Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

• Klíčová slova
• Disulfide bond forming protein, Electrochemical sensing, Membrane proteins, Oxidoreductase, Surface inhibition,
• MeSH
• adsorpce MeSH
• elektrochemické techniky * MeSH
• elektrody MeSH
• molekulární modely MeSH
• povrchové vlastnosti MeSH
• prášky, zásypy, pudry chemie   MeSH
• proteindisulfidreduktasa (glutathion) analýza   antagonisté a inhibitory   metabolismus   MeSH
• rekombinantní proteiny analýza   metabolismus   MeSH
• rtuť chemie   MeSH
• stříbro chemie   farmakologie   MeSH
• uhlík chemie   MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prášky, zásypy, pudry MeSH
• proteindisulfidreduktasa (glutathion) MeSH
• rekombinantní proteiny MeSH
• rtuť MeSH
• stříbro MeSH
• uhlík MeSH

The stabilization of the reduction state of proteins and peptides is very important for the monitoring of protein-protein, protein-DNA and protein-xenobiotic interactions. The reductive state of protein or peptide is characterized by the reactive sulfhydryl group. Glutathione in the reduced (GSH) and oxidized (GSSG) forms was studied by cyclic voltammetry. Tris(2-carboxyethyl)phosphine (TCEP) as the disulfide bond reductant and/or hydrogen peroxide as the sulfhydryl group oxidant were used. Cyclic voltammetry measurements, following the redox state of glutathione, were performed on a hanging mercury drop electrode (HMDE) in borate buffer (pH 9.2). It was shown that in aqueous solutions TCEP was able to reduce disulfide groups smoothly and quantitatively. The TCEP response at -0.25 V vs. Ag/AgCl/3 M KCl did not disturb the signals of the thiol/disulfide redox couple. The origin of cathodic and anodic signals of GSH (at -0.44 and -0.37 V) and GSSG (at -0.69 and -0.40 V) glutathione forms is discussed. It was shown that the application of TCEP to the conservation of sulfhydryl groups in peptides and proteins can be useful instrument for the study of peptides and proteins redox behavior.

• MeSH
• biokompatibilní materiály chemie   MeSH
• disulfidy analýza   chemie   MeSH
• elektrochemie metody   MeSH
• fosfiny chemie   MeSH
• glutathion analýza   chemie   MeSH
• glutathiondisulfid analýza   chemie   MeSH
• oxidace-redukce MeSH
• testování materiálů metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biokompatibilní materiály MeSH
• disulfidy MeSH
• fosfiny MeSH
• glutathion MeSH
• glutathiondisulfid MeSH
• tris(2-carboxyethyl)phosphine MeSH Prohlížeč

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.

• MeSH
• aspartátové endopeptidasy MeSH
• bromkyan MeSH
• disulfidy metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• glykosylace MeSH
• metabolismus sacharidů * MeSH
• molekulární sekvence - údaje MeSH
• Mucor enzymologie   MeSH
• peptidové fragmenty MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů * MeSH
• sekvenční homologie nukleových kyselin * MeSH
• serinové endopeptidasy MeSH
• trypsin MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• bromkyan MeSH
• disulfidy MeSH
• endopeptidasy MeSH
• glutamyl endopeptidase MeSH Prohlížeč
• peptidové fragmenty MeSH
• serinové endopeptidasy MeSH
• trypsin MeSH

Proper formation of disulfide bonds in proteins is a prerequisite to their stability and function. Information on disulfide pattern may therefore serve as an indication of the proper folding of recombinant proteins, and can also be used in protein homology modeling for the purpose of structure refinement. Protein handling and digestion at basic pH leads to disulfide bond scrambling. That is why the samples are usually treated and digested at low pH where no scrambling occurs. Unfortunately, the specific proteases used in protein research are active at high pH values. Here, we present a complete sample handling protocol, which allows processing of disulfide containing proteins at basic pH. We modified the standard SDS gel electrophoresis and protein digestion conditions by the addition of an oxidative agent, cystamine. This modification prevented disulfide scrambling, which we otherwise observed in the samples handled according to the general protocol. Lysozyme from hen egg was used as a model protein for the development of the method. We then applied our protocol to human leukocyte antigen CD69, for which the disulfide bonding is known, but only for its monomeric form. In addition, the disulfide arrangement was then 'de novo' identified in the recombinant murine leukocyte receptor NKR-P1A and in the larger glycosylated proteins beta-N-acetylhexosaminidases from Aspergillus oryzae and Penicillium oxalicum.

• MeSH
• disulfidy chemie   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proteiny chemie   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• proteiny MeSH

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-beta2-microglobulin mAb BBM.1 revealed the presence of an approximately equal 78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-Greceptor interactions and for the search for specific receptors that bind HLA-G.

• MeSH
• antigeny povrchové chemie   MeSH
• choriokarcinom imunologie   patologie   MeSH
• cystein chemie   MeSH
• cystin chemie   MeSH
• dimerizace MeSH
• dithiothreitol farmakologie   MeSH
• geny MHC třídy II MeSH
• histokompatibilita - antigeny třídy I chemie   MeSH
• HLA antigeny chemie   MeSH
• HLA-G antigeny MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• molekulová hmotnost MeSH
• nádorové buňky kultivované MeSH
• nádory dělohy imunologie   patologie   MeSH
• oxidace-redukce MeSH
• receptory imunologické genetika   metabolismus   MeSH
• receptory KIR MeSH
• receptory KIR2DL4 MeSH
• rekombinantní fúzní proteiny chemie   MeSH
• substituce aminokyselin MeSH
• těhotenství MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antigeny povrchové MeSH
• cystein MeSH
• cystin MeSH
• dithiothreitol MeSH
• histokompatibilita - antigeny třídy I MeSH
• HLA antigeny MeSH
• HLA-G antigeny MeSH
• KIR2DL4 protein, human MeSH Prohlížeč
• receptory imunologické MeSH
• receptory KIR MeSH
• receptory KIR2DL4 MeSH
• rekombinantní fúzní proteiny MeSH

We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl-tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild-type showed resistance to oxidation with H2 O2 , while the activities of cysteine-to-serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.

