In this study, two complementary approaches, affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations, have been employed for quantitative characterization and structure elucidation of the complex between hexaarylbenzene (HAB)-based receptor R and lithium ion Li(+) . First, by means of ACE, the apparent binding constant of LiR(+) complex (K LiR +) in methanol was determined from the dependence of the effective electrophoretic mobilities of LiR(+) complex on the concentration of lithium ions in the 25 mM Tris/50 mM chloroacetate background electrolyte (BGE) using non-linear regression analysis. Prior to regression analysis, the effective electrophoretic mobilities of the LiR(+) complex were corrected to reference temperature 25 °C and constant ionic strength 25 mM. The apparent binding constant of the LiR(+) complex in the above methanolic BGE was evaluated as logK LiR + = 1.15±0.09. Second, the most probable structures of nonhydrated LiR(+) and hydrated LiR(+)·3H(2)O complexes were derived by DFT calculations. The optimized structure of the hydrated LiR(+)·3H(2)O complex was found to be more realistic than the nonhydrated LiR(+) complex because of the considerably higher binding energy of LiR(+)·3H(2)O complex (500.4 kJ/mol) as compared with LiR(+) complex (427.5 kJ/mol).

• MeSH
• benzenové deriváty analýza   MeSH
• chromatografie afinitní MeSH
• elektroforéza kapilární MeSH
• ionty analýza   MeSH
• kvantová teorie * MeSH
• lithium analýza   MeSH
• molekulární struktura MeSH
• osmolární koncentrace MeSH
• teplota MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzenové deriváty MeSH
• ionty MeSH
• lithium MeSH

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

• MeSH
• chromatografie afinitní * MeSH
• cytosol enzymologie   MeSH
• ekdysteron metabolismus   MeSH
• enzymy imobilizované MeSH
• ribulosa-1,5-bisfosfát-karboxylasa izolace a purifikace   metabolismus   MeSH
• Spinacia oleracea enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ekdysteron MeSH
• enzymy imobilizované MeSH
• ribulosa-1,5-bisfosfát-karboxylasa MeSH

Cobalt binding proteins from mouse liver, which were expressed in response to CoCl2 poisoning, were separated using gel permeation chromatography and then immobilised metal ion affinity chromatography (IMAC) with immobilized cobalt ions. Conditions used in IMAC-Co2+ were optimised. The fractions eluted with 60 mM imidazole were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Differences between the samples were also evaluated by a two-dimensional electrophoresis. Samples from the Co2+-treated mice provided higher number of electrophoretic spots than those from the untreated mice. Relative molecular masses of these proteins are appropriately 37,000; 32,000 and 26,000 and their isoelectric points (pI) are 6.5-7.5.

• MeSH
• chromatografie afinitní metody   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• kobalt metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proteiny izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kobalt MeSH
• proteiny MeSH

Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)-enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β-cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)-enantiomers of ANPs-based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0-25 mM) of βCD. The apparent binding constants of the complexes of (R,S)-enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)-enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of (R,S)-enantiomers of ANPs with βCD have been found to be relatively weak - their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3-46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3-55.2 L/mol.

• Klíčová slova
• Acyclic nucleoside phosphonates, Affinity capillary electrophoresis, Binding constant, Nucleotide analogs, β-Cyclodextrin,
• MeSH
• beta-cyklodextriny chemie   MeSH
• elektroforéza kapilární metody   MeSH
• nukleosidy chemie   izolace a purifikace   MeSH
• organofosfonáty chemie   izolace a purifikace   MeSH
• stereoizomerie MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-cyklodextriny MeSH
• betadex MeSH Prohlížeč
• nukleosidy MeSH
• organofosfonáty MeSH

Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.

• Klíčová slova
• Affinity chromatography, Affinity depletion, Affinity enrichment, Mass spectrometry, Peptide, Protein, Proteomics,
• MeSH
• chromatografie afinitní * metody   MeSH
• lidé MeSH
• proteiny izolace a purifikace   analýza   chemie   MeSH
• proteomika * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteiny MeSH

Nearly all of biology depends on interactions between molecules: proteins with small molecules, proteins with other proteins, nucleic acids with small molecules, and nucleic acids with proteins that regulate gene expression, our concern in this Special Issue. All those kinds of interactions, and others, constitute the vast majority of biology at the molecular level. An understanding of those interactions requires that we quantify them to learn how they interact: How strongly? With which partners? How-and how well-are different partners distinguished? This review addresses the evolution of our current understanding of the molecular origins of affinity and specificity in regulatory protein-DNA interactions, and suggests that both these properties can be modulated by cooperativity.

