In protein-based formulations, conformational distortions and attractive interactions may cause insoluble and undesired aggregates. In the case of ionic peptides, including cationic or anionic, commonly electrostatic interactions are the main factors that control structure assembling. In this study, it was proposed that grafting of chitosan (CS) to γ-polyglutamic acid (γ-PGA) might exhibit much strong inhibiting effect on the formation of protein aggregates due to multiple amino groups and hydrophilic properties. To guarantee stable and safe biopharmaceutical formulation, the potency of a variety of stabilizers including sugars (glucose, sucrose), polyols (sorbitol, glycerol), surfactant (Tween 20), salting-out salt (PBS), and also different pH values have been evaluated on stabilizing or destabilizing the native state of CS-g-PGA copolymer using FTIR, CD, DLS, and SDS-PAGE. The comparable analysis revealed that the stability of CS-g-PGA was strongly dependent on pH owing to the polyelectrolyte characteristics of the polymers. Altogether these results implied that CS at optimized conditions might be an important precursor for the pharmaceutical industry and function as a new polymer for aggregation suppression and protein stabilization.

• Klíčová slova
• Chemical stabilization, Chitosan, Protein folding, pH depending structure, γ-polyglutamic acid,
• MeSH
• chitosan * chemie   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina polyglutamová * chemie   analogy a deriváty   MeSH
• rozpouštědla chemie   MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitosan * MeSH
• kyselina polyglutamová * MeSH
• poly(gamma-glutamic acid) MeSH Prohlížeč
• rozpouštědla MeSH

The radiolytic stability of hydrophobic extracting compounds CyMe4-BTBP and CyMe4-BTPhen and a hydrophilic masking agent (PhSO3H)2-BTPhen, widely employed for trivalent minor actinoid and lanthanoid separation, against γ radiation was tested. Even though the solvent with a promising fluorinated diluent BK-1 provides better extraction properties compared to octan-1-ol, its radiation stability is much lower, and no extraction was observed already after an absorbed dose of 150 kGy (CyMe4-BTBP) or 200 kGy (CyMe4-BTPhen). For the (PhSO3H)2-BTPhen hydrophilic masking agent, the results showed that the rate of radiolytic degradation was significantly higher in 0.25 M HNO3 than in 0.5 M HNO3. For both the hydrophobic and hydrophilic agents, degradation was slower in the presence of both organic and aqueous phases during irradiation.

• Publikační typ
• časopisecké články MeSH

γ-Conglutin, a lupin seed protein, is an intriguing protein both in terms of the complexity of its molecular structure and a broad spectrum of unique health-promoting properties manifested in animal and human trials. Moreover, this protein is an evolutionary cornerstone whose physiological significance for the plant has not been determined yet. Herein, a comprehensive characterization of γ-conglutin glycosylation is presented and includes (i) the identification of the N-glycan-bearing site, (ii) the qualitative and quantitative composition of glycan-building saccharides, as well as (iii) the effect of oligosaccharide removal on structural and thermal stability. The obtained results indicate the presence of glycans belonging to different classes attached to the Asn98 residue. In addition, the detachment of the oligosaccharide significantly affects secondary structure composition, which disturbs the oligomerization process. The structural changes were also reflected in biophysical parameters, i.e., at a pH value of 4.5, an increase in γ-conglutin thermal stability was observed for the deglycosylated monomeric form. Collectively, the presented results provide evidence of the high complexity of the post-translational maturation and suggest the possibility of a functional effect that glycosylation might have on γ-conglutin structure integrity.

• Klíčová slova
• N-glycosylation, lupin seed, mass spectrometry, post-translational modification, thermal stability, γ-conglutin,
• MeSH
• glykosylace MeSH
• lidé MeSH
• Lupinus * chemie   MeSH
• rostlinné proteiny * metabolismus   MeSH
• semena rostlinná chemie   MeSH
• zásobní proteiny semen metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• rostlinné proteiny * MeSH
• zásobní proteiny semen MeSH

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2(I) -O-, 3(I) -O-, and 6(I) -O-carboxymethyl-α-, β-, and γ-cyclodextrin and native α-, β-, and γ-cyclodextrin as chiral additives at 0-12 mmol/L for β-cyclodextrin and its derivatives and 0-50 mmol/L for α- and γ-cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin-Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin-Tröger's base complexes.

• Klíčová slova
• Capillary electrophoresis, Chiral separation, Cyclodextrin, Regioisomers, Stability constants,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl-D-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2.

• Klíčová slova
• class 2 cytokine receptors, fibronectin type III domain, interferon-γ receptor 2,
• MeSH
• aminokyselinové motivy MeSH
• disulfidy chemie   MeSH
• glykosylace MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• receptory interferonů chemie   MeSH
• sbalování proteinů MeSH
• stabilita proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• IFNGR2 protein, human MeSH Prohlížeč
• receptory interferonů MeSH

The sugar-phosphate backbone of RNA can exist in diverse rotameric substates, giving RNA molecules enormous conformational variability. The most frequent noncanonical backbone conformation in RNA is α/γ = t/t, which is derived from the canonical backbone by a crankshaft motion and largely preserves the standard geometry of the RNA duplex. A similar conformation also exists in DNA, where it has been extensively studied and shown to be involved in DNA-protein interactions. However, the function of the α/γ = t/t conformation in RNA is poorly understood. Here, we present molecular dynamics simulations of several prototypical RNA structures obtained from X-ray and NMR experiments, including canonical and mismatched RNA duplexes, UUCG and GAGA tetraloops, Loop E, the sarcin-ricin loop, a parallel guanine quadruplex, and a viral pseudoknot. The stability of various noncanonical α/γ backbone conformations was analyzed with two AMBER force fields, ff99bsc0χOL3 and ff99bsc0χOL3 with the recent εζOL1 and βOL1 corrections for DNA. Although some α/γ substates were stable with seemingly well-described equilibria, many were unstable in our simulations. Notably, the most frequent noncanonical conformer α/γ = t/t was unstable in both tested force fields. Possible reasons for this instability are discussed. Our work reveals a potentially important artifact in RNA force fields and highlights a need for further force field refinement.