• Klíčová slova
• aminoacyl-tRNA synthetase, cysteine reactivity, disulfide bond, hydrogen peroxide, oxidative stress, thermal stability,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• cystein genetika   metabolismus   MeSH
• disulfidy MeSH
• oxidace-redukce MeSH
• rostliny metabolismus   MeSH
• serin-tRNA-ligasa * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• disulfidy MeSH
• serin-tRNA-ligasa * MeSH

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells.

• Klíčová slova
• Anterior Gradient-2, Aptamers, Cancer, Protein mass spectrometry, Therapeutics, p53,
• MeSH
• ATPázy spojené s různými buněčnými aktivitami MeSH
• chemorezistence genetika   MeSH
• cystein genetika   metabolismus   MeSH
• disulfidy chemie   metabolismus   MeSH
• DNA-helikasy chemie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kyseliny sulfenové metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mukoproteiny MeSH
• multimerizace proteinu genetika   MeSH
• mutace MeSH
• nádory chemie   genetika   patologie   MeSH
• onkogenní proteiny MeSH
• oxidace-redukce * MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin genetika   MeSH
• signální transdukce MeSH
• transportní proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• ATPázy spojené s různými buněčnými aktivitami MeSH
• cystein MeSH
• disulfidy MeSH
• DNA-helikasy MeSH
• kyseliny sulfenové MeSH
• mukoproteiny MeSH
• onkogenní proteiny MeSH
• proteiny MeSH
• RUVBL2 protein, human MeSH Prohlížeč
• transportní proteiny MeSH

Per(2,3,6-tri-O-benzyl)-γ-cyclodextrin was debenzylated by DIBAL-H to produce a mixture of C6(I),C6(IV) and C6(I),C6(V) isomeric diols, which were separated and isolated. The C2-symmetrical C6(I),C6(V) diol was transformed into dithiol and dimerized to produce a γ-cyclodextrin duplex structure. A crystal structure revealed tubular cavity whose peripheries are slightly elliptically distorted. The solvent accessible volume of the cavity of the γ-CD duplex is about 740 Å(3). Due to this large inner space the duplex forms very stable inclusion complexes with steroids; bile acids examined in this study show binding affinities to the γ-cyclodextrin duplex in the range of 5.3 × 10(7) M(-1)-1.9 × 10(8) M(-1).

• MeSH
• chemie farmaceutická metody   MeSH
• dimerizace MeSH
• disulfidy chemie   MeSH
• gama-cyklodextriny chemická syntéza   chemie   MeSH
• imatinib mesylát chemie   MeSH
• kalorimetrie MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• krystalografie rentgenová MeSH
• kyselina lithocholová chemie   MeSH
• kyslík chemie   MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární konformace MeSH
• rozpouštědla chemie   MeSH
• steroidy chemie   MeSH
• sulfhydrylové sloučeniny chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• gama-cyklodextriny MeSH
• gamma-cyclodextrin MeSH Prohlížeč
• imatinib mesylát MeSH
• kyselina lithocholová MeSH
• kyslík MeSH
• rozpouštědla MeSH
• steroidy MeSH
• sulfhydrylové sloučeniny MeSH

The antimicrobial 40-amino-acid-peptide lucifensin was synthesized by native chemical ligation (NCL) using N-acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid-phase synthesis is imcompatible. Two models of ligation were developed: [15+25] Ala-Cys and [19+21] His-Cys. The [19+21] His-Cys method gives lower yield because of the lower stability of 18-peptide-His-Nbz-CONH2 peptide, as suggested by density functional theory calculation. Acetamidomethyl-deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1-6], [C3-5], and [C2-4], which limits its ability to form α-helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one-pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium.

• Klíčová slova
• CD spectroscopy, DTT reduction, antimicrobial activity, lucifensin, native chemical ligation, solid-phase peptide synthesis,
• MeSH
• antiinfekční látky * chemická syntéza   chemie   farmakologie   MeSH
• defensiny * chemická syntéza   chemie   farmakologie   MeSH
• grampozitivní bakterie růst a vývoj   MeSH
• kationické antimikrobiální peptidy * chemická syntéza   chemie   farmakologie   MeSH
• sekundární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiinfekční látky * MeSH
• defensiny * MeSH
• kationické antimikrobiální peptidy * MeSH
• lucifensin MeSH Prohlížeč

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

• Klíčová slova
• DDB1, HBx, Smc5/6 complex, hepatitis B virus,
• MeSH
• aminokyselinové motivy MeSH
• aminokyseliny MeSH
• DNA vazebné proteiny metabolismus   MeSH
• hepatitida B metabolismus   virologie   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• rekombinantní fúzní proteiny MeSH
• sekvence aminokyselin MeSH
• trans-aktivátory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virové regulační a přídatné proteiny MeSH
• virus hepatitidy B fyziologie   MeSH
• zinek metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminokyseliny MeSH
• DDB1 protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• hepatitis B virus X protein MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• trans-aktivátory MeSH
• virové regulační a přídatné proteiny MeSH
• zinek MeSH