• Klíčová slova
• biological constraints, cryptic thermodynamic factors, drug design, gestalt properties of proteins, host–guest chemistry, pre-organization, protein folding coupled to ligand binding,
• MeSH
• bakteriální proteiny metabolismus   MeSH
• biologické modely MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• fyziologie bakterií * MeSH
• interakce mikroorganismu a hostitele * MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

• Klíčová slova
• deubiquitinating enzyme, polyubiquitin, ubiquitin binding,
• MeSH
• alanin genetika   MeSH
• cystein genetika   MeSH
• deubikvitinasy chemie   genetika   MeSH
• endopeptidasy chemie   genetika   MeSH
• katalýza MeSH
• konformace proteinů MeSH
• lidé MeSH
• mutace genetika   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• specifické proteázy ubikvitinu chemie   genetika   MeSH
• substituce aminokyselin genetika   MeSH
• trans-aktivátory chemie   genetika   MeSH
• transportní proteiny chemie   genetika   MeSH
• ubikvitin chemie   genetika   MeSH
• ubikvitinace genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alanin MeSH
• cystein MeSH
• deubikvitinasy MeSH
• endopeptidasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SAGA complex, S cerevisiae MeSH Prohlížeč
• specifické proteázy ubikvitinu MeSH
• trans-aktivátory MeSH
• transportní proteiny MeSH
• ubikvitin MeSH
• UBP8 protein, S cerevisiae MeSH Prohlížeč
• USP4 protein, human MeSH Prohlížeč

[11C]-PK11195 (PK11195) has been widely used with positron emission tomography (PET) to assess levels of the translocator protein 18 kDa (TSPO) as a marker of neuroinflammation. Recent ligands, such as [11C]-PBR28 and [11C]-DPA713, have improved signal-to-noise ratio and specificity for TSPO over PK11195. However, these second generation radiotracers exhibit binding differences due to a single polymorphism (rs6971) that leads to three genotypes: C/C, C/T and T/T associated with high, mixed and low binding affinities, respectively. Here we report that [3H]-DPA-713 in the presence of cholesterol or PK11195 has an accelerated dissociation rate from TSPO in platelets isolated from individuals with the T/T genotype. This allosteric interaction was not observed in platelets isolated from individuals with the C/C or C/T genotype. The results provide a molecular rationale for low binding affinity of T/T TSPO and further support the exclusion of these subjects from PET imaging studies using second generation TSPO ligands.

• Klíčová slova
• Allosteric modulation, Residence time, Translocator protein 18 KDa (TSPO),
• MeSH
• genotyp MeSH
• lidé MeSH
• neurozobrazování metody   MeSH
• pozitronová emisní tomografie metody   MeSH
• radiofarmaka chemie   MeSH
• receptory GABA analýza   chemie   genetika   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• radiofarmaka MeSH
• receptory GABA MeSH
• TSPO protein, human MeSH Prohlížeč

The locations of three bilirubin (BR)-binding sites with different affinities were identified as subdomains IB, IIA and IIIA for five mammalian serum albumins (SAs): human (HSA), bovine (BSA), rat, (RSA), rabbit (RbSA) and sheep (SSA). The stereoselectivity of a high-affinity BR-binding site was identified in the BR/SA=1/1 system by circular dichroism (CD) spectroscopy, the sites with low affinity to BR were analyzed using difference CD. Site-specific ligand-competition experiments with ibuprofen (marker for subdomain IIIA) and hemin (marker for subdomain IB) did not reveal any changes for the BR/SA=1/1 system and showed a decrease of the bound BR at BR/SA=3/1. Both sites were identified as sites with low affinity to BR. The correlation between stereoselectivity and the arrangement of Arg-Lys residues indicated similarity between the BR-binding sites in subdomain IIIA for all of the SAs studied. Subdomain IB in HSA, BSA, SSA and RbSA has P-stereoselectivity while in RSA it has M-selectivity toward BR. A ligand-competition experiment with gossypol shows a decrease of the CD signal of bound BR for the BR/SA=1/1 system as well as for BR/SA=3/1. Subdomain IIA was assigned as a high-affinity BR-binding site. The P-stereoselectivity of this site in HSA (and RSA, RbSA) was caused by the right-hand localization of charged residues R257/R218-R222, whereas the left-hand orientation of R257/R218-R199 led to the M-stereoselectivity of the primary binding site in BSA (and SSA).

• Klíčová slova
• BR, BSA, Bilirubin, Binding site, CD, GS, HSA, High affinity, Ibf, Ligand-completion, Low affinity, PSB, RSA, RbSA, SA, SSA, Stereoselectivity, bilirubin, bovine serum albumin, circular dichroism, dCD, difference circular dichroism, gossypol, human serum albumin, ibuprofen, phosphate saline buffer, rabbit serum albumin, rat serum albumin, serum albumin, sheep serum albumin,
• MeSH
• aminokyselinové motivy MeSH
• bilirubin chemie   metabolismus   MeSH
• cirkulární dichroismus MeSH
• hemin chemie   metabolismus   MeSH
• ibuprofen chemie   metabolismus   MeSH
• králíci MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• ligandy * MeSH
• molekulární konformace MeSH
• ovce MeSH
• sérový albumin chemie   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• skot MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bilirubin MeSH
• hemin MeSH
• ibuprofen MeSH
• ligandy * MeSH
• sérový albumin MeSH

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward L: -fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.

• MeSH
• bakteriální adheziny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• fukosa chemie   metabolismus   MeSH
• kalorimetrie metody   MeSH
• kinetika MeSH
• kompetitivní vazba MeSH
• krystalizace MeSH
• kvarterní struktura proteinů MeSH
• lektiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• mutace * MeSH
• počítačová simulace MeSH
• Pseudomonas aeruginosa genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sekvenční delece MeSH
• terciární struktura proteinů MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adhesin, Pseudomonas MeSH Prohlížeč
• bakteriální adheziny MeSH
• fukosa MeSH
• lektiny MeSH
• rekombinantní proteiny MeSH