• MeSH
• aptamery nukleotidové chemie   MeSH
• konformace nukleové kyseliny * MeSH
• krystalografie rentgenová MeSH
• magnetická rezonanční spektroskopie MeSH
• RNA chemie   MeSH
• simulace molekulární dynamiky MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aptamery nukleotidové MeSH
• RNA MeSH

Microtubules, polymers of the heterodimeric protein αβ-tubulin, are indispensable for many cellular activities such as maintenance of cell shape, division, migration, and ordered vesicle transport. In vitro assays to study microtubule functions and their regulation by associated proteins require the availability of assembly-competent purified tubulin. However, tubulin is a thermolabile protein that rapidly converts into a nonpolymerizing state. For this reason, it is usually stored at -80 °C or liquid nitrogen to preserve its conformation and polymerization properties. In this chapter, we describe a method for freeze-drying of assembly-competent tubulin in the presence of nonreducing sugar trehalose, and methods enabling the evaluation of tubulin functions in rehydrated samples.

• Klíčová slova
• Freeze-drying, Microtubules, Stability, Trehalose, Tubulin,
• MeSH
• lidé MeSH
• lyofilizace MeSH
• stabilita proteinů MeSH
• trehalosa chemie   MeSH
• tubulin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• trehalosa MeSH
• tubulin MeSH

Although the general instability of the iron nitride γ'-Fe4N with respect to other phases at high pressure is well established, the actual type of phase transitions and equilibrium conditions of their occurrence are, as of yet, poorly investigated. In the present study, samples of γ'-Fe4N and mixtures of α Fe and γ'-Fe4N powders have been heat-treated at temperatures between 250 and 1000 °C and pressures between 2 and 8 GPa in a multi-anvil press, in order to investigate phase equilibria involving the γ' phase. Samples heat-treated at high-pressure conditions, were quenched, subsequently decompressed, and then analysed ex situ. Microstructure analysis is used to derive implications on the phase transformations during the heat treatments. Further, it is confirmed that the Fe-N phases in the target composition range are quenchable. Thus, phase proportions and chemical composition of the phases, determined from ex situ X-ray diffraction data, allowed conclusions about the phase equilibria at high-pressure conditions. Further, evidence for the low-temperature eutectoid decomposition γ'→α+ε' is presented for the first time. From the observed equilibria, a P-T projection of the univariant equilibria in the Fe-rich portion of the Fe-N system is derived, which features a quadruple point at 5 GPa and 375 °C, above which γ'-Fe4N is thermodynamically unstable. The experimental work is supplemented by ab initio calculations in order to discuss the relative phase stability and energy landscape in the Fe-N system, from the ground state to conditions accessible in the multi-anvil experiments. It is concluded that γ'-Fe4N, which is unstable with respect to other phases at 0 K (at any pressure), has to be entropically stabilised in order to occur as stable phase in the system. In view of the frequently reported metastable retention of the γ' phase during room temperature compression experiments, energetic and kinetic aspects of the polymorphic transition γ'⇌ε' are discussed.

• Klíčová slova
• high pressure, iron nitride, phase diagrams, phase equilibria, phase stability, phase transitions,
• Publikační typ
• časopisecké články MeSH

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.

• Klíčová slova
• H475Y(1561C→T) polymorphism, arene-binding site, crystal structure, folate hydrolase 1, zinc metalloprotease,
• MeSH
• antigeny povrchové chemie   genetika   MeSH
• glutamátkarboxypeptidasa II chemie   genetika   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kyselina polyglutamová chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• polymorfismus genetický MeSH
• stabilita enzymů MeSH
• vazebná místa genetika   MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• folyl-n-gamma-L-glutamic acid MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• kyselina polyglutamová MeSH

Peptide-like foldamers controlled by normal amide backbone hydrogen bonding have been extensively studied, and their folding patterns largely rely on configurational and conformational constraints induced by the steric properties of backbone substituents at appropriate positions. In contrast, opportunities to influence peptide secondary structure by functional groups forming individual hydrogen bond networks have not received much attention. Here, peptide-like foldamers consisting of alternating α,β,γ-triamino acids 3-amino-4-(aminomethyl)-2-methylpyrrolidine-3-carboxylate (AAMP) and natural amino acids glycine and alanine are reported, which were obtained by solution phase peptide synthesis. They form ordered secondary structures, which are dominated by a three-dimensional bridged triazaspiranoid-like hydrogen bond network involving the non-backbone amino groups, the backbone amide hydrogen bonds, and the relative configuration of the α,β,γ-triamino and α-amino acid building blocks. This additional stabilization leads to folding in both nonpolar organic as well as in aqueous environments. The three-dimensional arrangement of the individual foldamers is supported by X-ray crystallography, NMR spectroscopy, chiroptical methods, and molecular dynamics simulations.

• Publikační typ
• časopisecké články MeSH