Influence of a Hairpin Loop on the Thermodynamic Stability of a DNA Oligomer

DSC was used to evaluate the mechanism of the thermally induced unfolding of the single-stranded hairpin HP = 5′-CGGAATTCCGTCTCCGGAATTCCG-3′ and its core duplex D (5′-CGGAATTCCG-3′)2. The DSC melting experiments performed at several salt concentrations were successfully described for HP and D in terms of a three-state transition model HP↔I (intermediate state) ↔ S (unfolded single-stranded state) and two state transition model D↔2S, respectively. Comparison of the model-based thermodynamic parameters obtained for each HP and D transition shows that in unfolding of HP only the HP↔I transition is affected by the TCTC loop. This observation suggests that in the intermediate state its TCTC loop part exhibits significantly more flexible structure than in the folded state while its duplex part remains pretty much unchanged

Modeling of Some Calorimetric and Spectropolarimetric Titration Data

Microcalorimetric and spectropolarimetric titrations were used to investigate micellization of cationic surfactants and binding of netropsin to dodecameric DNA duplex. For description of both processes, model functions containing linear and non-linear parameters were derived. Model analysis was based on a weighted (multi)linear regression and a standard »Simplex« procedure. Close investigation of the interplay of adjustable parameters has shown that a proper choice of the model function can significantly reduce the correlations between parameters. Values of physical properties (enthalpy changes, apparent equilibrium constants...) obtained from such curve modeling are in very good agreement with the corresponding values obtained by other methods. Equations and the calculation procedure reported here could be easily generalized and used for the description of some other concentration dependent properties in similar systems

Differences in Unfolding Energetics of CcdB Toxins From V. fischeri and E. coli

Ccd system is a toxin-antitoxin module (operon) located on plasmids and chromosomes of bacteria. CcdB(F) encoded by ccd operon located on Escherichia coli plasmid F and CcdB(Vfi) encoded by ccd operon located on Vibrio fischeri chromosome are members of the CcdB family of toxins. Native CcdBs are dimers that bind to gyrase-DNA complexes and inhibit DNA transcription and replication. While thermodynamic stability and unfolding characteristics of the plasmidic CcdB(F) in denaturant solutions are reported in detail, the corresponding information on the chromosomal CcdB(Vfi) is rather scarce. Therefore, we studied urea-induced unfolding of CcdB(Vfi) at various temperatures and protein concentrations by circular dichroism spectroscopy. Global model analysis of spectroscopic data suggests that CcdB(Vfi) dimer unfolds to the corresponding monomeric components in a reversible two-state manner. Results reveal that at physiological temperatures CcdB(Vfi) exhibits lower thermodynamic stability compared to CcdB(F). At high urea concentrations CcdB(Vfi), similarly to CcdB(F), retains a significant amount of secondary structure. Differences in thermodynamic parameters of CcdB(Vfi) and CcdB(F) unfolding can reasonably be explained by the differences in their structural features

Differences in Unfolding Energetics of CcdB Toxins From V. fischeri and E. coli

Ccd system is a toxin-antitoxin module (operon) located on plasmids and chromosomes of bacteria. CcdB(F) encoded by ccd operon located on Escherichia coli plasmid F and CcdB(Vfi) encoded by ccd operon located on Vibrio fischeri chromosome are members of the CcdB family of toxins. Native CcdBs are dimers that bind to gyrase-DNA complexes and inhibit DNA transcription and replication. While thermodynamic stability and unfolding characteristics of the plasmidic CcdB(F) in denaturant solutions are reported in detail, the corresponding information on the chromosomal CcdB(Vfi) is rather scarce. Therefore, we studied urea-induced unfolding of CcdB(Vfi) at various temperatures and protein concentrations by circular dichroism spectroscopy. Global model analysis of spectroscopic data suggests that CcdB(Vfi) dimer unfolds to the corresponding monomeric components in a reversible two-state manner. Results reveal that at physiological temperatures CcdB(Vfi) exhibits lower thermodynamic stability compared to CcdB(F). At high urea concentrations CcdB(Vfi), similarly to CcdB(F), retains a significant amount of secondary structure. Differences in thermodynamic parameters of CcdB(Vfi) and CcdB(F) unfolding can reasonably be explained by the differences in their structural features

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)(3) using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; K(Tel22) ≈ <K(dsDNA)) and for highly selective quadruplex-specific ligands (Phen-DC3, 360A-Br; K(Tel22) > K(dsDNA)). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation

