study, a series of new functionalised Cp complexes of molybdenum was synthesised from derivatised fulvenes as π-ligand precursors. Indeed, the coordination sphere's modulation by various N,N-chelating ligands afforded species active toward leukemic cell line MOLT-4 with IC50 values depending on the character of the N,N-chelator used. However, following study clearly showed that functionalisation of the Cp ring with an amine moiety considerably improved cytotoxicity. These results are of crucial importance for the future design of highly active cytotoxic drugs, as modification of cyclopentadienyl is believed to have a minor effect on biological activity.

• Klíčová slova
• MOLT-4, antitumor agents, cyclopentadienyl ligand, cytotoxicity, fulvene,
• MeSH
• buněčné linie MeSH
• cyklopentany chemie   farmakologie   MeSH
• komplexní sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• molybden chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklopentany MeSH
• komplexní sloučeniny MeSH
• ligandy MeSH
• molybden MeSH
• protinádorové látky MeSH

A variety of colloidal chemical approaches has been developed in the last few decades for the controlled synthesis of nanostructured materials in either water or organic solvents. Besides the precursors, the solvents, reducing agents, and the choice of surfactants are crucial for tuning the composition, morphology and other properties of the resulting nanoparticles. The ligands employed include thiols, amines, carboxylic acids, phosphines and phosphine oxides. Generally, adding a single ligand to the reaction mixture is not always adequate to yield the desired features. In this review, we discuss in detail the role of the oleic acid/oleylamine ligand pair in the chemical synthesis of nanoparticles. The combined use of these ligands belonging to two different categories of molecules aims to control the size and shape of nanoparticles and prevent their aggregation, not only during their synthesis but also after their dispersion in a carrier solvent. We show how the different binding strengths of these two molecules and their distinct binding modes on specific facets affect the reaction kinetics toward the production of nanostructures with tailored characteristics. Additional functions, such as the reducing function, are also noted, especially for oleylamine. Sometimes, the carboxylic acid will react with the alkylamine to form an acid-base complex, which may serve as a binary capping agent and reductant; however, its reducing capacity may range from lower to much lower than that of oleylamine. The types of nanoparticles synthesized in the simultaneous presence of oleic acid and oleylamine and discussed herein include metal oxides, metal chalcogenides, metals, bimetallic structures, perovskites, upconversion particles and rare earth-based materials. Diverse morphologies, ranging from spherical nanoparticles to anisotropic, core-shell and hetero-structured configurations are presented. Finally, the relation between tuning the resulting surface and volume nanoparticle properties and the relevant applications is highlighted.

• MeSH
• aminy chemie   MeSH
• kyselina olejová * chemie   MeSH
• ligandy MeSH
• nanočástice * chemie   MeSH
• oxidy MeSH
• rozpouštědla chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aminy MeSH
• kyselina olejová * MeSH
• ligandy MeSH
• oleylamine MeSH Prohlížeč
• oxidy MeSH
• rozpouštědla MeSH

BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

• Klíčová slova
• Deorphanization, Functional evolution, HEK293 cells, Odorant receptor, Pest insect, Pheromone receptor, Xenopus oocyte,
• MeSH
• hmyzí proteiny chemie   genetika   MeSH
• ligandy MeSH
• nosatcovití chemie   genetika   MeSH
• receptory pachové chemie   genetika   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• ligandy MeSH
• receptory pachové MeSH

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

• Klíčová slova
• anti-EGFR antibody, cetuximab, concentration through kinetics, ligand-receptor interaction, radioimmunoassay, radioiodination,
• MeSH
• cetuximab chemie   farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• nádorové buněčné linie MeSH
• protinádorové látky imunologicky aktivní chemie   farmakologie   MeSH
• radiofarmaka chemie   farmakologie   MeSH
• radioimunoanalýza metody   normy   MeSH
• radioizotopy jodu chemie   farmakologie   MeSH
• radioligandová zkouška metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cetuximab MeSH
• ligandy MeSH
• protinádorové látky imunologicky aktivní MeSH
• radiofarmaka MeSH
• radioizotopy jodu MeSH

