Recent theoretical studies performed on the folding/unfolding mechanism of model telomeric human DNA, 5'-AGGGTTAGGGTTAGGGTTAGGG-3' (Tel22), have indicated that in presence K(+) ions Tel22 folds into two hybrid G-quadruplex structures characterized by one double and reversal TTA loops arranged a different way. They predicted new unfolding pathway from initial mixture G-quadruplexes via corresponding intermediate triplex final, fully unfolded state. Significantly, no experimental evidence...

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) triple distilled water 5.5-6.0) was examined by differential scanning calorimetry, UV CD spectroscopy fluorescence emission the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 at temperatures below 60 degrees C, protein exists a native state characterized pronounced tertiary structure, beta-rich secondary structure low degree ANS-binding. higher temperatures, it undergoes two-state...

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% polypeptide is found ordered in crystal. The MazE monomer consisting two β-hairpins connected by a short α-helix has no hydrophobic core on its own and represents only one half typical protein domain. complete structure formed association chains, creating between four-stranded β-sheets. This consists exclusively aliphatic...

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement various ht-DNA conformations in process. Therefore, thermodynamic driving forces and mechanisms G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically structurally netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) two bisquinolinium ligands (Phen-DC3, 360A-Br) fragment (Tel22)...

Toxin-antitoxin (TA) modules are small operons involved in bacterial stress response and persistence. higBA form a family of TA with an inverted gene organization toxin belonging to the RelE/ParE superfamily. Here, we present crystal structures chromosomally encoded Vibrio cholerae antitoxin (VcHigA2), (VcHigB2) their complex, which show significant differences structure mechanisms function compared module from plasmid Rts1, defining member family. The VcHigB2 is more closely related...

Abstract Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region antitoxin HigA2 Vibrio cholerae is neutralize HigB2 toxin through ultra-high-affinity interaction. Here, we show that same also mediate interactions with its operator DNA and, interplay folded helix-turn-helix domain, regulates transcription higBA2 operon. NMR, SAXS, ITC and in...

Many examples are known of regions intrinsically disordered proteins that fold into α-helices upon binding to their targets. These helical motifs (HBMs) can be partially also in the unbound state, and this so-called residual structure affect affinity kinetics. To investigate underlying mechanisms governing formation structure, we assembled a dataset experimental helix contents 65 peptides containing HBM fold-upon-binding. The average helicity is 17% increases 60% target binding. target-bound...

Intrinsically disordered proteins (IDPs) are that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into defined when interacting with specific binding partners. The energetic implications of such behavior have widely discussed, yet experimental thermodynamic data is scarce. We present here thorough and structural study the an IDP (antitoxin CcdA) its molecular target (gyrase poison CcdB). show binding-coupled folding CcdA driven by...

Abstract Why human telomere DNA fragments fold into different G‐quadruplex structures with parallel, hybrid, and antiparallel strand orientations depending on the temperature concentration of co‐solutes remains poorly understood. Similarly, formation intermediate along folding or interconversion pathways is not well Herein, we address these questions by introducing a conceptual framework, based global thermodynamic analysis DSC CD spectroscopy data, which led to detailed description...

Abstract A new folding intermediate of Oxytricha nova telomeric Oxy‐1.5 G‐quadruplex was characterized in aqueous solution using NMR spectroscopy, native gel electrophoresis, thermal differential spectra (TDS), CD and scanning calorimetry (DSC). experiments have revealed that this (i‐Oxy‐1.5) exists two symmetric bimolecular forms which all guanine bases are involved GG N1‐carbonyl base pairs. Kinetic analysis K + ‐induced structural transitions shows from i‐Oxy‐1.5 is much faster proceeds...

An established method for the quantitation of helix content in peptides using circular dichroism (CD) relies on linear spectroscopic model. This model assumes an average value helix-length correction all peptide conformers, irrespective length helical segment. Here we assess validity this approximation and introduce a more physically realistic ensemble-based analysis CD signal which is assigned specifically to each ensemble conformer. We demonstrate that underestimates helicity, with...

Monoclonal antibodies are the fastest growing class of therapeutics. However, aggregation limits their shelf life and can lead to adverse immune responses. Assessment optimization long-term antibody stability therefore key challenges in biologic drug development. Here, we present a platform based on analysis temperature-dependent data that dramatically shorten assessment thus accelerate formulations. For set used therapeutic areas from oncology rheumatology osteoporosis, obtain an accurate...

Expression of recombinant genes can be controlled using inducible promoters. However, the most commonly used IPTG- and arabinose-inducible promoters result in an 'all-or-nothing' response, leading to fully induced uninduced bacterial subpopulations. Here, we investigate whether appropriate modifications these promoter systems combined into a single vector system, enabling homogenous expression two interest that precisely tuned inducer concentration. We show positive feedback loops related...

Abstract Galectin-8 is a tandem-repeat galectin consisting of two distinct carbohydrate recognition domains and potential drug target. We have developed library galectin-8N inhibitors that exhibit high nanomolar K d values as determined by competitive fluorescence polarization assay. A detailed thermodynamic analysis the binding -galactosides to isothermal titration calorimetry reveals important differences in enthalpic and/or entropic contributions binding. Contrary expectations, 2- O...

Cytosine methylation, a key epigenetic modification in the regulation of gene expression, raises intriguing questions about its role formation and thermodynamic stability G‐quadruplex (G4) structures. We investigated impact 5‐methylcytosine residue (Cm) on well‐characterized bcl2Mid G4 structure that forms GC‐rich region B‐cell lymphoma 2 (BCL2) promoter, which influences expression. Using solution‐state NMR biophysical techniques, we discovered an unexpected sequence‐specific effect Cm...

Cytosine methylation, a key epigenetic modification in the regulation of gene expression, raises intriguing questions about its role formation and thermodynamic stability G‐quadruplex (G4) structures. We investigated impact 5‐methylcytosine residue (Cm) on well‐characterized bcl2Mid G4 structure that forms GC‐rich region B‐cell lymphoma 2 (BCL2) promoter, which influences expression. Using solution‐state NMR biophysical techniques, we discovered an unexpected sequence‐specific effect Cm...

Titration calorimetry was employed to study the thermodynamic parameters of micellization N-undecyl-, N-tridecyl-, N-tetradecyl-, and N-pentadecylpyridinium bromides tetradecyl- hexadecyltrimethylammonium in salt-free aqueous solutions at 25 °C. For this purpose a model equation for calorimetric titration curve based on mass action process derived. As adjustable it contains aggregation number n, degree micelle ionization p/n, enthalpy ΔHM. Critical concentrations cmc were determined by...

It is well-known that the addition of salts influences properties proteins in solution. The essential nature this phenomenon far from being fully understood, partly due to absence relevant thermodynamic information. To help fill gap, work isothermal titration calorimetry (ITC) was employed study ion-lysozyme association aqueous buffer solutions at pH = 4.0. ITC curves measured for NaCl, NaBr, NaI, NaNO3, NaSCN, KCl, CaCl2, and BaCl2 three different temperatures were described by a model...

Abstract Many intrinsically disordered proteins (IDP) that fold upon binding retain conformational heterogeneity in IDP‐target complexes. The thermodynamics of such fuzzy interactions is poorly understood. Herein we introduce a thermodynamic framework, based on analysis ITC and CD spectroscopy data, provides experimental descriptions IDP association terms folding contributions which can be predicted using sequence propensities molecular modeling. We show how modulate the entropy enthalpy by...