STRESS AT TEACHERS WORK

Ljudje smo vse bolj izpostavljeni stresu, tako v zasebnem kot tudi službenem okolju. Stres doživljamo različno in se nanj specifično odzivamo. Obravnavali smo dejavnike, ki povzročajo stres pri delu učitelja. Najprej smo stres obravnavali teoretično. Opisovali smo fenomen stresa, opredelili ključne pojme, specifične stresorje, navedli teorije o stresu, opisali posledice, ki jih stres povzroča in pristope za obvladovanje stresa. Nato smo izvedli raziskavo, v kateri smo s pomočjo ankete, ki smo jo podelili na različnih stopnjah izobraževalnega sistema (osnovna šola, srednja šola in višja strokovna šola), anketirali učitelje. Pridobljene podatke smo analizirali in med seboj primerjali. Z raziskavo smo ugotavljali, ali učitelji doživljajo različne oblike stresa in kakšna je njegova jakost. Želeli smo izvedeti, kako se odzivajo na stresne situacije, na različnih izobraževalnih stopnjah. Ugotovili smo, da se stres po stopnjah izobraževalnega sistema bistveno ne razlikuje, vendar se pojavlja v različnih jakostih in oblikah. Stres največkrat povzroča zlasti zahtevnost dela z otroki, konfliktni odnosi s sodelavci, konfliktni odnosi z vodstvom, zahtevni odnosi s starši in zahteve po stalnem usposabljanju. Ti škodljivi vplivi stresa zmanjšujejo učiteljevo motivacijo za delo, otežujejo medsebojne odnose, povzročajo izgorevanje pri delu, vendar po analizi sodeč ne vplivajo toliko na samo ustvarjalnost pri delu in ne povzročajo večje odsotnosti z dela. Učitelji se v vedno večji meri zavedajo stresa in vpliva le-tega na njihovo delo, zato se poslužujejo raznih tehnik oz. osebnih tehnik za zmanjšanje vpliva stresa na delovnem in osebnem področju. Iz pridobljenih podatkov smo ugotovili tudi, da institucije o ozaveščanju zaposlenih o stresu na delovnem mestu in obvladovanju le-tega ne poskrbijo dovolj. Stres v izobraževalnem procesu je postal stalni učiteljev spremljevalec.People are exposed to stress in private and formal environment. We experience stress differently and respond to it specifically. We discussed the factors caused by stress at teacher’s work. First we discussed stress theoretically. We have presented the phenomenon of stress, specific stressors and the theories of stress. We have also identified key concepts of stressdescribed the consequences that are caused by stress and approaches to stress management. Afterwards we conducted a research at different stages of the educational system by interviewing the teachers. The acquired data of the interviews were analysed and compared with each other. With this research we have stated whether the teachers are experiencing different forms of stress and what is the strength of the stress. We also wanted to know about their reactions in stressful situations at different stages of the educational system. We have stated that stress is not significantly different at educational system stages, but it is occurring in different strengths and forms. The complexity of working with children, conflict relations with colleagues, conflict relations with the management, complex relationships with parents and requirements of continuous training are mostly the cause of stress. These harmful effects of stress reduce teacher’s motivation causing difficult relations and burning out, but the analysis shows that these harmful effects do not have too much affect on the creativity at work and do not cause absence from work. Teachers are aware of stress and its impact on work and personality and that is why they are using different techniques or personal techniques to reduce the impact of stress at work and at personal level. From the obtained data we have concluded that institutions do not do enough to inform teachers of stress at work and how to manage it. Stress has become the permanent teacher’s companion in the educational processes

Conditional cooperativity in DNA minor-groove recognition by oligopeptides

The recognition of specific DNA sequences in processes such as transcription is associated with a cooperative binding of proteins. Some transcription regulation mechanisms involve additional proteins that can influence the binding cooperativity by acting as corepressors or coactivators. In a conditional cooperativity mechanism, the same protein can induce binding cooperativity at one concentration and inhibit it at another. Here, we use calorimetric (ITC) and spectroscopic (UV, CD) experiments to show that such conditional cooperativity can also be achieved by the small DNA-directed oligopeptides distamycin and netropsin. Using a global thermodynamic analysis of the observed binding and (un)folding processes, we calculate the phase diagrams for this system, which show that distamycin binding cooperativity is more pronounced at lower temperatures and can be first induced and then reduced by increasing the netropsin or/and Na+ ion concentration. A molecular interpretation of this phenomenon is suggested