MOTIVATION: Structure-based methods for detecting protein-ligand binding sites play a crucial role in various domains, from fundamental research to biomedical applications. However, current prediction methodologies often rely on holo (ligand-bound) protein conformations for training and evaluation, overlooking the significance of the apo (ligand-free) states. This oversight is particularly problematic in the case of cryptic binding sites (CBSs) where holo-based assessment yields unrealistic performance expectations. RESULTS: To advance the development in this domain, we introduce CryptoBench, a benchmark dataset tailored for training and evaluating novel CBS prediction methodologies. CryptoBench is constructed upon a large collection of apo-holo protein pairs, grouped by UniProtID, clustered by sequence identity, and filtered to contain only structures with substantial structural change in the binding site. CryptoBench comprises 1107 structures with predefined cross-validation splits, making it the most extensive CBS dataset to date. To establish a performance baseline, we measured the predictive power of sequence- and structure-based CBS residue prediction methods using the benchmark. We selected PocketMiner as the state-of-the-art representative of the structure-based methods for CBS detection, and P2Rank, a widely-used structure-based method for general binding site prediction that is not specifically tailored for cryptic sites. For sequence-based approaches, we trained a neural network to classify binding residues using protein language model embeddings. Our sequence-based approach outperformed PocketMiner and P2Rank across key metrics, including area under the curve, area under the precision-recall curve, Matthew's correlation coefficient, and F1 scores. These results provide baseline benchmark results for future CBS and potentially also non-CBS prediction endeavors, leveraging CryptoBench as the foundational platform for further advancements in the field. AVAILABILITY AND IMPLEMENTATION: The CryptoBench dataset, including the benchmark model, is available on Open Science Framework-https://osf.io/pz4a9/. The code and tutorial are available at the GitHub repository-https://github.com/skrhakv/CryptoBench/.

• MeSH
• benchmarking MeSH
• databáze proteinů MeSH
• konformace proteinů MeSH
• ligandy MeSH
• proteiny * chemie   metabolismus   MeSH
• software * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• výpočetní biologie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny * MeSH

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant, whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown.

• Klíčová slova
• cancer, ligand binding, phosphoglycerate kinase, protein phosphorylation, protein stability,
• MeSH
• adenosindifosfát metabolismus   MeSH
• cytosol * metabolismus   MeSH
• fosfoglycerátkinasa * metabolismus   genetika   chemie   MeSH
• fosforylace MeSH
• kinetika MeSH
• lidé MeSH
• ligandy MeSH
• mutace MeSH
• nádory * genetika   metabolismus   patologie   MeSH
• simulace molekulární dynamiky * MeSH
• stabilita proteinů MeSH
• termodynamika MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosindifosfát MeSH
• fosfoglycerátkinasa * MeSH
• ligandy MeSH

The development of singlet oxygen photosensitizers, which target specific cellular organelles, constitutes a pertinent endeavor to optimize the efficiency of photodynamic therapy. Targeting of the cell membrane eliminates the need for endocytosis of drugs that can lead to toxicity, intracellular degradation, or drug resistance. In this context, we utilized copper-free click chemistry to prepare a singlet oxygen photosensitizing complex, made of a molybdenum-iodine nanocluster stabilized by triazolate apical ligands. In phosphate-buffered saline, the complex formed nanoaggregates with a positive surface charge due to the protonatable amine function of the apical ligands. These nanoaggregates targeted cell membranes and caused an eminent blue-light phototoxic effect against HeLa cells at nanomolar concentrations, inducing apoptotic cell death, while having no dark toxicity at physiologically relevant concentrations. The properties of this complex were compared to those of a negatively charged parent complex to highlight the dominant effect of the nature of apical ligands on biological properties of the nanocluster. These two complexes also exerted (photo)antibacterial effects on several pathogenic strains in the form of planktonic cultures and biofilms. Overall, we demonstrated that the rational design of apical ligands toward cell membrane targeting leads to enhanced photodynamic efficiency.

• MeSH
• buněčná membrána MeSH
• HeLa buňky MeSH
• jod * farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• molybden * farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jod * MeSH
• ligandy MeSH
• molybden * MeSH